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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    CONCISE INTERNATIONAL CHEMICAL ASSESSMENT DOCUMENT NO. 5


    LIMONENE

    INTER-ORGANIZATION PROGRAMME FOR THE SOUND MANAGEMENT OF CHEMICALS
    A cooperative agreement among UNEP, ILO, FAO, WHO, UNIDO, UNITAR and
    OECD

    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.


    First draft prepared by
    Dr A. Falk Filipsson, National Institute for Working Life, Solna,
    Sweden,
    Mr J. Bard, Aseda, Sweden, and
    Ms S. Karlsson, National Chemicals Inspectorate, Solna, Sweden


    Published under the joint sponsorship of the United Nations
    Environment Programme, the International Labour Organisation, and the
    World Health Organization, and produced within the framework of the
    Inter-Organization Programme for the Sound Management of Chemicals.


    World Health Organization                    Geneva, 1998

         The International Programme on Chemical Safety (IPCS),
    established in 1980, is a joint venture of the United Nations
    Environment Programme (UNEP), the International Labour Organisation
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    assessment of the risk to human health and the environment from
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    technical assistance in strengthening national capacities for the
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         The Inter-Organization Programme for the Sound Management of
    Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and
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    Nations Industrial Development Organization, and the Organisation for
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    Environment and Development to strengthen cooperation and increase
    coordination in the field of chemical safety.  The purpose of the IOMC
    is to promote coordination of the policies and activities pursued by
    the Participating Organizations, jointly or separately, to achieve the
    sound management of chemicals in relation to human health and the
    environment.

    WHO Library Cataloguing in Publication Data

    Limonene.

         (Concise international chemical assessment document ; 5)

         1.Terpenes - toxicity  2.Environmental exposure
         3.Food contamination  I.International Programme on Chemical
         Safety  II.Series

         ISBN 92 4 153005 7       (NLM Classification: QV 633)
         ISSN 1020-6167

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    TABLE OF CONTENTS

         FOREWORD

    1. EXECUTIVE SUMMARY

    2. IDENTITY AND PHYSICAL/CHEMICAL PROPERTIES

    3. ANALYTICAL METHODS

    4. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

    5. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

    6. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

         6.1. Environmental levels
         6.2. Human exposure

    7. COMPARATIVE KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

    8. EFFECTS ON LABORATORY MAMMALS AND  IN VITRO TEST SYSTEMS

         8.1. Single exposure
         8.2. Irritation and sensitization
         8.3. Short-term exposure
         8.4. Long-term exposure
              8.4.1. Subchronic exposure
              8.4.2. Chronic exposure and carcinogenicity
         8.5. Genotoxicity and related end-points
         8.6. Reproductive and developmental toxicity
         8.7. Immunological and neurological effects

    9. EFFECTS ON HUMANS

    10. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

         10.1. Aquatic environment
         10.2. Terrestrial environment

    11. EFFECTS EVALUATION

         11.1. Evaluation of health effects
              11.1.1. Hazard identification and dose-response assessment
                     
              11.1.2. Criteria for setting guidance values for limonene
                     
              11.1.3. Sample risk characterization
         11.2. Evaluation of environmental effects

    12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

    13. HUMAN HEALTH PROTECTION AND EMERGENCY ACTION

         13.1. Human health hazards
         13.2. Advice to physicians
         13.3. Storage
         13.4. Spillage

    14. CURRENT REGULATIONS, GUIDELINES, AND STANDARDS

         INTERNATIONAL CHEMICAL SAFETY CARD

         REFERENCES

         APPENDIX 1 - SOURCE DOCUMENT

         APPENDIX 2 - CICAD PEER REVIEW

         APPENDIX 3 - CICAD FINAL REVIEW BOARD

         RÉSUMÉ D'ORIENTATION

         RESUMEN DE ORIENTACION
    

    FOREWORD

         Concise International Chemical Assessment Documents (CICADs) are
    the latest in a family of publications from the International
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         CICADs are concise documents that provide summaries of the
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    conclusions drawn.

         The primary objective of CICADs is characterization of hazard and
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         Risks to human health and the environment will vary considerably
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    of locally measured or predicted exposure scenarios.  To assist the
    reader, examples of exposure estimation and risk characterization are
    provided in CICADs, whenever possible.  These examples cannot be
    considered as representing all possible exposure situations, but are
    provided as guidance only.  The reader is referred to EHC 1701 for
    advice on the derivation of health-based guidance values.


              

    1 International Programme on Chemical Safety (1994)  Assessing
     human health risks of chemicals: derivation of guidance values for
    health-based exposure limits. Geneva, World Health Organization
    (Environmental Health Criteria 170).

         While every effort is made to ensure that CICADs represent the
    current status of knowledge, new information is being developed
    constantly.  Unless otherwise stated, CICADs are based on a search of
    the scientific literature to the date shown in the executive summary. 
    In the event that a reader becomes aware of new information that would
    change the conclusions drawn in a CICAD, the reader is requested to
    contact the IPCS to inform it of the new information.

    Procedures

         The flow chart shows the procedures followed to produce a CICAD. 
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    FIGURE 1

    1.  EXECUTIVE SUMMARY

         This CICAD on limonene  (d-limonene,  l-limonene, and
     d/l-limonene) was based primarily on a review prepared in 1993 for
    the Nordic Expert Group (Karlberg & Lindell, 1993).  A second review
    produced under the auspices of the Nordic Council of Ministers
    (Josefsson, 1993), a preliminary, non-peer-reviewed information source
    on environmental exposure and effects (US EPA, 1994), and searches of
    relevant databases covering the years 1993-1995 were used for the
    identification of additional data for the assessment of limonene.  In
    a final search of the literature from 1996 to 1997, no data that would
    change the conclusions made in the CICAD were identified.  Information
    concerning the nature and availability of the source document is
    presented in Appendix 1.  Information on the peer review of this CICAD
    is presented in Appendix 2.  This CICAD was approved for publication
    at a meeting of the Final Review Board, held in Brussels, Belgium, on
    18-20 November 1996.  Participants at the Final Review Board meeting
    are listed in Appendix 3.  The International Chemical Safety Card
    (ICSC 0918) for  d-limonene, produced by the International Programme
    on Chemical Safety (IPCS, 1993), has also been reproduced in this
    document.  Emphasis was given to  d-limonene owing to the large
    amount of available data on this isomeric form.

         Limonene occurs naturally in certain trees and bushes.  Limonene
    and other monoterpenes are released in large amounts mainly to the
    atmosphere, from both biogenic and anthropogenic sources.  Limonene is
    used as a solvent in degreasing metals prior to industrial painting,
    for cleaning in the electronic and printing industries, and in paint
    as a solvent.  Limonene is also used as a flavour and fragrance
    additive in food, household cleaning products, and perfumes.

         Limonene is a skin irritant in both experimental animals and
    humans.  In rabbits,  d-limonene was found to be an eye irritant. 
    Studies in guinea-pigs revealed that air-oxidized  d-limonene, but
    not  d-limonene itself, induced contact allergy.  Because  d- and
     l-limonene are enantiomers, this could also be true for  l-limonene
    and dipentene (the mixture).  Handling and purity of the chemical, and
    possibly addition of antioxidants, may thus be crucial for the
    allergenic capacity of limonene.

         The critical organ in animals (except for male rats), following
    peroral or intraperitoneal administration, is the liver.  Studies in
    which experimental animals were exposed by inhalation to limonene have
    not been identified.  Exposure to limonene affects the amount and
    activity of different liver enzymes, liver weight, cholesterol levels,
    and bile flow.  These changes have been observed in mice, rats, and
    dogs.  Available data are insufficient to determine the critical organ
    in humans.

         In male rats, exposure to  d-limonene causes damage to the
    kidneys and renal tumours.  The male rat specific protein
    alpha2µ-globulin is considered to play a crucial role in the
    development of neoplastic as well as non-neoplastic kidney lesions. 

    Thus, these kidney lesions are considered not relevant for human risk
    assessment.   d-Limonene has been studied in a battery of short-term
     in vitro tests and found to be non-genotoxic.  There is no evidence
    that limonene has teratogenic or embryotoxic effects in the absence of
    maternal toxicity. In general,  d-limonene could be considered (with
    the exception of its irritative and sensitizing properties) to be a
    chemical with fairly low toxicity.

         Food is the principal source of exposure to limonene, based on
    available data.  A guidance value for the ingestion of limonene was
    calculated to be 0.1 mg/kg body weight per day.  At current estimated
    levels of exposure, limonene in foodstuffs does not appear to
    represent a significant risk to human health.

         In the atmosphere, limonene and other terpenes react rapidly with
    photochemically produced hydroxyl and nitrate radicals and ozone.  The
    oxidation of terpenes such as limonene contributes to aerosol and
    photochemical smog formation.  In soil, limonene is expected to have
    low mobility; in the aquatic environment, it is expected to bind
    strongly to sediment.  Limonene is resistant to hydrolysis. 
    Biodegradation occurs under aerobic, but not anaerobic, conditions.

         Terrestrial organisms are most likely exposed to limonene via the
    air.  The few studies on terrestrial species (i.e. insects) using
    vapour exposure revealed effects of limonene at parts per million
    levels.  Measured environmental concentrations are typically around
    0.1-2 ppb (0.6-11 µg/m3).  At polluted sites, limonene concentrations
    in soil may exceed effect levels of soil-living organisms (e.g.
    earthworms).  In the aquatic environment, limonene shows high acute
    toxicity to fish and  Daphnia. Limonene concentrations in surface
    waters are generally much lower than experimentally determined acute
    toxicity levels, and therefore it is likely that limonene poses a low
    risk for acute toxic effects on aquatic organisms.  No studies were
    found on chronic effects.

    2.  IDENTITY AND PHYSICAL/CHEMICAL PROPERTIES

         Limonene is a colourless liquid at room temperature.  The
    structural formula for limonene is given below.  The chemical exists
    as two optical isomers,  d- and  l-limonene, and the racemic mixture
    dipentene.  The purity of commercial  d-limonene is about 90-98%.  

         Physical and chemical data on limonene presented in Table 1 were
    taken from Karlberg & Lindell (1993), unless otherwise stated. 
    Impurities are mainly other monoterpenes, such as myrcene 
    (7-methyl-3-methylene-1,6-octadiene), alpha-pinene 
    (2,6,6-trimethyl-bicyclo[3.1.1]hept-2-ene), alpha-pinene 
    (6,6-dimethyl-2-methylene-bicyclo[3.1.1]heptane), sabinene 
    (2-methyl-5-(1-methylethyl)-bicyclo[3.1.0]hexan-2-ol), and 
    Gamma3-carene ((1S-cis)-3,7,7-trimethyl-bicyclo[4.1.0]hept-2-ene). 
    The vapour pressure of limonene is high and its solubility in water is
    low, giving a high value of the Henry's law constant, which predicts a
    high rate of vaporization of limonene.

    CHEMICAL STRUCTURE 1



        Table 1: Physical/chemical properties of limonene.a

                                                                                                                                      

                                            d-Limonene                       l-Limonene                        Dipentene
                                                                                                                                      

    CAS no.                                  5989-27-5                        5989-54-8                        138-86-3
    Chemical name                (R)-1-methyl-4-(1-methylethenyl) (S)-1-methyl-4-(1-methylethenyl)   1-methyl-4-(1-methylethenyl)
                                            cyclohexene                      cyclohexene                      cyclohexene
    Empirical formula                         C10H16                           C10H16                           C10H16
    Molecular weight                          136.23                           136.23                           136.23
    Melting point (°C)                        -74.35                           -74.35                            -95.9
    Boiling point (°C)                      175.5-176.0                      175.5-176.0                      175.5-176.0
    Density (g/cm3 at 20°C)                   0.8411                           0.8422                           0.8402
    Vapour pressure (Pa at 20°C)                190                              190                              190
    Water solubility (mg/litre at 25°C)        13.8b                              -                                -
    Henry's law constant (kPa m3/mol at 25°C)  34.8c                              -                                -
    Log Kow                                    4.23d                              -                        4.83e (limonene)
                                                                                                                                      

    a Conversion factors: 1 ppm = 5.56 mg/m3; 1 mg/m3 = 0.177 ppm.
    b Massaldi & King, 1973; Assessment Tool for the Evaluation of Risk (ASTER) database, Environmental Research Laboratory, US
      Environmental Protection Agency, Duluth, MN, 1991.
    c Calculated value (ENVIROFATE database, Office of Toxic Substances, US Environmental Protection Agency, and Syracuse Research
      Corporation [SRC], New York, NY, 1995).
    d Calculated value (US EPA, 1990a, 1994).
    e Calculated value (US EPA, 1994; Log Octanol-Water Partition Coefficient Program [LOGKOW], Syracuse Research Corporation [SRC],
      New York, NY).

