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 Organization, or the World Health Organization.

Concise International Chemical Assessment Document 67

First draft prepared by Mr Philip Copestake, Toxicology Advice & Consulting Ltd, Surrey, United Kingdom

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, 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, 2005

The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organization (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals.

The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, the United Nations Institute for Training and Research, and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 UN Conference on 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

Selected alkoxyethanols : 2-Butoxyethanol

(Concise international chemical assessment document ; 67)

1.Ethylene glycols - adverse effects 2.Ethylene glycols - toxicity 3.Environmental exposure
4. Risk assessment I.International Programme on Chemical Safety II.Series.

ISBN 92 4 153067 7           (LC/NLM Classification: QV 82)

ISSN 1020-6167

©World Health Organization 2005

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Technically and linguistically edited by Marla Sheffer, Ottawa, Canada, and printed by Wissenchaftliche Verlagsgesellschaft mbH, Stuttgart, Germany

FOREWORD

Concise International Chemical Assessment Documents (CICADs) are published by the International Programme on Chemical Safety (IPCS) — a cooperative programme of the World Health Organization (WHO), the International Labour Organization (ILO), and the United Nations Environment Programme (UNEP). CICADs have been developed from the Environmental Health Criteria documents (EHCs), more than 200 of which have been published since 1976 as authoritative documents on the risk assessment of chemicals.

International Chemical Safety Cards on the relevant chemical(s) are attached at the end of the CICAD, to provide the reader with concise information on the protection of human health and on emergency action. They are produced in a separate peer-reviewed procedure at IPCS. They may be complemented by information from IPCS Poison Information Monographs (PIM), similarly produced separately from the CICAD process.

CICADs are concise documents that provide summaries of the relevant scientific information concerning the potential effects of chemicals upon human health and/or the environment. They are usually based on selected national or regional evaluation documents or on existing EHCs. Before acceptance for publication as CICADs by IPCS, these documents undergo extensive peer review by internationally selected experts to ensure their completeness, accuracy in the way in which the original data are represented, and the validity of the conclusions drawn.

The primary objective of CICADs is characterization of hazard and dose–response from exposure to a chemical. CICADs are not a summary of all available data on a particular chemical; rather, they include only that information considered critical for characterization of the risk posed by the chemical. The critical studies are, however, presented in sufficient detail to support the conclusions drawn. For additional information, the reader should consult the identified source documents upon which the CICAD has been based.

Risks to human health and the environment will vary considerably depending upon the type and extent of exposure. Responsible authorities are strongly encouraged to characterize risk on the basis 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 170.¹

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 IPCS to inform it of the new information.

Procedures

The flow chart shows the procedures followed to produce a CICAD. These procedures are designed to take advantage of the expertise that exists around the world — expertise that is required to produce the high-quality evaluations of toxicological, exposure, and other data that are necessary for assessing risks to human health and/or the environment. The IPCS Risk Assessment Steering Group advises the Coordinator, IPCS, on the selection of chemicals for an IPCS risk assessment based on the following criteria:

• there is the probability of exposure; and/or

• there is significant toxicity/ecotoxicity.

Thus, it is typical of a priority chemical that:

• it is of transboundary concern;

• it is of concern to a range of countries (developed, developing, and those with economies in transition) for possible risk management;

• there is significant international trade;

• it has high production volume;

• it has dispersive use.

Advice from Risk Assessment Steering Group Criteria of priority: there is the probability of exposure; and/or there is significant toxicity/ ecotoxicity. Thus, it is typical of a priority chemical that it is of transboundary concern; it is of concern to a range of countries (developed, developing, and those with economies in transition) for possible risk management; there is significant international trade; the production volume is high; the use is dispersive. Special emphasis is placed on avoiding duplication of effort by WHO and other international organizations. A prerequisite of the production of a CICAD is the availability of a recent high-quality national/regional risk assessment document = source document. The source document and the CICAD may be produced in parallel. If the source document does not contain an environmental section, this may be produced de novo, provided it is not controversial. If no source document is available, IPCS may produce a de novo risk assessment document if the cost is justified. Depending on the complexity and extent of controversy of the issues involved, the steering group may advise on different levels of peer review: standard IPCS Contact Points above + specialized experts above + consultative group

The Steering Group will also advise IPCS on the appropriate form of the document (i.e. a standard CICAD or a de novo CICAD) and which institution bears the responsibility of the document production, as well as on the type and extent of the international peer review.