    

    3.  ANALYTICAL METHODS

         Airborne limonene may be collected by charcoal tube sampling
    followed by desorption with carbon disulfide (Searle, 1989) or
    alternatively on Tenax (Janson & Kristensson, 1991) or on multisorbent
    sampling tubes (Chan et al., 1990) followed by thermal desorption. 
    Limonene is usually analysed by gas chromatography with flame
    ionization detection or mass spectrometry.  For limonene in blood,
    liquids, and tissues, a head-space technique could be used.  The
    detection limit in air is 5 µg/m3 (Searle, 1989) and in blood,
    1.4 µg/litre (Falk Filipsson et al., 1993).  As limonene is easily
    oxidized in air, it is also important to analyse the oxidation
    products.  Hydroperoxides of  d-limonene can be analysed by gas
    chromatography if the sample is injected on-column (Karlberg et al.,
    1994).  A high-performance liquid chromatography method for limonene
    has also been developed (Nilsson et al., 1996).

    4.  SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

         Limonene, like other monoterpenes, occurs naturally in certain
    trees and bushes.  It is found in peel from citrus fruits, in dill,
    caraway, fennel, and celery, and in turpentine.  Typical
    concentrations of monoterpenes in air in conifer forests are 1-10
    µg/m3, but variations are large (Strömvall, 1992).  Mean emission
    rates of limonene from different plant species (i.e. lemon, orange,
    pistachio, and walnut) in the Central Valley of California ranged from
    0.4 to 2.5 mg/g dry leaf weight per hour (Arey et al., 1991). 
    Monoterpenes are released in significant amounts mainly to the
    atmosphere.  Biogenic emissions are in the order of, or may exceed,
    those from anthropogenic sources (Dimitriades, 1981; Altshuller, 1983;
    Lamb et al., 1987).  Global annual emissions of biogenic monoterpenes
    range from 147 to 827 million tonnes (Fehsenfeld et al., 1992).

         Limonene is used as a substitute for chlorinated hydrocarbons,
    chlorofluorocarbons, and other solvents.  It is used in degreasing
    metals (30% limonene) prior to industrial painting, for cleaning in
    the electronic industry (50-100% limonene), for cleaning in the
    printing industry (30-100% limonene), and in paint as a solvent. 
    Limonene is also used as a solvent in histological laboratories and as
    a flavour and fragrance additive in food, household cleaning products,
    and perfumes.  d-Limonene has been used as a gallstone solubilizer in
    humans (Igimi et al., 1976, 1991).

         The annual worldwide production of  d-limonene and orange
    oil/essence oil (95%  d-limonene) in 1991 was approximately 45 kt
    (Florida Chemical Co., 1991).  Citrus plantings under way are expected
    to increase that figure to 73 kt annually within a decade (IARC,
    1993).  Production volumes in Japan were about 40 kt in each of 1992
    and 1993 (Chemical Daily, 1994, 1995).  In 1984, the US consumption of
     d-limonene was 250 t.1  The number of industrial plants in the USA
    handling  d-limonene in 1983 was 87, and the estimated number of
    employees exposed to the chemical was 140 000.2  The corresponding
    numbers of industrial plants and exposed employees were 2 and 1843 for
     l-limonene and 103 and 185 000 for dipentene, respectively.  In
    1974, the corresponding numbers for dipentene were 70 and 45 000,
    respectively.  The increased use of dipentene has probably continued
    after 1983, especially because of its use as a substitute for
    chlorinated hydrocarbons, chlorofluorocarbons, and other solvents, but
    no production data were identified.  According to the product register
    set up by the Swedish National Chemicals Inspectorate, between 69 and


              

    1 Source: Environmental Chemicals Data and Information Network
    (ECDIN). Ispra, Italy, CEC Joint Research Centre (1993).

    2 Source: Registry of Toxic Effects of Chemical Substances (RTECS).
    US Department of Health and Human Services, National Institute of
    Occupational Safety and Health (NIOSH) (1994).

    80 t of  d-limonene in 48 products (15 for consumers) were used
    during 1994 in Sweden.  The corresponding numbers for dipentene were
    74-88 t in 106 products (26 for consumers).  No use of  l-limonene
    was reported.

    5.  ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

         Monoterpenes such as limonene are released in large amounts
    mainly to the atmosphere.  The chemical and physical properties of
    limonene also indicate that the substance will be distributed mainly
    to air.

         When released to ground, limonene is expected to have low to very
    low mobility in soil, based on its physical/chemical properties.  The
    soil adsorption coefficient  (Koc), calculated on the basis of the
    solubility (13.8 mg/litre at 25°C) and the log octanol/water partition
    coefficient (4.232), ranges from 1030 to 4780.3  The Henry's law
    constant indicates that limonene will rapidly volatilize from both dry
    and moist soil; however, its strong adsorption to soil may slow this
    process.3

         In the aquatic environment, limonene is expected to adsorb to
    sediment and suspended organic matter and to rapidly volatilize to the
    atmosphere, based on its physical/chemical properties.3  The
    estimated half-life for volatilization of limonene from a model river
    (1 m deep, flow 1 m/s, and wind speed 3 m/s) is 3.4 hours.3  The
    bioconcentration factor, calculated on the basis of water solubility
    and the log octanol/water partition coefficient, is 246-262,3
    suggesting that limonene may bioaccumulate in fish and other aquatic
    organisms.

         Limonene does not have functional groups for hydrolysis, and its
    cyclohexene ring and ethylene group are known to be resistant to
    hydrolysis (US EPA, 1994).  Therefore, hydrolysis of limonene is not
    expected, neither in terrestrial nor in aquatic environments.  The
    hydrolytic half-life of  d-limonene has been estimated to be >1000
    days.4  Biotic degradation of limonene has been shown with some
    species of microorganisms, such as  Penicillium digitatum, 
     Corynespora cassiicola,  Diplodia gossypina (Abraham et al., 1985),
    and a soil strain of  Pseudomonas sp. (PL strain) (Dhavalikar &
    Bhattacharayya, 1966; Shulka & Bhattacharayya, 1968).  As these
    studies were not designed to determine the biodegradability of
    limonene, the results provided only indications of possible
    biodegradation.  However, limonene was readily biodegradable (41-98%
    degradation by biochemical oxygen demand in 14 days) under aerobic
    conditions in a standard test (OECD 301 C "Modified MITI Test (I)";
    OECD, 1981) (MITI, 1992).  Also, in a test simulating aerobic sewage
    treatment (OECD 303 A "Simulation Test - Aerobic Sewage Treatment:
    Coupled Units Test"; OECD, 1981), limonene disappeared almost
    completely (>93.8%) during 14 days of incubation (Schwartz et al.,

              

    3 Source: Hazardous Substances Data Bank. Bethesda, MD, National
    Library of Medicine (1995).

    4 Source: ASTER (Assessment Tool for the Evaluation of Risk)
    database. Duluth, MN, US Environmental Protection Agency,
    Environmental Research Laboratory.

    1990).  However,  this test was not suitable for such a volatile
    substance as limonene.  The disappearance of limonene was likely due
    in part to volatilization, but it could not be determined to what
    extent the removal was due to biodegradation and sorption compared
    with volatilization.

         Biodegradation has also been assessed under anaerobic conditions. 
    In a test on methanogenic degradation (batch bioassay inoculated with
    granular sludge, 30°C), there was no indication of any metabolism of
    limonene, possibly because of toxicity to the microorganisms 
    Sierra-Alvarez et al., 1990).  Complex chlorinated terpenes, similar to
    toxaphene (a persistent, mobile, and toxic insecticide, with global
    distribution) and its degradation products, were produced by 
    photo-initiated reactions in an aqueous system initially containing 
    limonene and other monoterpenes, simulating pulp bleaching conditions 
    (Larson & Marley, 1988).

         In the atmosphere, limonene is expected to rapidly undergo 
    gas-phase reactions with photochemically produced hydroxyl radicals,
    ozone, and nitrate radicals (Table 2). Based on experimentally
    determined rate constants, calculated lifetimes for the reaction of
     d-limonene with photochemically produced hydroxyl radicals range
    from 0.3 to 2 hours (Winer et al., 1976, 1984; Atkinson & Carter,
    1984; Atkinson et al., 1984; Atkinson, 1990).  The corresponding
    lifetimes for the reaction with ozone are in the range of 0.2-2.6
    hours (Atkinson & Carter, 1984; Atkinson et al., 1984, 1990; Winer et
    al., 1984; Klöpffer et al., 1988; Nolting & Zetzsch, 1988; Atkinson,
    1990).  Based on experimentally determined rate constants, calculated
    lifetimes for the nighttime reaction of  d-limonene with nitrate
    radicals range from 0.9 to 9 minutes (Atkinson & Carter, 1984;
    Atkinson et al., 1984; Winer et al., 1984; Atkinson, 1990).  The
    daytime atmospheric lifetime of  d-limonene has been estimated to
    range from 12 to 48 minutes, depending upon the local hydroxyl radical
    and ozone concentrations (Altshuller, 1983).

         Products formed from the hydroxyl radical reaction with limonene
    are 4-acetyl-1-methylcyclohexene (Arey et al., 1990; Grosjean et al.,
    1992; Hakola et al., 1994), a keto-aldehyde (Arey et al., 1990; Hakola
    et al., 1994), formaldehyde, 3-oxobutanal, glyoxal, and a C10
    dicarbonyl (Grosjean et al., 1992).  The same carbonyls, along with
    formic acid and C8 and C9 carboxylic acids, may also form in
    reactions with ozone (Grosjean et al., 1992).  Ozonolysis of limonene
    may also result in bis(hydroxymethyl)peroxide, a precursor to
    hydroxymethyl hydroperoxide (Gäb et al., 1985), and hydrogen peroxide
    (Becker et al., 1990).  Hydroxymethyl hydroperoxide,
    bis(hydroxymethyl)peroxide, and hydrogen peroxide have various toxic
    effects on plant cells and enzymes (Gäb et al., 1985; Becker et al.,
    1990).  The reaction of  d-limonene with ozone in the dark results in
    the formation of 4-acetyl-1-methylcyclohexene and formaldehyde
    (Grosjean et al., 1993).  Reactions with nitrogen oxides produce
    aerosol formation as well as lower molecular weight products, such as
    formaldehyde, acetaldehyde, formic acid, acetone, and peroxyacetyl
    nitrate (Altshuller, 1983).



        Table 2: Rate constants and lifetimes of d-limonene in gas-phase reactions with hydroxyl radicals (OH),
    ozone (O3), and nitrate radicals (NO3).

                                                                                                                                   

                    Concentration        Lifetime       Rate constant
    Substance       (molecules/cm3)a     (hours)        (cm3 molecule-1 s-1)   Reference
                                                                                                                                   

    OH              1x106 (0.04 ppt)     0.32           9.0x10-10              Winer et al., 1976
                    4x106 (0.16 ppt)     0.5            1.4x10-10              Atkinson et al., 1984; Winer et al., 1984
                    1x106 (0.04 ppt)     1.6            1.7x10-10              Atkinson, 1990
                    1x106 (0.04 ppt)     2              1.4x10-10              Atkinson & Carter, 1984
                    1x106 (0.04 ppt)     2              1.4x10-10              Atkinson et al., 1984; Winer et al., 1984
    O3              200 ppb              0.18           6.4x10-16              Atkinson et al., 1984; Winer et al., 1984
                    7x1011               0.5b           5.4x10-16              Klöpffer et al., 1988
                    30 ppb               0.6            6.4x10-16              Atkinson et al., 1984; Winer et al., 1984
                    7x1011               0.62           6.4x10-16              Atkinson, 1990
                    7x1011               0.67           6.0x10-16              Atkinson & Carter, 1984
                    7x1011               1.9            2.09x10-16             Atkinson et al., 1990
                    7x1011               2.6            1.53x10-16             Nolting & Zetzsch, 1988
    NO3             100 ppt              0.015          7.7x10-12              Atkinson et al., 1984; Winer et al., 1984
                                         (0.9 min)
                    2.4x108              0.08           1.4x10-11              Atkinson & Carter, 1984
                                         (5 min)
                    2.4x108              0.09           1.3x10-11              Atkinson, 1990
                                         (5.3 min)
                    10 ppt               0.15           7.7x10-12              Atkinson et al., 1984; Winer et al., 1984
                                         (9 min)
                                                                                                                                   

    a Unless otherwise indicated.
    b Half-life (in hours).
    

         Terpenes such as limonene contribute to aerosol and photochemical
    smog formation (Gäb et al., 1985; Sekiya et al., 1988).  Emissions of
    biogenic hydrocarbons such as limonene and other terpenes to the
    atmosphere may either decrease ozone concentrations when nitrogen
    oxide concentrations are low or, if emissions take place in polluted
    air (i.e. containing high nitrogen oxide levels), lead to an increase
    in ozone concentrations (Altshuller, 1983; Fehsenfeld et al., 1992).