The first draft is usually based on an existing national, regional, or international review. When no appropriate source document is available, a CICAD may be produced de novo. Authors of the first draft are usually, but not necessarily, from the institution that developed the original review. A standard outline has been developed to encourage consistency in form. The first draft undergoes primary review by IPCS to ensure that it meets the specified criteria for CICADs.

The second stage involves international peer review by scientists known for their particular expertise and by scientists selected from an international roster compiled by IPCS through recommendations from IPCS national Contact Points and from IPCS Participating Institutions. Adequate time is allowed for the selected experts to undertake a thorough review. Authors are required to take reviewers’ comments into account and revise their draft, if necessary. The resulting second draft is submitted to a Final Review Board together with the reviewers’ comments. At any stage in the international review process, a consultative group may be necessary to address specific areas of the science. When a CICAD is prepared de novo, a consultative group is normally convened.

The CICAD Final Review Board has several important functions:

• to ensure that each CICAD has been subjected to an appropriate and thorough peer review;
• to verify that the peer reviewers’ comments have been addressed appropriately;
• to provide guidance to those responsible for the preparation of CICADs on how to resolve any remaining issues if, in the opinion of the Board, the author has not adequately addressed all comments of the reviewers; and
• to approve CICADs as international assessments.

Board members serve in their personal capacity, not as representatives of any organization, government, or industry. They are selected because of their expertise in human and environmental toxicology or because of their experience in the regulation of chemicals. Boards are chosen according to the range of expertise required for a meeting and the need for balanced geographic representation.

Board members, authors, reviewers, consultants, and advisers who participate in the preparation of a CICAD are required to declare any real or potential conflict of interest in relation to the subjects under discussion at any stage of the process. Representatives of nongovernmental organizations may be invited to observe the proceedings of the Final Review Board. Observers may participate in Board discussions only at the invitation of the Chairperson, and they may not participate in the final decision-making process.

This CICAD² on 2-butoxyethanol is an update of the CICAD published in 1998 (IPCS, 1998), which was based upon reviews prepared by NIOSH (1990) and ATSDR (1996) of the USA. Human health aspects of the CICAD have been extensively revised, as important new information on carcinogenicity and an assessment of the mode of action for tumour development observed in these studies have become available. Additional detailed information on potential exposure has also been incorporated as a basis for the sample risk characterization.³ The update was prepared by Toxicology Advice & Consulting Ltd of the United Kingdom and is based primarily on documentation prepared as part of the Canadian Priority Substances Program under CEPA (Environment Canada & Health Canada, 2002). The objective of assessments on priority substances under CEPA is to assess potential effects of indirect exposure in the general environment on human health as well as environmental effects. Data identified as of October 1999 were considered in the source document. A comprehensive literature search of several online databases was conducted in February 2003 to identify any key references published subsequent to those incorporated in the source document. Information on the nature of the peer review and availability of the source document is presented in Appendix 2. Information on the peer review of this CICAD is presented in Appendix 3. This CICAD was approved as an international assessment at a meeting of the Final Review Board, held in Hanoi, Viet Nam, on 28 September – 1 October 2004. Participants at the Final Review Board meeting are listed in Appendix 4. The International Chemical Safety Cards for 2-butoxyethanol (ICSC 0059) and 2-butoxyethyl acetate (ICSC 0839), produced by IPCS (2003, 2005a), have also been reproduced in this document.

2-Butoxyethanol (CAS No. 111-76-2) is a colourless liquid that is miscible with water and most organic solvents. It has not been reported to occur as a natural product.

2-Butoxyethanol is used widely as a solvent in surface coatings, such as spray lacquers, quick-dry lacquers, enamels, varnishes, varnish removers, and latex paint. It is also used in metal and household cleaners.

Based upon limited data, ambient exposures in air are generally in the µg/m³ range. Indirect exposure of the general population to 2-butoxyethanol is most likely from inhalation and dermal absorption during the use of products containing the chemical. Levels of airborne 2-butoxyethanol in occupational settings are typically in the mg/m³ range.

2-Butoxyethanol is readily absorbed following inhalation, oral, and dermal exposure. The chemical is metabolized primarily via alcohol and aldehyde dehydrogenases, with the formation via BALD of BAA, the principal metabolite, although other metabolic pathways have also been identified.

2-Butoxyethanol has moderate acute toxicity and is irritating to the eyes and skin; it is not a skin sensitizer. The principal effect exerted by 2-butoxyethanol and its metabolite BAA is haematotoxicity. In vitro studies indicate that human red blood cells are not as sensitive as rat red blood cells to the haemolytic effects of 2-butoxyethanol and BAA and that the latter is the more potent haemolytic agent. In rats, adverse effects on the central nervous system, kidneys, and liver occur at higher exposure concentrations than do haemolytic effects. In animals, adverse effects on reproduction and development have been observed only at maternally toxic doses. Long-term studies in laboratory animals gave some evidence of carcinogenicity in mice (increased incidences of haemangiosarcomas of the liver or hepatocellular carcinomas in males and squamous cell papillomas or carcinomas of the forestomach in females) and equivocal evidence in female rats (a marginal increase in the incidence of benign or malignant pheochromocytomas of the adrenal gland). The results of in vitro tests for mutagenicity of 2-butoxyethanol were inconsistent; 2-butoxyethanol was not genotoxic in vivo.

Based on limited data from case reports and one clinical study, similar acute effects — including haemolytic effects as well as effects on the central nervous system — are observed in humans and rats exposed to 2-butoxyethanol, although the effects are observed at much higher exposure concentrations in humans than in rats. A TC, making use of chemical-specific adjustment factors, for haemolytic effects of 11 mg/m³ has been developed, based on BMCs. A TC of 0.04 mg/m³ for lesions in the forestomach of mice was also established.

Levels of 2-butoxyethanol in ambient air in Canada are less than the TCs derived for effects on the blood or forestomach. For example, the mean concentration of 2-butoxyethanol in outdoor air reported in a multimedia exposure study was 8.4 µg/m³, with a maximum of 243 µg/m³. However, exposure to 2-butoxyethanol during use of products containing the substance could potentially exceed the TCs, based on limited data on emissions from products currently available. Conservative estimates of short-term indoor air concentrations resulting from emissions of some common household products ranged up to 62 mg/m³.

Based upon extremely conservative assumptions, the highest predicted concentrations of 2-butoxyethanol in surface waters in the immediate vicinity of effluent streams may, in some cases, exceed PNECs. However, more realistic assumptions based on the available data suggest that risk to aquatic organisms is low. Owing to the short half-life of 2-butoxyethanol in the atmosphere, measured or predicted concentrations of this chemical in air are considered to have no environmental significance.

2-Butoxyethanol (CAS No. 111-76-2; C₆H₁₄O₂; relative molecular mass, 118.2), also known as monobutyl glycol ether and butyl cellosolve, is a synthetic glycol ether. It is a colourless liquid with a mild ether odour; the odour threshold is approximately 0.5 mg/m³ (Amoore & Hautala, 1983). At ambient temperature, 2-butoxyethanol is miscible with water and most organic solvents. 2-Butoxyethanol has a boiling point of 171 °C, a vapour pressure of 0.1 kPa at 20 °C, and a log K_ow of 0.83. A calculated Henry’s law constant is 0.551 Pa·m³/mol (ASTER, 1996). Additional physical and chemical properties are presented in the International Chemical Safety Card for 2-butoxyethanol reproduced in this document.

The structural formula of 2-butoxyethanol is given below:

The conversion factors⁴ for 2-butoxyethanol in air (at 20 °C and 101.3 kPa) are as follows: 1 ppm in air = 4.91 mg/m³; 1 mg/m³ = 0.204 ppm.

Laboratory analysis for 2-butoxyethanol in environmental samples is usually by GC in combination with FID, ECD, or MS detection; infrared absorption spectrophotometry is also sometimes used. The detection limits of these analytical methods in air include 0.15 mg/m³ for a 48-litre sample (OSHA, 1990) and 0.01–0.02 mg for 2- to 10-litre samples (NIOSH, 1994). Multidimensional GC–MS has been used to improve the detection limit to 5–7 µg per sample (Kennedy et al., 1990).