    6.  ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

    6.1  Environmental levels

         Data on environmental levels of limonene are presented in Table
    3.  The concentrations of limonene and other monoterpenes in air vary
    considerably.  Recorded concentrations in rural areas depend on many
    factors, such as the type of vegetation, temperature, time of the day,
    and time of the year (Strömvall, 1992).  Biogenic monoterpene
    emissions are assumed to be very low in the late autumn and winter
    months compared with summer (Altshuller, 1983).  Measured
    concentrations (between 1979 and 1992) of limonene in the air of rural
    forest areas in Europe, Canada, the USA, Nepal, the Republic of
    Georgia, and Japan ranged from 1.6 × 10-4 to 2.2 ppb (0.9 ng/m3  to
    12.2 µg/m3) (Shaw et al., 1983; Hutte et al., 1984; Roberts et al.,
    1985; Jüttner, 1986, 1988; Petersson, 1988; Helmig et al., 1989;
    Clement et al., 1990; Janson & Kristensson, 1991; Ciccoioli et al.,
    1992, 1993; Helmig & Arey, 1992; Peters et al., 1994).  Based upon
    these data, typical concentrations of limonene in air from rural areas
    range from 0.1 to 0.2 ppb (0.6-1.1 µg/m3).

         On the basis of measured concentrations (between 1973 and 1990)
    of limonene in the air from urban or suburban areas in Europe, the
    USA, and Russia that ranged from not detectable to 5.7 ppb (31.7
    µg/m3) (Bertsch et al., 1974; Ioffe et al., 1977, 1979; Hutte et al.,
    1984; De Bortoli et al., 1986; Jüttner, 1988; Ciccoioli et al., 1992;
    Helmig & Arey, 1992), typical concentrations of limonene in
    urban/suburban air are likely to range from 0.1 to 2 ppb (0.6-11.1
    µg/m3).  Concentrations of limonene in air emissions from kraft pulp
    industries, stone groundwood production, and various waste and
    landfill sites have ranged from approximately 0.3 to 41 000 ppb (1.7
    µg/m3 to 240 mg/m3) (Young & Parker, 1983, 1984; Koe & Ng, 1987;
    Strömvall, 1992; Eitzer, 1995).

         Limonene has been detected in groundwater and surface waters,
    ice, sediments, and soil.  Mean limonene concentrations in two
    polluted Spanish rivers were 590 and 1600 ng/litre (Gomez-Belinchon et
    al., 1991).  Samples of water collected from the Gulf of Mexico
    contained limonene at a concentration of 2-40 ng/litre (Sauer, 1981). 
    Limonene has also been detected at Terra Nova Bay, Antarctica; water
    and pack ice samples contained limonene at concentrations up to 20 and
    15 ng/litre, respectively (Desideri et al., 1991).  Limonene
    concentrations up to 920 µg/g in soil and from 1 to 130 µg/litre in
    groundwater were measured in a polluted area at a former site for the
    production of charcoal and pine tar products in Florida (McCreary et
    al., 1983).  Limonene was also detected but not quantified in fish
    (i.e. carp) collected from Las Vegas Wash, Nevada (Hiatt, 1983).



        Table 3: Concentrations of limonene in various media.

                                                                                                                                                

    Medium          Concentration                          Location and sampling date                                       Reference
                                                                                                                                                

    Air, rural      0.036 µg/m3 (6.4x10-3 ppb)             Whitaker's Forest, Sierra Nevada Mountains, California,
                                                             June 1990                                                      Helmig & Arey, 1992
                    0.49 ng/litre (8.7x10-2 ppb)           Monte Cimini, Italy, (forest site)                               Ciccoioli et al., 1992
                    detected                               Eggegebirge, North Rhine-Westfalia, Germany, 1988
                                                             (forest site)                                                  Helmig et al., 1989
                    40 ppbCa (25 µg/m3)a                   Forest site in Republic of Georgia, July 1979                    Shaw et al., 1983
                    0.030 ppb                              Rocky Mountains, Colorado, average day July-Dec. 1982            Roberts et al., 1985
                    0.072 ppb                              Rocky Mountains, Colorado, average night July-Dec. 1982          Roberts et al., 1985
                    0.002-0.13 ppb                         Rocky Mountains, Colorado, range night July-Dec. 1982            Roberts et al., 1985
                    detected                               Western Colorado                                                 Hutte et al., 1984
                    0.34 µg/m3 (6.0x10-2 ppb)              Eastern Germany, July (forest site)                              Ciccoioli et al., 1993
                    1.16 µg/m3 (0.20 ppb)                  Nepal, September-October, 1991                                   Ciccoioli et al., 1993
                    1.3-7.3 µg/m3 (0.23-1.3 ppb)           Forest, Jönköping, Sweden, night June-July, 1983                 Petersson, 1988
                    0.1-2.2 ppb (0.6-12.2 µg/m3)           Forest, Northwest Qubec, Canada, July 1989                       Clement et al., 1990
                    detected                               Southern Black Forest, Germany, Nov.-Jan. (1984-1985)            Jüttner, 1986
                    0.9-89 ng/m3 (1.6x10-4 - 1.6x10-2 ppb) Southern Black Forest, Germany, March-Dec. 1985                  Jüttner, 1988
                    <0.05-0.25 ng/litre
                       (<8.8x10-3 - 4.4 x10-2 ppb)         Speulderbos Forest, Netherlands, summer 1992                     Peters et al., 1994
                    0-0.5 ppb                              Järllsa Sweden, June 1989                                        Janson & Kristensson, 
                                                                                                                              1991
                                                                                                                                                
    Air, urban/     ndb-0.36 µg/m3 (nd-6.4x10-2 ppb)       Urban Riverside, California, June 1990                           Helmig & Arey, 1992
      suburban      0.14 ng/litre (2.5x10-2 ppb)           Montelibretti, Italy (suburban site)                             Ciccoioli et al., 1992
                    0-5.7 ppb (0-31.7 µg/m3)               Houston, Texas                                                   Bertsch et al., 1974
                    <1-11 µg/m3 (<0.2-1.9 ppb)             Rural, suburban and urban sites in Northern Italy, 1983-1984
                                                             (mean 1 µg/m3, or 0.2 ppb)                                     De Bortoli et al., 1986
                    detected                               Leningrad, Russia, summer-autumn, 1976                           Ioffe et al., 1977
                    detected                               Denver, Colorado, USA, Jan.-Feb. 1984                            Hutte et al., 1984
                    nd-2.0 ng/m3 (nd-3.5x10-4 ppb)         Tübingen, Germany, March-April 1985 (suburban)                   Jüttner, 1988
                    detected                               Six larger citiesc in USSR, 1977                                 Ioffe et al., 1979
                                                                                                                                                

    Table 3 (continued)

                                                                                                                                                

    Medium          Concentration                          Location and sampling date                                       Reference
                                                                                                                                                

    Air, emissions  1.7-10 100 µg/m3 (0.3-1.8x103 ppb)     8 municipal solid waste composting facilities, USA               Eitzer, 1995
                    2-240 mg/m3 (3.5x102 - 4.1x104 ppb)    8 landfill sites, UK (mean approx. 101 mg/m3, or 1.8x104 ppb)    Young & Parker, 1983,
                                                                                                                              1984
                    detected                               Refuse waste, Singapore                                          Koe & Ng, 1987
                    1.9-14 µg/m3 (0.34-2.5 ppb)            Emission plumes from kraft pulp industries, Sweden               Strömvall, 1992
                    3.8-39 µg/m3 (0.67-6.9 ppb)            Ambient air downwind from stone groundwood production,
                                                             Sweden, 1989                                                   Strömvall, 1992
                                                                                                                                                
    Water, sea      2-40 ng/litre                          Gulf of Mexico,d 1977                                            Sauer, 1981
                    0.55 ng/litre (mean)                   Barcelona, Mediterranean Sea, Spain, 1986                        Gomez-Belinchon et
                                                                                                                              al., 1991
                    4.4 ng/litre (mean)                    Vilanova-Sitges, Mediterranean Sea, Spain, 1986                  Gomez-Belinchon et
                                                                                                                              al., 1991
                    nd-20 ng/litre                         Terra Nova Bay, Antarctica, 1988-1989, seawater
                                                             (mean 5.4 ng/litre)                                            Desideri et al., 1991
                    nd-82 ng/litre                         Terra Nova Bay, Antarctica, 1988-1989, particulate               Desideri et al., 1991
                    84 ng/litre                            Resurrection Bay, Alaska, June 1985                              Button & Jüttner, 1989
                    0.47 ng/litre                          Resurrection Bay, Alaska, June 1986                              Button & Jüttner, 1989
                                                                                                                                                
    Water, river    590 ng/litre (mean)                    Llobregat River, Barcelona, Spain, 1985-1986                     Gomez-Belinchon et
                                                                                                                              al., 1991
                    1600 ng/litre (mean)                   Besós River, Barcelona, Spain, 1985-1986                         Gomez-Belinchon et 
                                                                                                                              al., 1991
                    detected                               Black Warrior River, Tuscaloosa, USA, 1975                       Bertsch et al., 1975
                    detected                               River Lee, London, UK                                            Waggott, 1981
                    detected                               River Glatt, Switzerland, 1975                                   Zürcher & Giger, 1976
                                                                                                                                                
    Water, estuary  25-633 ng/litre                        Southampton Water estuary, UK                                    Bianchi et al., 1991
                                                                                                                                                

    Table 3 (continued)

                                                                                                                                                

    Medium          Concentration                          Location and sampling date                                       Reference
                                                                                                                                                
    Water,          70 ng/litre (max.)                     Otis Air Base, Massachusetts (sewage-contaminated water)         Barber et al., 1988
    groundwater     1-130 µg/litre                         Former site for production of charcoal and pine tar
                                                             products, Gainsville, Florida                                  McCreary et al., 1983
                                                                                                                                                
    Water,          0.03 µg/litre                          13 cities in USA (detected in 1 of 13 cities)                    Keith et al., 1976
    drinking-water  detected                               UK (detected in 5 of 14 samples)                                 Fielding et al., 1981
                    187 µg/kg (1.87x105 ng/litre)          Canada, bottled drinking-water (detected in 1 of 182 samples)    Page et al., 1993
                                                                                                                                                
    Water,          nd-20 µg/litre                         Influent waste water, sewage works, Göteborg, Sweden,            Paxéus et al., 1992
                                                             1989-1991                                          
    wastewater,     10-220 ppb (10x103 - 220x103 ng/litre) Kraft mill aerated lagoons, USA                                  Wilson & Hrutfiord, 1975
    and landfill    nd                                     Effluent wastewater, sewage works, Göteborg, Sweden,             Paxéus et al., 1992
                                                             1989-1991                                         
    leachate        detected                               Industrial landfill leachate, USA                                Venkataramani & Ahlert,
                                                                                                                              1984
                                                                                                                                                
    Ice             4-15 ng/litre                          Terra Nova Bay, Antarctica, 1988-1989, pack ice,                 Desideri et al., 1991
                                                             (mean 8 ng/litre)                             
                                                                                                                                                
    Sediment        105-807 ng/kg                          Southampton Water estuary, UK                                    Bianchi et al., 1991
                                                                                                                                                
    Soil            nd-920 µg/g                            Former site for production of charcoal and pine tar              McCreary et al., 1983
                                                             products, Gainsville, Florida, USA               
    Litter          4.0 µg/g (mean)                        Litter of single leaf pinyon woodlands, Western Great            Wilt et al., 1988
                                                             Basin, USA                                         
                                                                                                                                                
    Fish            detected                               Carp from Las Vegas Wash, USA                                    Hiatt, 1983
                    nd                                     Rainbow  trout from Colorado River, USA                          Hiatt, 1983
                                                                                                                                                
    a Average concentration of terpenes, on a particulate carbon basis.
    b Not detected.
    c Baku, Kemerovo, Leningrad, Murmansk, Tashkent, and Tblisi.
    d Near the mouth of the Mississippi River and on the Louisiana Shelf.
    

    6.2  Human exposure

         Examples of estimated exposure to limonene in the general and
    occupational environments are presented here, on the basis of
    identified data primarily from the USA and Sweden.  Countries are
    strongly encouraged, however, to estimate exposure on the basis of
    local data, possibly in a manner similar to that outlined here.

         The intake in food may be unavoidable, as limonene occurs
    naturally in citrus fruits and spices and is used as a flavour and
    fragrance additive.  However, there is a considerable interindividual
    variation in intake, owing to different diet patterns.  Based on daily
    US consumption of  d-limonene per capita, the intake of  d-limonene
    from food for the general population was estimated to be 0.27 mg/kg
    body weight per day (Flavor and Extract Manufacturers Association,
    1991).