GC methods combined with FID, ECD, or MS detection and HPLC methods coupled with ultraviolet or radiochemical detection have been developed for the analysis of 2-butoxyethanol and its metabolite BAA in urine and blood (Smallwood et al., 1984, 1988; Groeseneken et al., 1986, 1989; Johanson et al., 1986, 1988; Rettenmeier et al., 1993; Sakai et al., 1993, 1994; Corley et al., 1994). The detection limits for BAA range from 0.03 to 0.1 mg/l. 2-Butoxyethanol and BAA in rat and human blood can be analysed by a GC–MS derivatization method with a detection limit range of 16–18 ng/g blood (Bormett et al., 1995). NIOSH (1990) reviewed the available data and developed guidelines for the biological monitoring of BAA.

2-Butoxyethanol does not occur naturally. It is usually produced by reacting ethylene oxide with butyl alcohol, but also by the direct alkylation of ethylene glycol with an agent such as dibutyl sulfate (Rowe & Wolf, 1982).

2-Butoxyethanol is widely used as a solvent in surface coatings, such as spray lacquers, quick-dry lacquers, enamels, varnishes, varnish removers, and latex paint (Leaf, 1985; Sax & Lewis, 1987; Stemmler et al., 1997). It is also used as a coupling agent in metal and household cleaners; as an intermediate in 2-butoxyethyl acetate production; and in herbicides, automotive brake fluids, printing inks, spot removers, and cosmetics (Leaf, 1985; Stemmler et al., 1997; ATSDR, 1998). The average concentration of 2-butoxyethanol in household products marketed in the USA in 1977 was 2.8%. "Low-pollutant" paints contained up to 6% 2-butoxyethanol in a study conducted in Germany (Plehn, 1990). Levels of 2-butoxyethanol in industrial and household window-cleaning agents have been reported to range from 1% to 30% (v/v) (Vincent et al., 1993; ATSDR, 1998). Concentrations up to 10% have been reported in cosmetic products, including hair dyes, nail polish, and nail polish removers (Health Canada, 1998a). 2-Butoxyethanol has also been used as an ice fog suppressant (USEPA, 1979). In 1994, 176 900 tonnes of 2-butoxyethanol were produced in the USA (USITC, 1996). Within the European Community, the total production capacity of 2-butoxyethanol was approximately 70 000–90 000 tonnes in the same year (ECETOC, 1994; CEFIC, 1995). Canadian production of 2-butoxyethanol was 182.7 tonnes in 1995 and 235.3 tonnes in 1996 (Environment Canada, 1997a,b).

2-Butoxyethanol may be released into air or water by facilities that manufacture, process, or use the chemical (ATSDR, 1998; USNLM, 2002). Products containing 2-butoxyethanol may also release the substance into the air. Solvent-based building materials such as paints will release 2-butoxyethanol to air as they dry. There is potential for the release of 2-butoxyethanol from hazardous waste sites, although quantitative data have not been identified. 2-Butoxyethanol has been detected in samples of groundwater and surface water taken near municipal landfills and hazardous waste sites (ATSDR, 1998). Concentrations of 2-butoxyethanol in aqueous samples from a municipal and an industrial landfill in the USA ranged from <0.4 to 84 mg/l (Beihoffer & Ferguson, 1994). 2-Butoxyethanol was detected at a concentration of 0.23 µg/m³ in the emissions of a municipal waste incineration plant in Germany (Jay & Stieglitz, 1995). The Canadian Chemical Producers’ Association (CCPA, 1997, 1999a,b) reported total environmental emissions to air by member companies between 1992 and 1998 ranging from 1 to 3 tonnes per year. According to data reported under CEPA, 319 tonnes of 2-butoxyethanol were released into the air in Canada in 1996, while 63 tonnes were released as waste, 6.5 tonnes were released into landfills, and 2 tonnes were released into water (Environment Canada, 1997b).

In the atmosphere, 2-butoxyethanol is expected to exist in the vapour phase. Owing to 2-butoxyethanol’s water solubility, wet deposition is likely to occur (ATSDR, 1998). The chemical will not persist in the atmosphere; it has an atmospheric half-life of approximately 17 h, based on an estimated rate constant for reaction with hydroxyl radicals (USNLM, 2002). Tuazon et al. (1998) reported that the gas-phase reaction products of 2-butoxyethanol with hydroxyl radicals in the presence of nitric oxide were n-butyl formate, 2-hydroxyethyl formate, propanal, 3-hydroxybutyl formate, an organic nitrate, and one or more hydroxycarbonyl products. Stemmler et al. (1997) irradiated synthetic air mixtures containing 2-butoxyethanol, methyl nitrite, and nitric oxide in a Teflon bag reactor at room temperature. The major oxidation products were butyl formate, ethylene glycol monoformate, butoxyacetaldehyde, 3-hydroxybutyl formate, and propionaldehyde, whereas minor products were 2-propyl-1,3-dioxolane, ethylene glycol monobutyrate, 2-hydroxybutyl formate, acetaldehyde, propyl nitrate, and butyraldehyde. Howard et al. (1991) estimated a half-life of 2-butoxyethanol in the air of 3.28–32.8 h.

The miscibility of 2-butoxyethanol with water, low log K_ow, and Henry’s law constant suggest that volatilization from water, adsorption, and bioconcentration are not important fate processes and that the chemical should not significantly bioconcentrate in aquatic organisms (OECD, 1997). A bioconcentration factor of 2.5 was calculated (SRC, 1988).

Aerobic biodegradation rates suggest that the half-life of 2-butoxyethanol in surface water will range from 1 to 4 weeks (Howard et al., 1991).

Because of its low K_oc, 2-butoxyethanol should be highly mobile in soil and potentially could transfer to groundwater (OECD, 1997). Howard et al. (1991) estimated half-lives of 2-butoxyethanol of 2–8 weeks in groundwater and 1–4 weeks in soil, based on unacclimated aqueous aerobic biodegradation.

2-Butoxyethanol is not likely to undergo direct hydrolysis in the aquatic environment, and it is likely to be readily biodegraded (ATSDR, 1998). Five-day theoretical BOD values range from 5% (without acclimation) to 73% (with acclimation); 10-day theoretical BOD values range from 57% to 74%. The maximum theoretical BOD value reported is 88% for 20 days (USNLM, 2002). Biodegradation is likely to be the most important mechanism for the removal of 2-butoxyethanol from aerobic soil and water.

A Level III fugacity model has been used to estimate the environmental partitioning of 2-butoxyethanol when released into air, water, or soil. Values for input parameters were as follows: molecular mass, 118 g/mol; vapour pressure, 296 Pa⁵; water solubility, 63 500 mg/l; log K_ow, 0.84⁶; Henry’s law constant, 0.551 Pa·m³/mol; half-life in air, 17 h; half-life in water, 550 h; half-life in soil, 550 h; and half-life in sediment, 1700 h. Modelling was based upon an assumed emission rate of 1000 kg/h, although the emission rate used would not affect the estimated percent distribution. If 2-butoxyethanol is emitted into air, EQC Level III fugacity modelling predicts that about 66% would be present in air, about 20% in water, and about 14% in soil. If 2-butoxyethanol is emitted into water, more than 99% would be present in water. Following its release to soil, about 75% would be present in soil and about 25% in water.

Few data on levels of 2-butoxyethanol in the environment have been identified. One study was conducted to determine concentrations of 2-butoxyethanol in multiple media in Canada to which humans are exposed, including drinking-water and indoor and outdoor air. Thirty-five participants were randomly selected from the Greater Toronto Area in Ontario, six from Queens Subdivision in Nova Scotia, and nine from Edmonton, Alberta. For each participant, samples of drinking-water and beverages and indoor, outdoor, and personal air were collected over a single 24-h period; however, samples of food were not analysed for 2-butoxyethanol. In outdoor air, levels of 2-butoxyethanol were above the limit of detection (0.84 µg/m³) in 32% of the 50 samples. The maximum concentration found was 243 µg/m³, and the mean concentration⁷ was 8.4 µg/m³. In indoor air, 2-butoxyethanol was detected in 66% of the 50 samples. The maximum and mean concentrations were 438 µg/m³ and 27.5 µg/m³, respectively. 2-Butoxyethanol was detected in 70% of the 50 personal air samples. Concentrations ranged from below the limit of detection to 275 µg/m³, with a mean concentration of 31 µg/m³. 2-Butoxyethanol was detected in 68% of the 50 drinking-water samples (detection limit 0.02 µg/l). Concentrations ranged from below the limit of detection to 0.94 µg/l, with a mean concentration of 0.21 µg/l. 2-Butoxyethanol was detected in 56% of the 50 beverage samples (detection limit 6.80 µg/l). Concentrations ranged up to 73.8 µg/l, and the mean concentration was 6.46 µg/l (Conor Pacific, 1998).