         Indoor concentrations of limonene (no specification of
    enantiomer) in northern Italy ranged from 10 to 480 µg/m3 (mean 140
    µg/m3) (De Bortoli et al., 1986), whereas concentrations ranged from
    1.6 to 78 µg/m3 (mean 18 µg/m3) in 17 residences in Ruston,
    Washington (Montgomery & Kalman, 1989).  In an investigation from Los
    Angeles, California, the arithmetic mean limonene level in indoor air
    was 40 µg/m3 (Wallace et al., 1991).  In 754 randomly selected
    residences in Canada, indoor concentrations of limonene ranged from 9
    to 30 µg/m3 (Fellin & Otson, 1993); concentrations were higher during
    the winter season when ventilation was lower.

         The intake of limonene from indoor and outdoor air for the
    general population is estimated to be 10 and 0.1 µg/kg body weight per
    day, respectively.  This is based on the daily inhalation volume for
    adults of 22 m3, a mean body weight for males and females of 64 kg,
    the assumption that 4 of 24 hours are spent outdoors (IPCS, 1994), and
    arithmetic mean limonene levels in indoor and outdoor air of 0.04 and
    0.002 mg/m3, respectively, in a study from Los Angeles (Wallace et
    al., 1991).

         Data on concentrations of limonene in drinking-water are limited. 
    However, the intake of limonene from drinking-water is likely to be
    negligible owing to its low solubility.  Dermal exposure to limonene
    by the general population is mainly from contact with household
    cleaning products in which limonene is a fragrance additive.  The
    dermal uptake of  d-limonene by humans is likely to be low compared
    with that via inhalation (Falk et al., 1991).

         Inhalation is the principal route of occupational exposure to
    limonene.  According to the National Exposure Database in Norway,
    concentrations of limonene between 1985 and 1992 in the occupational
    environment ranged from 0 to 886 mg/m3 (mean 28 mg/m3) (Fjelstad &
    Wolbæk, 1992).  In a study from Sweden, occupational concentrations
    ranged from 0.9 to 400 mg/m3 (Carlsson et al., 1991).  There is also
    a potential for dermal exposure to limonene in the occupational
    environment, although quantitative data are not available.

         The estimated intake of limonene from occupational exposure was
    calculated on the same basis as for indoor and outdoor air, assuming
    that 8 of 24 hours are spent in the workplace each day, with an air
    concentration of 150 mg/m3, which is the occupational exposure limit
    value in Sweden (National Board of Occupational Safety and Health,
    1993).  The intake of limonene associated with working at the
    occupational exposure limit was estimated as 17 mg/kg body weight per
    day.

    7.  COMPARATIVE KINETICS AND METABOLISM IN LABORATORY ANIMALS
        AND HUMANS

          d-Limonene has a high partition coefficient between blood and
    air (lambdablood/air = 42) and is easily taken up in the blood at the
    alveolus (Falk et al., 1990).  The net uptake of  d-limonene in
    volunteers exposed to the chemical at concentrations of 450, 225, and
    10 mg/m3 for 2 hours during light physical exercise averaged 65%
    (Falk Filipsson et al., 1993).  Orally administered  d-limonene is
    rapidly and almost completely taken up from the gastrointestinal tract
    in humans as well as in animals (Igimi et al., 1974; Kodama et al.,
    1976).  Infusion of labelled  d-limonene into the common bile duct of
    volunteers revealed that the chemical was very poorly absorbed from
    the biliary system (Igimi et al., 1991).  In shaved mice, the dermal
    absorption of [3H] d/l-limonene from bathing water was rapid,
    reaching the maximum level in 10 minutes (von Schäfer & Schäfer,
    1982).  In one study (one hand exposed to 98%  d-limonene for 2
    hours), the dermal uptake of  d-limonene in humans was reported to be
    low compared with that by inhalation (Falk et al., 1991); however,
    quantitative data were not provided.

          d-Limonene is rapidly distributed to different tissues in the
    body and is readily metabolized.  Clearance from the blood was 1.1
    litre/kg body weight per hour in males exposed for 2 hours to
     d-limonene at 450 mg/m3 (Falk Filipsson et al., 1993).  A high
    oil/blood partition coefficient and a long half-life during the slow
    elimination phase suggest high affinity to adipose tissues (Falk et
    al., 1990; Falk Filipsson et al., 1993).  In rats, the tissue
    distribution of radioactivity was initially high in the liver,
    kidneys, and blood after the oral administration of [14C] d-limonene
    (Igimi et al., 1974); however, negligible amounts of radioactivity
    were found after 48 hours.  Differences between species regarding the
    renal disposition and protein binding of  d-limonene have been
    observed.  For rats, there is also a sex-related variation 
    (Lehman-McKeeman et al., 1989; Webb et al., 1989).  The concentration 
    of  d-limonene equivalents was about 3 times higher in male rats than 
    in females, and about 40% was reversibly bound to the male rat specific
    protein, alpha2µ-globulin (Lehman-McKeeman et al., 1989; 
    Lehman-McKeeman & Caudill, 1992).

         The biotransformation of  d-limonene has been studied in many
    species, with several possible pathways of metabolism (Figure 1). 
    Metabolic differences between species have been observed with respect
    to the metabolites present in both plasma and urine.  About 25-30% of
    an oral dose of  d-limonene in humans was found in urine as
     d-limonene-8,9-diol and its glucuronide; about 7-11% was eliminated
    as perillic acid (4-(1-methylethenyl)-1-cyclohexene-1-carboxylic acid)
    and its metabolites (Smith et al., 1969; Kodama et al., 1976). 
     d-Limonene-8,9-diol is probably formed via  d-limonene-8,9-epoxide
    (Kodama et al., 1976; Watabe et al., 1981).  In another study,
    perillic acid was reported to be the principal metabolite in plasma in
    both rats and humans (Crowell et al., 1992).  Other reported pathways
    of limonene metabolism involve ring hydroxylation and oxidation of the
    methyl group (Kodama et al., 1976).

         Following the inhalation exposure of volunteers to  d-limonene
    at 450 mg/m3 for 2 hours, three phases of elimination were observed
    in the blood, with half-lives of about 3, 33, and 750 minutes,
    respectively (Falk Filipsson et al., 1993).  About 1% of the amount
    taken up was eliminated unchanged in exhaled air, whereas about 0.003%
    was eliminated unchanged in the urine.  When male volunteers were
    administered (per os) 1.6 g [14C] d-limonene, 50-80% of the
    radioactivity was eliminated in the urine within 2 days (Kodama et
    al., 1976).  Limonene has been detected, but not quantified, in breast
    milk of non-occupationally exposed mothers (Pellizzari et al., 1982).

    FIGURE 2

    8.  EFFECTS ON LABORATORY MAMMALS AND  IN VITRO TEST SYSTEMS

    8.1  Single exposure

         The acute toxicity of  d-limonene in rodents is fairly low after
    oral, intraperitoneal, subcutaneous, and intravenous administration,
    based on the magnitude of the LD50 values (Table 4).  LD50 values
    were approximately 5 g/kg body weight for the oral administration of
     d-limonene or  d/l-limonene to rats and for dermal application of
     d/l-limonene to rabbits and 6 g/kg body weight for oral
    administration to mice (Tsuji et al., 1974, 1975b; Opdyke, 1978). 
    Studies on the acute inhalation toxicity of limonene were not
    identified.

         Effects observed following the acute exposure of rodents to
    limonene include increased bile flow at 85 mg/kg body weight (Kodama
    et al., 1976), inhibition of  S-3-hydroxy-3-methylglutaryl-CoA
    reductase activity at 409 mg/kg body weight (Clegg et al., 1980),
    enzyme induction at 600 and 1200 mg/kg body weight  (Ariyoshi et al.,
    1975), and decreased motor activity, hypothermia, and potentiation of
    hexobarbital-induced sleep at 3 ml/kg body weight (Tsuji et al.,
    1974).

    8.2  Irritation and sensitization

          d-Limonene is considered a skin irritant (Cronin, 1980;
    Fischer, 1986).  The skin irritancy of limonene in guinea-pigs (Klecak
    et al., 1977) and rabbits (Lacy et al., 1987; Okabe et al., 1990) is
    considered moderate and low, respectively.  In an  in vivo study of
    rabbit skin irritation,  d-limonene was ranked 3.5 of 8 on the basis
    of the primary irritation index (Bagley et al., 1996); effects were
    graded according to OECD Test Guideline 404 (OECD, 1993).  In a study
    in rabbits,  d-limonene caused irritation to the eyes (Tsuji et al.,
    1974).

         Although  d-limonene was once considered the main allergen in
    citrus fruits, data from more recent studies in animals have revealed
    air-oxidized  d-limonene, rather than unoxidized  d-limonene, to be
    the sensitizing agent.  When limonene (unspecified form and unknown
    purity of the test material) was tested in four different
    sensitization assays with guinea-pigs (Open Epicutaneous Test,
    Maximization Test, Draize's Test, and a test with Freund's Complete
    Adjuvant), it was sensitizing in all but Draize's Test (Klecak et al.,
    1977).  In another study in mice,  d-limonene did not induce
    sensitization (Maisey & Miller, 1986).  Hydroperoxides and other
    oxidation products of  d-limonene formed on exposure to the air have
    proved to be potent contact allergens when tested with Freund's
    Complete Adjuvant in guinea-pigs, whereas unoxidized  d-limonene did
    not cause any sensitization (Karlberg et al., 1991, 1992).



        Table 4: Acute toxicity of limonene.

                                                                                                                                 

    Species (sex)      Route of administration     Type of limonene     LD50 (g/kg body weight)     Reference
                                                                                                                                 

    rabbit             dermal                      d/l                  >5                          Opdyke, 1978
    rat                oral                        d/l                  5.3                         Opdyke, 1978
    rat (m/f)          oral                        d                    4.4/5.1                     Tsuji et al., 1975b
    rat (m/f)          intraperitoneal             d                    3.6/4.5                     Tsuji et al., 1975b
    rat (m/f)          intravenous                 d                    0.125/0.11                  Tsuji et al., 1975b
    mouse (m/f)        oral                        d                    5.6/6.6                     Tsuji et al., 1975b
    mouse (m/f)        oral, 7 days                d                    5.3/6.8a                    Tsuji et al., 1974
    mouse (m/f)        intraperitoneal, 3 days     d                    3.1/3.0a                    Tsuji et al., 1974
    mouse (m + f)      intraperitoneal             d                    1.3                         Tsuji et al., 1975b
    mouse (m/f)        intraperitoneal, 10 days    d                    0.59/0.50a                  Tsuji et al., 1974
    mouse (m + f)      subcutaneous                d                    >41.5                       Tsuji et al., 1975b
    mouse (m + f)      subcutaneous, 7 days        d                    >21.5                       Tsuji et al., 1974
                                                                                                                                 

    a Calculated from ml/kg body weight.
    

    8.3  Short-term exposure

         Increases in hepatic cytochrome P-450 content have been observed
    in female rats administered limonene (isomer unspecified; 40 mg/kg
    body weight per day for 3 days) by intraperitoneal injection (Austin
    et al., 1988) and in rats administered 5%  d-limonene in the diet for
    2 weeks (Maltzman et al., 1991).  Increased epoxide hydratase activity
    was observed in rats administered 1% or 5%  d-limonene in the diet
    for 2 weeks (Maltzman et al., 1991).  Increases in phase II enzymes
    (glutathionyltransferase and UDP-glucuronyltransferase) during the
    exposure of rats to 5% limonene in food have also been described
    (Maltzman, 1991).  Increased relative liver weight (from 5 to 20
    times) has been observed in rats administered  d-limonene at a dose
    of 75-300 mg/kg body weight; at 300 mg/kg body weight, the increase
    was significant (Kanerva et al., 1987b).  In cats, infusion of 97%
     d-limonene into the bile system to dissolve gallstones caused acute
    and chronic inflammatory changes (Schenk et al., 1980).

    8.4  Long-term exposure

    8.4.1  Subchronic exposure

         Peroral administration of  d-limonene to rats at a dose of 400
    mg/kg body weight for 30 days resulted in a 20-30% increase in the
    amount and activity of different liver enzymes (cytochrome P-450,
    cytochrome b5, aminopyrine demethylase, and aniline hydroxylase),
    increased relative liver weight, and decreased cholesterol levels
    (Ariyoshi et al., 1975).  Administration of  d-limonene (0, 2, 5, 10,
    30, and 75 mg/kg body weight per day) by gavage to groups of 10 male
    rats, 5 days/week for 13 weeks (Webb et al., 1989), resulted in the
    pathological formation of granular casts at the outer zone of the
    renal medulla.  The no-observed-effect level (NOEL), based upon
    histological examination of the kidneys, was considered to be 5 mg/kg
    body weight per day.  The LOEL for increased liver and kidney weight
    was 75 mg/kg body weight per day, the highest dose tested.  The  NOEL
    for effects in the liver was 10 mg/kg body weight; the 
    no-observed-adverse-effect level (NOAEL) for effects in the liver was 
    30 mg/kg body weight per day.  Linear regression analysis revealed a 
    dose-related trend in the increased relative weights of the kidney and
    liver at 30 and 75 mg/kg body weight per day.  No histopathological
    changes were observed in the liver in these two studies.  The amount
    and activity of different liver enzymes were not investigated, and
    thus the increase in relative liver weight may be due to enzyme
    induction.