In samples of drinking-water collected between 1989 and 1995 from four sites in Ontario, Canada, concentrations of 2-butoxyethanol were above the limit of detection (not specified) in one sample from each site (i.e. in 9–17% of total samples at each site). The highest concentration measured was 5.0 µg/l (OMEE, 1996). 2-Butoxyethanol was detected (but not quantified) in a sample of paper and paperboard food packaging materials in the United Kingdom (Castle et al., 1997).

Reported levels of 2-butoxyethanol in samples of ambient air collected between 1990 and 1993 (including samples taken from Nepal, Europe, and Antarctica) have ranged from below the limits of detection to 100 µg/m³ (Ciccioli et al., 1993, 1996; Daisey et al., 1994; Brinke, 1995; Shields et al., 1996). In Canada, the mean concentration of 2-butoxyethanol in samples of ambient air collected in the vicinity of an automotive plant were reported as 2.3 µg/m³ (when concentrations in samples where 2-butoxyethanol was not detected were assumed to be equivalent to one half the limit of detection), and the maximum concentration was 7.3 µg/m³ (OMEE, 1994). 2-Butoxyethanol was detected at a concentration of 23 µg/l in one of seven groundwater samples collected near the Valley of Drums, Kentucky, USA (ATSDR, 1998). Samples from the Hayashida River in Japan, where effluent entered the river from the leather industry, contained 2-butoxyethanol at 1310 and 5680 µg/l (Yasuhara et al., 1981). Information on levels in soils or sediments has not been identified. 2-Butoxyethanol levels below 100 µg/l have been reported in samples of industrial wastewater effluents in the USA (ATSDR, 1998).

ChemCAN4 (version 0.95) modelling has been used to estimate environmental concentrations of 2-butoxyethanol. This model is a Level III fugacity-based regional model developed to estimate the environmental fate of chemicals in Canada. According to a survey conducted under CEPA, the highest reported recent release of 2-butoxyethanol in Canada was 319 tonnes, from facilities in British Columbia, Ontario, and Quebec in 1996 (Environment Canada, 1997a). To make a conservative estimate of environmental concentrations of 2-butoxyethanol, it was assumed, for modelling purposes, that all of this was released into southern Ontario. "Ontario – Mixed Wood Plain" was therefore selected as the geographic region for ChemCAN4 modelling of 2-butoxyethanol. The 2-butoxyethanol input rate was 36.4 kg/h, all to the atmosphere. Chemical input values were as follows: molecular mass, 118 g/mol; vapour pressure, 296 Pa⁸; water solubility, 63 500 mg/l; log K_ow, 0.84⁹; Henry’s law constant, 0.551 Pa·m³/mol; half-life in air, 17 h; half-life in water, 550 h; half-life in soil, 550 h; and half-life in sediment, 1700 h. Modelling was based upon an assumed emission rate of 1000 kg/h, although the emission rate used would not affect the estimated per cent distribution. For Ontario – Mixed Wood Plain, environmental characteristics were as follows: total surface area, 169 000 km²; percentage of area covered by water, 43.8%; average air height, 2 km; average water depth, 20 m; average soil depth, 10 cm; residence time in air, 1.71 days; residence time in water, 618 days; and environmental temperature, 7.4 °C. Environmental concentrations of 2-butoxyethanol in southern Ontario predicted by ChemCAN4 modelling (assuming all release is to the atmosphere) are as follows: 1.623 ng/m³ in air; 3.02 × 10⁻⁴ µg/l in water; 4.28 × 10⁻³ ng/g dry weight in soil; and 1.64 × 10⁻⁴ ng/g dry weight in sediments. The ChemCAN4 model estimates average concentrations throughout the region; therefore, actual concentrations in the vicinity of releases could be higher than those estimated by the model.