    8.4.2  Chronic exposure and carcinogenicity

         The oral administration of  d-limonene (0.4, 1.2, or 3.6 ml/kg
    body weight per day) to dogs for 6 months caused nausea and vomiting
    (Tsuji et al., 1975a).  A 35% increase in alkaline phosphatase and
    cholesterol in serum and slightly increased total and relative liver
    weights were observed in dogs after peroral administration of
     d-limonene at a dose of 1.2 ml/kg body weight per day for 6 months
    (about 1000 mg/kg body weight per day) (Webb et al., 1990).

         In a 2-year study,  d-limonene was administered (per os) 5
    days/week to groups of 50 F344/N rats (0, 75, or 150 mg/kg body weight
    per day to males, and  0, 300, or 600 mg/kg body weight per day to
    females) and B6C3F1 mice (0, 250, or 500 mg/kg body weight per day to
    males, and 0, 500, or 1000 mg/kg body weight per day to females) (NTP,
    1990).  Slightly  lower body weights were observed for rats in the
    high-dose groups and female mice in the high-dose group; however, no
    clinical symptoms could be related to the administration of
     d-limonene.  For female rats in the high-dose group, survival was
    reduced after 39 weeks (NTP, 1990).  There was clear evidence of
    carcinogenic activity of  d-limonene in male rats, based upon a 
    dose-related increase in the incidence of hyperplasia and adenoma/
    adenocarcinoma in renal tubular cells.  However, there was no evidence
    of carcinogenicity in female rats or in male and female mice.  The
    carcinogenic response in the kidney of male rats has been linked to a
    unique renal perturbation involving alpha2µ-globulin.

         To determine whether  d-limonene would cause a sustained
    increase in renal cell proliferation and exhibit promoting activity
    for the development of renal adenomas in male F344 rats, the animals
    were administered (by stomach tube)  d-limonene (150 mg/kg body
    weight per day) as a promoter 5 days/week for 30 weeks (Dietrich &
    Swenberg, 1991).   N-ethyl- N-hydroxyethylnitrosamine (500 ppm) was
    used as an initiator in the drinking-water for 2 weeks.  In addition,
    male alpha2µ-globulin-deficient rats were exposed in the same manner
    to determine if the male rat specific urinary protein alpha2µ-globulin
    is required for  d-limonene to cause these effects.  Exposure to
     d-limonene alone caused a significant increase in the number of
    atypical tubules and atypical hyperplasias in F344 rats, compared with
    vehicle controls.  There was no increase in the incidence of tumours
    or preneoplastic lesions in the alpha2µ-globulin-deficient rats
    exposed to  d-limonene, whereas a 10-fold increase in the incidence
    of renal adenoma and atypical hyperplasia was observed in F344 rats
    exposed to  d-limonene, compared with controls.  There was a
    significant decrease in the incidence of liver tumours in animals
    exposed to  N-ethyl- N-hydroxyethylnitrosamine and  d-limonene,
    compared with  N-ethyl- N-hydroxyethylnitrosamine exposure alone.

    8.5  Genotoxicity and related end-points

         On the basis of available data, there is no evidence that
     d-limonene or its metabolites are genotoxic or mutagenic.  Limonene
    and its epoxides were not mutagenic when tested at concentrations of 
    0.3-3333 µg/plate in  in vitro assays using different strains of
     Salmonella typhimurium, in the presence or absence of metabolic
    activation (Florin et al., 1980; Watabe et al., 1981; Haworth et al.,
    1983; Connor et al., 1985; NTP, 1990).   d-Limonene did not increase
    the frequency of forward mutation at the TK+/- locus in mouse L5178Y
    cells (NTP, 1990), induce cytogenetic damage in Chinese hamster ovary
    cells (Anderson et al., 1990), or malignantly transform Syrian hamster
    embryo cells (Pienta, 1980).  In one  in vitro study, following
    exposure with benzo (a)pyrene,  d-limonene (21.9 µmol/litre)
    inhibited the formation of transformed cell colonies in tracheal
    epithelium isolated from rats (Steele et al., 1990).

         No evidence of mutagenicity was reported in an  in vivo spot
    test with mice, involving the intraperitoneal injection of limonene at
    215 mg/kg body weight per day on days 9-11 during gestation (Fahrig,
    1984).

    8.6  Reproductive and developmental toxicity

         Studies on the reproductive toxicity of limonene were not
    identified.  There is no evidence that limonene has teratogenic or
    embryotoxic effects in the absence of maternal toxicity.  In rats, the
    oral administration of  d-limonene (2869 mg/kg body weight per day)
    on days 9-15 of gestation resulted in decreased body weight and deaths
    among the dams.  Delayed ossification and decreased total body and
    organ weights (thymus, spleen, and ovary) were observed in the
    offspring (Tsuji et al., 1975b).  In mice, the oral administration of
     d-limonene (2869 mg/kg body weight per day) on days 7-12 of
    gestation resulted in reduced growth in the mothers and a
    significantly increased incidence of skeletal anomalies and delayed
    ossification in the offspring (Kodama et al., 1977a).  The oral
    administration of  d-limonene (250, 500, or 1000 mg/kg body weight
    per day) to rabbits on days 6-18 of gestation had no dose-related
    effects on the offspring.  At the highest dose, there were some deaths
    and reduced weight gain among the dams; at the intermediate dose,
    growth was decreased (Kodama et al., 1977b).

    8.7  Immunological and neurological effects

         Reports relating limonene to type I allergy were not identified. 
    In a study designed to assess the immunological effects of
     d-limonene on B- and T-cell responses, BALB/c mice were administered
    (by forced intragastric feeding)  d-limonene (0.1 ml) daily for 9
    weeks (Evans et al., 1987).  Mice given keyhole limpet haemocyanin
    prior to exposure to  d-limonene had suppressed primary and secondary
    anti-keyhole limpet haemocyanin responses.  Mice exposed to

     d-limonene prior to the administration of keyhole limpet haemocyanin
    had significantly increased antibody and mitogen-induced proliferative
    responses.  However, the purity of the  d-limonene in this study was
    not checked, and oxidation products may have been the active
    substances.

          Effects on the central nervous system following exposure to
    limonene have been reported in experimental studies with animals;
    however, it is difficult to ascertain whether these effects were the
    result of general intoxication or a more direct effect of the
    chemical.  The peroral administration of  d-limonene (3 ml) to rats
    and mice resulted in decreased motor activity (Tsuji et al., 1974).  A
    similar effect was also observed in mice orally administered a
    limonene dose of 1000 mg/kg body weight per day for 13 weeks (NTP,
    1990).

    9.  EFFECTS ON HUMANS

         Case reports or epidemiological studies on the effects of
    limonene on human health were not identified.  Available data have
    been derived from studies with volunteers.  In older investigations,
    multiple exposures and confounding factors such as mechanical damage,
    irritation, other allergens, and infections due to wet work (Beerman
    et al., 1938; Schwartz, 1938; Birmingham et al., 1951) may have
    contributed to the effects reported following exposure to limonene. 
    None of eight subjects reported any discomfort, irritation, or
    symptoms related to central nervous system effects during a 2-hour
    inhalation exposure to  d-limonene at 10, 225, or 450 mg/m3;
    however, a slight decline in vital capacity was observed following
    exposure to the highest concentration (Falk Filipsson et al., 1993).

         In a study in which the sensitivity of four patch testing systems
    (Finn chamber, Hill Top patch, Van der Bend chamber, and Webril patch)
    was evaluated in volunteers,  d-limonene (perfume-grade) reacted
    strongly in all types of patches within 10-15 minutes of exposure
    (York et al., 1995).  Skin irritation was assessed before application,
    as well as immediately and 1, 24, 48, and 72 hours after removal of
    the patch, using a scoring system based broadly on that used for
    rabbit irritation studies (OECD, 1993), but modified to account for
    the nature of reactions on human skin.  There was evidence of sensory
    effects and urticarial responses on removal of the patches. 
    Significant irritation persisted for 24 hours, and these reactions
    persisted for 48 and 72 hours in many volunteers (York et al., 1995). 
    Dermal exposure to  d-limonene (98%) for 2 hours in one subject
    caused burning, itching, aching, and a long-lasting purpuric rash
    (Falk et al., 1991).

          d-Limonene infused directly into the bile system of human
    volunteers to dissolve gallstones caused pain in the upper abdomen,
    nausea, vomiting, and diarrhoea, as well as increases in serum
    aminotransferases and alkaline phosphatase (Igimi et al., 1976, 1991). 
    The oral administration of 20 g  d-limonene to volunteers resulted in
    diarrhoea, painful constrictions, and proteinuria, but no biochemical
    changes (total protein, bilirubin, cholesterol, aspartate
    aminotransferase, alanine aminotransferase, alkaline phosphatase) in
    the liver (Igimi et al., 1976).  Reports of contact allergy to
    dipentene have appeared (Calnan, 1979; Rycroft, 1980).  In one
    investigation, 15 of 22 people with an allergy to oil of turpentine
    also reacted to dipentene (Cachao et al., 1986).  Patch testing in
    consecutive dermatitis patients from Sweden and Belgium revealed
    positive reactions in 1.5-2% of the subjects tested with oxidized 
     d-limonene, a finding similar to that observed with other common
    sensitizers, such as formaldehyde (A.-T. Karlberg, personal
    communication, 1996).   d-Limonene reduced non-immunological contact
    urticaria caused by cinnamic aldehyde, with competitive receptor
    inhibition suggested as the mechanism of suppression (Guin et al.,
    1984).  No sensitizing effect was observed when 25 volunteers were
    exposed to  d-limonene in a Human Maximization Test (Grief, 1967).

    10.  EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

    10.1  Aquatic environment

         The acute toxicity of  d-limonene ranges from slight to high for
    aquatic organisms (Table 5).  The lowest acute toxicity values (EC50
    or LC50) identified were approximately 0.4 mg/litre for  Daphnia (US
    EPA, 1990b) and 0.7 mg/litre for fish (US EPA, 1990a,b).  The 
    no-observed-effect concentration (NOEC) for green algae is
    approximately 4 mg/litre (US EPA, 1990a).  The acute toxicity (EC50
    or LC50) of dipentene to  Daphnia and fish is about 50-70 times
    lower than that for  d-limonene (US EPA, 1990b).  No studies were
    identified on the chronic toxicity of limonene to aquatic organisms.

    10.2  Terrestrial environment

         The toxicity of limonene has been studied in various terrestrial
    organisms (Table 6).  Limonene generally has moderate acute toxicity
    in insects and mites.  The acute toxicity of  d-limonene to
    earthworms  (Eisenia foetida Savigny) was high (LC50 = 6.0 ppm;
    mg/kg) (Karr et al., 1990).  Sublethal effects (i.e. abnormal
    rebounding of medial giant fibre pathway [MGF] impulses and
    spontaneous lateral giant fibre pathway [LGF] spiking) were observed
    following exposure of earthworms to 4.2 ppm (mg/kg) limonene (Karr et
    al., 1990).  Limonene has low subacute toxicity to bobwhite quail
     (Colinus virginianus) exposed via the diet (LC50 > 5620 ppm;
    mg/kg) (US EPA, 1994).

        Table 5: Toxicity of limonene to aquatic organisms.

                                                                                                                                 

    Species                                  End-point; exposure                Results (mg/litre)      Reference
                                                                                                                                 

    Algae

    Green algaea                             96-h NOEC; static                  4.08                    US EPA, 1990a
                                                                                                                                 

    Crustaceans

    Water flea (Daphnia magna)b              48-h LC50; flow-through            0.577 (0.496-0.672)     US EPA, 1990b
                                             48-h EC50; flow-through            0.421 

    Water flea (D. magna)c                   acute LC50                         39 ppm                  US EPA, 1994
    Water flea (D. magna)a                   48-h LC50; flow-through            31 (27.5-34.8)          US EPA, 1990b
                                             48-h EC50; flow-through            28.2

    Water flea (Daphnia pulex)b              48-h EC50; flow-through            0.730                   US EPA, 1990a
    Water flea (D. pulex)c                   48-h EC50; static                  69.6                    Passino & Smith, 1987
    Daphniab                                 21-d NOEC; structure-activity      0.15                    US EPA, 1990a
                                             relationship (SAR) analysis
                                                                                                                                 
    Fish

    Fathead minnow (Pimephales promelas)b    96-h LC50; flow-through            0.702 (0.619-0.796)     US EPA, 1990b
    Fathead minnow  (P. promelas)b           96-h LC50; flow-through            0.720 (0.618-0.839)     US EPA, 1990b
                                             96-h EC50; flow-through            0.688 (0.606-0.782)

    Fathead minnow (P. promelas)a            96-h LC50; flow-through            38.5 (35.4-41.8)        US EPA, 1990a,b
                                             96-h EC50; flow-through            28.2

    Fishc                                    acute LC50                         80 ppm                  US EPA, 1994
    Fishb                                    96-h LC50; flow-through            0.711                   US EPA, 1990a
    Golden orfe (Leuciscus idus)a            48-h LC50                          32                      Roth, 1990
                                                                                                                                 

    Table 5 (continued)

                                                                                                                                 

    Species                                  End-point; exposure                Results (mg/litre)      Reference
                                                                                                                                 

    Insects

    Water hyacinth weevil (Neochetina        Mortality (73%, range 40-100%),    50% limonene            Haag, 1986
    eichhorniae, 60%, N. bruchi, 40%)b       weevils were dipped in limonene 

    Mosquito fly (Culex quinquefasciatus)c   2nd-instar larvae (23-33°C),       6.6-26.1 ppm            Mohsen et al., 1989
                                             72-h LC50; static
                                             4th-instar larvae (23-33°C),       7.8-30.6 ppm            Mohsen et al., 1989
                                             72-h LC50; static 
                                                                                                                                 

    a d/l-Limonene.
    b d-Limonene.
    c Optical isomer not specified.
    

        Table 6: Toxicity of limonene to terrestrial organisms.

                                                                                                                                                

    Species                     End-point; exposure                                     Results                           Reference
                                                                                                                                                

    Insects

    Cat flea                    Adult LD50; contact                                     160 (157-163) µg/cm2              Hink & Fee, 1986
    (Ctenocephalides felis)a,b  Adult LD50; vapour                                      259 (234-281) µg/cm2
                                Pupae LD50; contact                                     376 (259-468) µg/cm2
                                Larvae LD50; contact                                    226 (221-231) µg/cm2
                                Eggs; lethal to all eggs; contact                       65 µg/cm2
    Variegated cutworm          Larvae; significant inhibition of pupation; dietary
    (Peridroma saucia)b         exposure                                                0.2% limonene in artificial feed  Harwood et al., 1990
    German cockroach            Adult 24-h LD50; topical                                700 (610-810) µg/insect           Karr & Coats, 1988
    (Blattella germanica L.)b   Adult 24-h LC50; fumigation                             23.3 (17.5-31.0) ppm
                                Adult; no mortality; oral                               25% limonene in feed
                                Nymph; no mortality; oral                               25% limonene in feed
                                Adult; no mortality; 72-h contact with treated          limonene (conc. not given)
                                surface
    German cockroach            Effect on growth rate; diet                             1-25% limonene in diet            Karr & Coats, 1992
    (B. germanica L.)b          EC50, oothecae yielding young; topical exposure         0.68 mg/ootheca
                                No effect on reproduction; via diet                     25% limonene in diet
                                Topical exposure                                        0.84 mg/cockroach
                                Vapour exposure                                         5 mg/litre in air
    Rice weevil (Sitophilus     Adult 24-h LC50; fumigation                             19.0 (13.2-27.3) ppm              Karr & Coats, 1988
    oryzae L.)b
    House fly (Musca            25-h LD50; topical                                      90 (70-130) µg/insect             Karr & Coats, 1988
    domestica L.)b
    Western corn rootworm       Egg 72-h LC50; contact with treated substrate           1.8 (0.8-2.9)% limonene           Karr & Coats, 1988
    (Diabrotica virgifera       Larvae 72-h LC50; contact with treated soil             12.2 (4.5-32.6) ppm
    virgifera LeConte)b
                                                                                                                                                

    Table 6 (continued)

                                                                                                                                                

    Species                     End-point; exposure                                     Results                           Reference
                                                                                                                                                

    Spiders and allies

    Spruce spider mite          24-h LC50; vapour                                       24.5 ppm                          Cook, 1992
    (Oligonychus ununguis       Significant decrease in oviposition                     5 ppm
    (Jacobi)),c adult female
                                                                                                                                                
    Segmented worms

    Earthworm (Eisenia          48-h LC50                                               6.0 (5.1-7.1) ppm                 Karr et al., 1990
    foetida Savigny)b           Sublethal effects                                       4.2 ppm
                                                                                                                                                

    Birds

    Bobwhite quail (Colinus     Subacute LC50; dietary exposure                         >5 620 ppm                        US EPA, 1994
    virginianus)d
                                                                                                                                                

    a Fleas were exposed to filter papers treated with limonene, either directly or to vapours from the filter papers.
    b d-Limonene.
    c l-Limonene.
    d Optical isomers not specified.
    

    11.  EFFECTS EVALUATION

    11.1  Evaluation of health effects

    11.1.1  Hazard identification and dose-response assessment

         Limonene is a skin irritant in experimental animals and humans. 
     d-Limonene is an eye irritant in rabbits.  Studies in guinea-pigs
    have revealed that air-oxidized  d-limonene, but not  d-limonene
    itself, induced contact allergy.  Similar results are likely with
     l-limonene and dipentene.

         The critical organ in animals (except for male rats) following
    peroral or intraperitoneal administration is the liver.  Exposure to
    limonene affects the amount and activity of different liver enzymes,
    liver weight, cholesterol levels, and bile flow, with effects having
    been observed in mice, rats, and dogs.  In male rats, exposure to
     d-limonene results in damage to the kidneys and an increased
    incidence of renal tumours.  As the male rat specific protein
    alpha2µ-globulin is considered to play a crucial role in the
    development of the neoplastic and non-neoplastic kidney lesions, they
    are considered not relevant for human risk assessment.

         A dose-related nephropathy was observed in the kidneys of male
    rats after oral administration of  d-limonene (NTP, 1990).  This
    lesion, consisting of  degeneration of epithelial cells in the
    convoluted tubules, granular casts in the outer stripe of the outer
    medulla, and epithelial regeneration, is characteristic of hyaline
    droplet nephropathy associated with the accumulation of 
    alpha2µ-globulin in the cytoplasm of tubular cells (Alden et al., 
    1984; Halder et al., 1985) in response to a variety of hydrocarbon 
    compounds (Swenberg et al., 1992).  Some compounds fit deeply into a 
    hydrophobic pocket of alpha2µ-globulin.  When hydrogen bonding between 
    the chemical and protein occurs, the digestibility of alpha2µ-globulin 
    by proteases is inhibited, leading to accumulation of the male rat
    specific protein in lysosomes of the P2 segment of the nephron
    (Lehman-McKeeman et al., 1990).  Although such chemicals fall into
    rather diverse classes, molecular modelling studies have demonstrated
    a strong structure-activity relationship with respect to 
    alpha2µ-globulin binding (Borghoff et al., 1991).  The accumulation of
    alpha2µ-globulin is cytotoxic, resulting in single-cell necrosis
    (Dietrich & Swenberg, 1991).  The exfoliated renal epithelium is
    restored by compensatory cell proliferation.  The increase in cell
    proliferation associated with alpha2µ-globulin is reversible.  Damage
    of this type has not been observed in female rats, male rats that do
    not produce alpha2µ-globulin, or other mammals, such as mice,
    hamsters, guinea-pigs, dogs, and monkeys (Alden, 1986; Kanerva &
    Alden, 1987a; Swenberg et al., 1989; Webb et al., 1989, 1990; NTP,
    1990; Ridder et al., 1990; Dietrich & Swenberg, 1991).  The processes
    leading to nephropathy and the development of renal cancer by such
    compounds are among the best understood for non-genotoxic chemicals
    and strongly indicate that it is a male rat specific process.  Acute
    and chronic renal effects induced in male rats by limonene will be

    unlikely to occur in any species not producing alpha2µ-globulin or a
    very closely related protein in the large quantities typically seen in
    the male rat (US EPA, 1991; Swenberg, 1993).

          d-Limonene has been studied in a variety of short-term 
     in vitro tests and has been found to be non-genotoxic.  There is no
    evidence that limonene has teratogenic or embryotoxic effects in the
    absence of maternal toxicity.

    11.1.2  Criteria for setting guidance values for limonene

         In numerous experimental studies, exposure to limonene has been
    shown to affect the liver.  Owing to a lack of data on  d-limonene
    exposure in humans, this organ cannot with certainty be stated as the
    critical organ in humans.  Based on available data, food is believed
    to be the principal source of exposure (96%) to limonene; the
    contribution from ambient air is approximately 4%.  The dermal uptake
    of limonene has not been estimated.

         To calculate a tolerable intake for humans, the animal study was
    chosen in which effects on the liver were observed at the lowest
    exposure level (Webb et al., 1989).  In this study, gavage
    administration of  d-limonene (5 days/week for 13 weeks) to rats
    caused increased relative liver weight at 30 and 75 mg/kg body weight
    per day. The NOEL for the liver was considered to be 10 mg/kg body
    weight per day.  Using uncertainty factors of 10 for intraspecies
    differences and 10 for interspecies differences, a tolerable intake
    for ingestion of  d-limonene by humans of 0.1 mg/kg body weight per
    day may be calculated from the NOEL.  A guidance value for inhalation
    exposure to  d-limonene was not developed, as inhalation is an
    insignificant route of exposure compared with ingestion.

    11.1.3  Sample risk characterization

         Exposure estimates vary as a function of use patterns, and the
    risk characterization presented here is provided only as an example,
    primarily for illustrative purposes.  In general,  d-limonene could
    be considered (with the exception of its irritative and sensitizing
    properties) to be a chemical with fairly low toxicity.  The calculated
    tolerable intake of 0.1 mg/kg body weight per day is of a similar
    magnitude as the estimated daily US consumption of  d-limonene of
    0.27 mg/kg body weight per day (Flavor and Extract Manufacturers
    Association, 1991).

    11.2  Evaluation of environmental effects

         Limonene and other terpenes are released in large amounts mainly
    to the atmosphere.  When released to soil or water, limonene is
    expected to evaporate to air to a significant extent, owing to its
    high volatility.  Thus, the atmosphere is the predominant
    environmental sink of limonene, where it is expected to rapidly
    undergo gas-phase reactions with photochemically produced hydroxyl
    radicals, ozone, and nitrate radicals.  The oxidation of terpenes,
    such as limonene, contributes to aerosol and photochemical smog

    formation.  Ozonolysis of limonene may also lead to the formation of
    hydrogen peroxide and organic peroxides, which have various toxic
    effects on plant cells and may be part of the damage to forests
    observed in the last decades (Peters et al., 1994).  Emissions of
    biogenic hydrocarbons such as limonene and other terpenes to the
    atmosphere may either decrease ozone concentrations when nitrogen
    oxide concentrations are low or, if emissions take place in polluted
    air (i.e. containing high nitrogen oxide levels), lead to an increase
    in ozone concentrations.

         Terrestrial organisms are most likely to be exposed to limonene
    via the air.  The few studies on terrestrial species (i.e. insects)
    using vapour exposure reveal effects of limonene at parts per million
    levels.  Measured environmental concentrations are typically around
    0.1-2 ppb (0.6-11 µg/m3), indicating a low risk for acute toxic
    effects on terrestrial organisms from direct exposure to limonene in
    air.  At polluted sites, limonene concentrations in soil (up to 920
    mg/kg soil) may exceed effect levels of soil-living organisms (e.g.
    earthworm, acute LC50 = 6.0 ppm; mg/kg).

         In the aquatic environment, limonene exhibits high acute toxicity
    to fish and  Daphnia. It may also bioaccumulate.  The lowest acute
    toxicity value identified was 0.4 mg/litre (48-hour EC50 for
     Daphnia). Because concentrations of limonene in surface waters of
    "polluted" and "unpolluted" areas are at least about 250 and 20 000
    times lower than this acute toxicity value, respectively, it is likely
    that limonene poses a low risk for acute toxic effects on aquatic
    organisms.  No studies were identified on chronic effects, and
    therefore risks associated with chronic exposures of aquatic organisms
    to limonene in "polluted" waters cannot be determined.

    12.  PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

         The International Agency for Research on Cancer (IARC, 1993) has
    classified  d-limonene in Group 3 (not classifiable as to its
    carcinogenicity to humans) based on a lack of available data on
    carcinogenicity to humans and limited evidence for carcinogenicity in
    experimental animals.

         The 41st meeting of the Joint FAO/WHO Expert Committee on Food
    Additives (JECFA, 1993b) withdrew the existing acceptable daily intake
    for  d-limonene of 0-1.5 mg/kg body weight per day (JECFA, 1993a) and
    in its place allocated "not specified."  On the basis of the available
    data, the total daily intake of the chemical arising from its use at
    the levels necessary to achieve the desired effect and from its
    acceptable background levels in food did not, in the opinion of the
    Committee, represent a health hazard.  For that reason, and for the
    reasons stated in the individual evaluations, the establishment of an
    acceptable daily intake expressed in numerical form was not deemed
    necessary.

         Information on international hazard classification and labelling
    is included in the International Chemical Safety Card reproduced in
    this document.

    13.  HUMAN HEALTH PROTECTION AND EMERGENCY ACTION

         Human health hazards, together with preventative and protective
    measures and first aid recommendations, are presented in the
    International Chemical Safety Card (ICSC 0918) reproduced in this
    document.

    13.1   Human health hazards

         Limonene is flammable but essentially non-toxic.  Repeated or
    prolonged contact with the oxidized chemical causes skin
    sensitization.

    13.2  Advice to physicians

         In case of poisoning, the treatment is supportive.  Like other
    volatile oils, if the patient lives for 48 hours, complete recovery is
    likely; laboratory evidence of renal damage may persist for several
    months (Dreisbach & Robertson, 1987).

    13.3  Storage

         Limonene is flammable, with a flash point of 45°C.  Keep the
    container in a cool, dry, well ventilated area, out of direct
    sunlight.  Keep the container tightly closed to prevent oxidation of
    the chemical.

    13.4  Spillage

         In the case of a large spill, emergency personnel need to use
    non-sparking tools to avoid fire and explosion hazards.

    14.  CURRENT REGULATIONS, GUIDELINES, AND STANDARDS

         Information on national regulations, guidelines, and standards is
    available from the International Register of Potentially Toxic
    Chemicals (IRPTC) legal file.

         The reader should be aware that regulatory decisions about
    chemicals taken in a certain country can be fully understood only in
    the framework of the legislation of that country.  The regulations and
    guidelines of all countries are subject to change and should always be
    verified with appropriate regulatory authorities before application.



        INTERNATIONAL CHEMICAL SAFETY CARD
    D-LIMONENE                                                                                                       ICSC:0918
                                                                  D-LIMONENE
                                                     (R)-4-Isopropenyl-1-methylcyclohexene
                                                                 (+)-Limonene
                                                                     C10H16
                                                            Molecular mass: 136.23
    CAS #       5989-27-5
    RTECS #     GW6360000
    ICSC #      0918
                                                                                                                                        
    TYPES OF                   ACUTE HAZARDS/                  PREVENTION                      FIRST AID/
    HAZARD/                    SYMPTOMS                                                        FIRE FIGHTING
    EXPOSURE
                                                                                                                                        
    FIRE                       Flammable.                      No open flames, NO sparks,      Powder, AFFF, foam, carbon dioxide.
                                                               and NO smoking.

    EXPLOSION                  Above 48°C explosive            Above 48°C use a closed         In case of fire: keep drums,
                               vapour/air mixtures may be      system, ventilation, and        etc., cool by spraying with
                               formed.                         explosion-proof electrical      water.
                                                               equipment.

    EXPOSURE                                                   STRICT HYGIENE!

    * INHALATION                                               Ventilation.                    Fresh air, rest.

    * SKIN                     Redness.                        Protective gloves. Protective   Remove contaminated clothes.
                                                               clothing.                       Rinse and then wash skin with
                                                                                               water and soap.

    * EYES                     Redness.                        Safety spectacles.              First rinse with plenty of
                                                                                               water for several minutes
                                                                                               (remove contact lenses if
                                                                                               easily possible), then take
                                                                                               to a doctor.

    * INGESTION                                                Do not eat, drink, or smoke     Rinse mouth.
                                                               during work.
                                                                                                                                        

    INTERNATIONAL CHEMICAL SAFETY CARD (continued)

                                                                                                                                        

    SPILLAGE DISPOSAL                             STORAGE                                   PACKAGING & LABELLING
                                                                                                                                        

    Collect leaking and spilled liquid in         Fireproof. Cool. Well closed.
    sealable containers as far as
    possible. Absorb remaining liquid in
    sand or inert absorbent and remove to
    safe place.

                                                                                                                                        

    IMPORTANT DATA      PHYSICAL STATE; APPEARANCE:                          EFFECTS OF SHORT-TERM EXPOSURE:
                        COLOURLESS LIQUID, WITH CHARACTERISTIC ODOUR.        The substance may irritate slightly the eyes
                                                                             and the skin.

                        CHEMICAL DANGERS:                                    EFFECTS OF LONG-TERM OR REPEATED EXPOSURE:
                        Reacts violently with a mixture of iodine            Repeated or prolonged contact may cause skin
                        pentafluoride and tetrafluoroethylene, causing       sensitization if the substance has been
                        fire and explosion hazard.                           oxidized.

                        OCCUPATIONAL EXPOSURE LIMITS (OELs):
                        TLV not established.

                        ROUTES OF EXPOSURE:
                        The substance can be absorbed into the body by
                        inhalation of its vapour, through the skin and
                        by ingestion.

                        INHALATION RISK:
                        No indication can be given about the rate in
                        which a harmful concentration in the air is
                        reached on evaporation of this substance at
                        20°C.

                                                                                                                                        

    INTERNATIONAL CHEMICAL SAFETY CARD (continued)

                                                                                                                                        

    PHYSICAL            Boiling point:                         178°C
    PROPERTIES          Melting                               -75°C
                        Relative density (water = 1):          0.84
                        Solubility in water:                   none
                        Vapour pressure, kPa at 14.4°C:        0.4
                        Relative vapour density (air = 1):     4.7
                        Flash point:                           48°C
                        Octanol/water partition coefficient
                        as log Pow:                            4.2

                                                                                                                                        

    ENVIRONMENTAL       
    DATA                

                                                                                                                                        

    NOTES

    ICSC: 0918 1.1                                                                                  Transport Emergency Card: TEC (R)-75
                                                                                                                   NFPA Code: H2; F3; R2
                                                                                                                                        
    

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    APPENDIX 1 - SOURCE DOCUMENT

    Karlberg A-T, Lindell B (1993) Limonene. In: Beije B, Lundberg P, eds.
     Criteria documents from the Nordic Expert Group 1993. Solna,
    National Institute of Occupational Health, Nordic Council of
    Ministers, pp. 207-246  (Arbete och Hälsa 35).

         Copies of the  Arbete och Hälsa document on limonene (ISSN:
    0346-7821; ISBN: 91-7045-240-7), prepared by the Nordic Expert Group,
    may be obtained from:

         National Institute for Working Life
         Publications Department
         S-171 84 Solna
         Sweden

         In the peer review procedure of documents prepared in the series
     Criteria documents from the Nordic Expert Group (focused on human
    health effects only), one member of the Nordic Expert Group serves as
    primary reviewer for the first draft.  A second draft is forwarded to
    all members of the Nordic Expert Group, who in turn consult
    appropriate specialists to review the document.  The specialists are
    chosen either because they have an extended knowledge of the substance
    itself or because they are specialists in the critical effect area of
    the substance evaluated.  The second review is performed by a review
    board, including the Nordic Expert Group participants with the  ad 
     hoc specialists.  After revision, the document is checked again by
    the members of the Nordic Expert Group and the  ad hoc experts for
    further comments.  The review board meeting is repeated if necessary.

    APPENDIX 2 - CICAD PEER REVIEW

         The draft CICAD on limonene was sent for review to institutions
    and organizations identified by IPCS after contact with IPCS national
    Contact Points and Participating Institutions, as well as to
    identified experts. Comments were received from:

         Department of Health, London, United Kingdom

         Department of Public Health, Albert Szent-Gyorgyi University
         Medical School, Szeged, Hungary

         Direccion General de Salud Ambiental, Subsecretario de Regulacion
         y Fomento, Sanitario, San Luis Potosi, Mexico

         Environmental Health Directorate, Health Canada, Ottawa, Canada

         International Agency for Research on Cancer, Lyon, France

         Ministry of Health, National Centre of Hygiene, Medical Ecology
         and Nutrition, Sofia, Bulgaria

         Ministry of Health and Welfare, International Affairs Division,
         Government of Japan, Tokyo, Japan

         National Institute for Working Life, Solna, Sweden

         National Institute of Public Health, Oslo, Norway

         United States Department of Health and Human Services (National
         Institute of Environmental Health Sciences)

         United States Environmental Protection Agency (Office of
         Pollution Prevention and Toxics; Office of Drinking Water)

    APPENDIX 3 - CICAD FINAL REVIEW BOARD

    Brussels, Belgium, 18-20 November 1996

    Members

    Dr A. Aitio, Institute of Occupational Health, Helsinki, Finland

    Dr K. Bentley, Director, Environment Policy Section, Commonwealth
    Department of Human Services and Health, Canberra, Australia

    Mr R. Cary, Toxicology and Existing Substances Regulation Unit, Health
    and Safety Executive, Merseyside, United Kingdom

    Dr J. de Fouw, National Institute of Public Health and Environmental
    Protection, Bilthoven, The Netherlands

    Dr C. DeRosa, Director, Division of Toxicology, Agency for Toxic
    Substances and Disease Registry, Atlanta, GA, USA

    Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood, Abbots
    Ripton, Huntingdon, Cambridgeshire, United Kingdom

    Dr W. Farland, Director, National Center for Environmental Assessment,
    Office of Research and Development, US Environmental Protection
    Agency, Washington, DC, USA  (Chairperson)

    Dr T.I. Fortoul, Depto. Biologia Celular y Tisular, National
    University of Mexico and Environmental Health Directorate of the
    Health Ministry, Mexico D.F., Mexico

    Dr H. Gibb, National Center for Environmental Assessment, US
    Environmental Protection Agency, Washington, DC, USA

    Dr R.F. Hertel, Federal Institute for Health Protection of Consumers &
    Veterinary Medicine, Berlin, Germany

    Mr J.R. Hickman, Environmental Health Directorate, Health Canada,
    Ottawa, Ontario, Canada

    Dr T. Lakhanisky, Head, Division of Toxicology, Institute of Hygiene
    and Epidemiology, Brussels, Belgium  (Vice-Chairperson)

    Dr I. Mangelsdorf, Documentation and Assessment of Chemicals,
    Fraunhofer Institute for Toxicology and Aerosol Sciences, Hanover,
    Germany

    Ms E. Meek, Head, Priority Substances Section, Environmental Health
    Directorate, Health Canada, Ottawa, Ontario, Canada

    Dr K. Paksy, National Institute of Occupational Health, Budapest,
    Hungary

    Mr D. Renshaw, Department of Health, London, United Kingdom

    Dr J. Sekizawa, Division of Chemo-Bio Informatics, National Institute
    of Hygienic Sciences, Tokyo, Japan

    Dr H. Sterzl-Eckert, GSF-Forschungszentrum für Umwelt und Gesundheit
    GmbH, Institut für Toxikologie, Oberschleissheim, Germany

    Professor S. Tarkowski, Department of Environmental Health Hazards,
    The Nofer Institute of Occupational Medicine, Lodz, Poland

    Dr M. Wallen, National Chemicals Inspectorate (KEMI), Solna, Sweden

    Observers

    Professor F.M.C. Carpanini,1 Director, Centre for Ecotoxicology and
    Toxicology of Chemicals (ECETOC), Brussels, Belgium

    Mr R. Haigh,1 Head of Unit, Health and Safety Directorate, European
    Commission, Luxembourg

    Mr B.U. Hildebrandt, Federal Ministry for the Environment, Nature
    Conservation and Nuclear Safety, Bonn, Germany

    Mr P. Hurst,1 Chemical and Consumer Policy Officer, Conservation
    Policy Division, World Wide Fund for Nature, Gland, Switzerland

    Dr A. Lombard (Representative of CEFIC), ELF-ATOCHEM, Paris, France

    Dr P. McCutcheon,1 Environment, Consumer Protection and Nuclear
    Safety, European Commission, Brussels, Belgium

    Dr R. Montaigne, Counsellor, Technical Affairs Department, European
    Chemical Industry Council (CEFIC), Brussels, Belgium

    Dr M. Pemberton, ICI Acrylics, Lancashire, United Kingdom

    Dr A. Smith, Organisation for Economic Co-operation and Development,
    Environment Division, Paris, France


              

    1 Invited but unable to attend.

    Secretariat

    Dr M. Baril, International Programme on Chemical Safety, World Health
    Organization, Geneva, Switzerland

    Dr L. Harrison, International Programme on Chemical Safety, World
    Health Organization, Geneva, Switzerland

    Dr M. Mercier, Director, International Programme on Chemical Safety,
    World Health Organization, Geneva, Switzerland

    Dr P. Toft, Associate Director, International Programme on Chemical
    Safety, World Health Organization, Geneva, Switzerland

    RÉSUMÉ D'ORIENTATION

         Les informations contenues dans le présent CICAD (document
    international succinct sur l'évaluation des risques chimiques) sur le
    limonène  (d-limonène,  l-limonène et  d/l limonène) proviennent
    principalement d'une étude effectuée en 1993 pour le Nordic Expert
    Group (Karlberg & Lindell, 1993).  D'autres données pour l'évaluation
    du limonène ont été rassemblées à partir d'une deuxième étude menée
    sous les auspices du Conseil des Ministres des Pays nordiques
    (Josefsson, 1993), d'un rapport préliminaire (qui n'a pas fait l'objet
    d'une évaluation par les pairs) sur la présence et les effets du
    limonène dans l'environnement (US EPA, 1994), et de recherches
    effectuées dans les bases de données pertinentes pour les années 
    1993-1995.  Une dernière recherche bibliographique portant sur les
    années 1996-1997 n'a pas fourni d'éléments de nature à modifier les
    conclusions formulées dans le CICAD.  L'appendice 1 donne des
    informations sur la nature et la disponibilité des sources
    documentaires.  Des informations concernant l'examen par les pairs du
    présent CICAD figurent à l'appendice 2.  La publication de ce CICAD a
    été approuvée à une réunion du Comité d'Evaluation finale qui s'est
    tenue à Bruxelles (Belgique) du 18 au 20 novembre 1996.  La liste des
    participants à cette réunion figure à l'appendice 3.  La fiche
    internationale de sécurité chimique concernant le  d-limonène (ICSC
    0918), établie par le Programme international sur la sécurité chimique
    (IPCS, 1993), est également reproduite dans le présent document. 
    L'accent a été mis sur le  d-limonène en raison de l'abondance des
    données disponibles sur cet isomère.

         Le limonène se trouve à l'état naturel dans certains arbres et
    arbustes.  Le limonène et d'autres monoterpènes sont libérés en
    grandes quantités, principalement dans l'atmosphère, à partir de
    sources biologiques naturelles et de sources artificielles.  Le
    limonène est utilisé comme solvant dans le dégraissage des métaux
    avant peinture, comme agent nettoyant dans les industries de
    l'électronique et de l'imprimerie et comme solvant pour peintures.  Il
    est également employé comme aromatisant dans les aliments, les
    produits d'entretien ménagers et les parfums.

         Le limonène est un irritant de la peau, tant pour l'homme que
    pour les animaux de laboratoire.  Chez le lapin, le  d-limonène est
    un irritant oculaire.  Des études effectuées sur des cobayes ont
    montré que le  d-limonène oxydé par l'air, mais non le  d-limonène
    lui-même, induisait des  allergies de contact.  Étant donné que le 
     d-limonène et le  l-limonène sont des énantiomères, il pourrait en
    être de même pour le  l-limonène et le dipentène (mélange des deux). 
    Le caractère allergène du limonène pourrait donc dépendre dans une
    large mesure des manipulations qu'il a subies, de sa pureté et de
    l'adjonction éventuelle d'antioxygènes.

         L'organe critique chez l'animal (sauf chez le rat mâle), après
    administration par voie orale ou intrapéritonéale, est le foie.  On
    n'a pas trouvé d'études au cours desquelles des animaux auraient été
    exposés au limonène par inhalation.  L'exposition au limonène a des
    effets sur la quantité et l'activité de diverses enzymes hépatiques,
    ainsi que sur le poids du foie, la cholestérolémie et la sécrétion
    biliaire.  Ces effets ont été observés chez la souris, le rat et le
    chien.  Les données disponibles sont insuffisantes pour déterminer
    quel est l'organe critique chez l'homme.

         Chez le rat mâle, l'exposition au  d-limonène provoque des
    lésions et des tumeurs rénales.  Il est admis qu'une protéine
    spécifique du rat mâle, l'alpha2µ-globuline, joue un rôle crucial dans
    le développement des lésions rénales néoplasiques ou non néoplasiques
    chez cet animal, de sorte que leur étude n'est pas jugée pertinente
    pour l'évaluation du risque chez l'homme.  Le  d-limonène a été
    soumis à une série d'épreuves  in vitro à court terme dans lesquelles
    il s'est révélé non génotoxique.  Il n'y a pas de preuve que le
    limonène exerce des effets tératogènes ou embryotoxiques en l'absence
    de toxicité maternelle.  De façon générale, on peut admettre que le
     d-limonène est un composé de toxicité relativement faible (si l'on
    excepte ses propriétés irritantes et sensibilisantes).

         D'après les données disponibles, les aliments sont la principale
    source d'exposition au limonène.  Une valeur guide de 0,1 mg/kg de
    poids corporel par jour a été établie pour l'ingestion.  D'après les
    estimations actuelles concernant le niveau d'exposition, la présence
    de limonène dans les aliments ne semble pas présenter un risque
    significatif pour la santé humaine.

         Dans l'atmosphère, le limonène et d'autres terpènes réagissent
    rapidement avec les radicaux hydroxyle et nitrate résultant de
    réactions photochimiques, ainsi qu'avec l'ozone.  L'oxydation des
    terpènes, dont le limonène, contribue à la formation d'aérosols et de
    smogs photochimiques.  Dans le sol, la mobilité du limonène devrait
    théoriquement être faible; dans le milieu aquatique, il devrait se
    lier fortement aux sédiments.  Le limonène est résistant à
    l'hydrolyse.  Il est biodégradable en conditions d'aérobiose, mais non
    en milieu anaérobie.

         Pour les organismes terrestres, l'air est la voie d'exposition la
    plus probable.  Les quelques études effectuées sur des espèces
    terrestres (des insectes) exposées aux vapeurs de limonène ont révélé
    que celui-ci produisait des effets à des concentrations de l'ordre de
    quelques parties par million.  Les concentrations mesurées dans
    l'environnement sont généralement de l'ordre de 0,1-2 ppb (0,6-
    11 µg/m3).  En cas de pollution, les concentrations dans le sol
    peuvent dépasser les niveaux pour lesquels on constate des effets sur
    les organisme vivants (par exemple, les vers de terre).  En milieu
    aquatique, des signes de toxicité aiguë peuvent être observés sur les
    poissons et les daphnies.  Les concentrations de limonène dans les
    eaux de surface sont généralement bien inférieures aux doses toxiques

    aiguës déterminées expérimentalement; il est donc peu probable que le
    limonène présente un risque de toxicité aiguë pour les organismes
    aquatiques.  Aucune étude n'a été découverte sur ses effets
    chroniques.

    RESUMEN DE ORIENTACION

         Esta reseña de la evaluación química internacional del limoneno
     (d-limoneno,  l-limoneno y  d/ l-limoneno) se basa principalmente
    en un examen preparado en 1993 para el Grupo Nórdico de Expertos
    (Karlberg & Lindell, 1993).  Un segundo examen efectuado bajo los
    auspicios del Consejo Nórdico de Ministros (Josefsson, 1993), una
    fuente de información preliminar, no revisada por expertos, sobre la
    exposición y los efectos ambientales (EPA de los Estados Unidos, 1994)
    y múltiples consultas en las bases de datos pertinentes sobre los años
    1993 a 1995 permitieron identificar datos adicionales para evaluar el
    limoneno.  En una última consulta de las publicaciones aparecidas en
    1996 y 1997 no se hallaron datos que modificaran las conclusiones
    enunciadas en la reseña de la evaluación química internacional.  En el
    apéndice 1 figura información sobre la naturaleza y disponibilidad del
    documento de base.  En el apéndice 2 se da información sobre el examen
    colegiado de la presente reseña.  Esta reseña fue aprobada para su
    publicación en una reunión del Comité de Revisión Final celebrada en
    Bruselas (Bélgica) del 18 al 20 de noviembre de 1996.  Los
    participantes en la reunión figuran en el apéndice 3.  Se ha
    reproducido asimismo la ficha internacional de seguridad química (ICSC
    0918) del  d-limoneno, elaborada por el Programa Internacional de
    Seguridad de las Sustancias Químicas (IPCS, 1993).  La atención
    especial prestada al  d-limoneno obedece a la gran cantidad de datos
    disponibles sobre esta forma isomérica.

         El limoneno está presente en forma natural en algunos árboles y
    arbustos.  Fuentes biógenas y antropógenas liberan limoneno y otros
    monoterpenos en grandes cantidades, principalmente en la atmósfera. 
    El limoneno se utiliza como disolvente para desengrasar los metales
    antes de la pintura industrial, para la limpieza en la industria
    electrónica y de la imprenta, y como disolvente en la pintura.  Se
    emplea asimismo como aditivo de sabor y aroma en alimentos, productos
    de limpieza de uso doméstico y perfumes.

         El limoneno es un irritante de la piel, tanto en animales de
    experimentación como en seres humanos.  Se ha comprobado que en los
    conejos el  d-limoneno irrita los ojos.  Estudios efectuados con
    cobayos han revelado que el  d-limoneno oxidado por el aire, pero no
    propiamente el  d-limoneno, produce dermatitis de contacto.  Como el
     d-limoneno y el  l-limoneno son enantiómeros, lo anterior podría
    ser cierto también para el  l-limoneno y el dipenteno (la forma
    racémica).  La manipulación y la pureza de la sustancia química, y
    posiblemente la adición de antioxidantes, pueden influir por tanto de
    manera crucial en la alergenicidad del limoneno. 

         El órgano crítico en los animales (excepto en las ratas macho),
    tras la administración oral o intraperitoneal de la sustancia, es el
    hígado.  No se sabe de estudios en que los animales de experimentación
    hayan sido expuestos al limoneno por inhalación.  La exposición al
    limoneno altera la cantidad y la actividad de distintas enzimas
    hepáticas, el peso del hígado, los niveles de colesterol y la

    secreción biliar.  Se han observado esos cambios en ratones, ratas y
    perros.  No se dispone de suficientes datos para determinar cuál es el
    órgano crítico en el ser humano. 

         En las ratas macho, la exposición al  d-limoneno causa lesiones
    y tumores renales.  Se cree que una proteína específica de la rata
    macho, la alpha2µ-globulina, cumple una función crucial en el
    desarrollo de lesiones renales tanto neoplásicas como no neoplásicas. 
    Por consiguiente, esas lesiones renales se consideran sin importancia
    para la evaluación del riesgo en el ser humano.  El  d-limoneno se
    estudió en una batería de pruebas  in vitro de corta duración
    demostrándose que no es genotóxico.  No hay constancia de que el
    limoneno tenga efectos teratogénicos o embriotóxicos en ausencia de
    toxicidad materna.  En general, el  d-limoneno puede considerarse una
    sustancia química con una toxicidad bastante baja (salvo por sus
    propiedades irritantes y sensibilizantes).

         Según los datos de que se dispone, los alimentos son la principal
    fuente de exposición al limoneno.  El valor de orientación calculado
    para la ingestión de limoneno es de 0,1 mg por kg de peso corporal
    diarios.  A los actuales niveles de exposición estimados, el limoneno
    presente en los productos alimenticios no parece constituir un riesgo
    significativo para la salud humana.

         En la atmósfera, el limoneno y otros terpenos reaccionan
    rápidamente con los radicales hidroxilo y nitrato y con el ozono
    producidos fotoquímicamente.  La oxidación de los terpenos como el
    limoneno contribuye a la formación de aerosoles y de niebla
    fotoquímica.  En principio, en el suelo el limoneno tiene poca
    movilidad, y en el medio acuático se une fuertemente al sedimento.  El
    limoneno es resistente a la hidrólisis.  La biodegradación se produce
    en condiciones aerobias, pero no en condiciones anaerobias.

         La vía de exposición al limoneno más probable en los organismos
    terrestres es el aire.  Los pocos estudios realizados sobre especies
    terrestres (esto es, insectos) mediante exposición al vapor han
    revelado efectos en niveles de partes por millón.  Las concentraciones
    ambientales medidas oscilan generalmente entre 0,1 y 2 ppmm (0,6-11
    µg/m3).  En lugares contaminados, las concentraciones de limoneno en
    el suelo pueden sobrepasar los niveles por encima de los cuales
    aparecen efectos en los organismos que viven en el suelo (por ejemplo,
    los gusanos).  En el medio acuático, el limoneno presenta una elevada
    toxicidad aguda para los peces y para  Daphnia.  Las concentraciones
    de limoneno en aguas superficiales suelen ser mucho más bajas que los
    niveles de toxicidad aguda determinados experimentalmente, por lo que
    es probable que el limoneno plantee un bajo riesgo de efectos tóxicos
    agudos para los organismos acuáticos.  No se conocen estudios sobre
    los efectos crónicos.





    See Also:
       Toxicological Abbreviations
       Limonene (WHO Food Additives Series 30)