Other than the multimedia exposure study described above (Conor Pacific, 1998), available data on concentrations of 2-butoxyethanol in residential indoor air are limited to detection of 2-butoxyethanol at a concentration of 8 µg/m³ in one of six samples of indoor air collected over 4- to 7-day periods in 1983–1984 from homes in northern Italy (De Bortoli et al., 1986). Concentrations of 2-butoxyethanol in the other five samples were below the limit of detection, which was not specified.

2-Butoxyethanol was measured in indoor air samples (three per site) at concentrations up to 33 µg/m³ during March and April 1991 at 70 office buildings in 25 states plus the District of Columbia across the USA (Shields et al., 1996). A specific limit of detection was not reported for 2-butoxyethanol; however, a general limit of detection of 0.5 µg/m³ was reported for VOCs. Geometric mean concentrations calculated based on the assumption of half of this general limit of detection (0.25 µg/m³) for samples in which the concentration of 2-butoxyethanol was below the limit of detection are reported for three categories of building. 2-Butoxyethanol was detected in 24% of the samples from 50 telecommunications offices at concentrations up to 33 µg/m³; the geometric mean concentration was 0.1 µg/m³. The compound was detected in 44% of the samples from nine data centres at concentrations up to 16 µg/m³, with a geometric mean concentration of 0.2 µg/m³. 2-Butoxyethanol was also detected in 73% of the samples from 11 administrative offices at concentrations up to 32 µg/m³, with a geometric mean concentration of 1.0 µg/m³ (Shields et al., 1996). In contrast, detectable concentrations of 2-butoxyethanol were not present in 70 samples of outdoor air collected in the immediate vicinities of these office buildings.

Indoor air was sampled between June and September 1990 in 12 office buildings in the San Francisco Bay area of northern California, USA. Concentrations of 2-butoxyethanol ranged from below the limit of detection (2 µg/m³) to 130 µg/m³. An arithmetic mean concentration was not reported. The geometric mean concentration was 7.9 µg/m³ in indoor air, compared with 1.9 µg/m³ in the air outside these buildings (Daisey et al., 1994; Brinke, 1995). However, the number of samples collected at each location, the frequencies of detection in indoor air, and key details of the sampling and analytical methods were not reported.

Based on information from the United States National Occupational Exposure Survey (NIOSH, 1983), the number of workers potentially exposed to 2-butoxyethanol in the workplace in the USA during 1981–1983 was estimated at about 1.7 million. Data on the occurrence of airborne 2-butoxyethanol in the workplace obtained from facilities in the USA indicate that, in general, most mean time-weighted average exposures are below 34 mg/m³ (NIOSH, 1990; ATSDR, 1998). Time-weighted average 2-butoxyethanol exposures have ranged from 5.4 to 26 mg/m³, with an average of 17 mg/m³, for silk screening; average exposures of 33 mg/m³ for silk screeners and 13 mg/m³ for silk screen spray painters have also been reported (NIOSH, 1990; ATSDR, 1998). In a study of various industrial operations, geometric mean atmospheric exposures to 2-butoxyethanol ranged from 1.5 to 17.7 mg/m³ for printing, from 3.4 to 93.6 mg/m³ for painting, and from 0.2 to 1774 mg/m³ in a mirror manufacturing plant (Veulemans et al., 1987). Workers employed in varnish production facilities have been reported to have individual exposures ranging from <0.5 to 39 mg/m³ (Angerer et al., 1990; Sohnlein et al., 1993). In a study of automobile cleaners using products containing 2-butoxyethanol, time-weighted average personal exposures ranged from <0.5 to 36 mg/m³ (Vincent et al., 1993).

Available data on levels of 2-butoxyethanol in environmental media in Canada upon which estimates of population exposure may be based are limited to air and drinking-water. These data are further limited by the lack of identification of reliable, quantitative, representative data on levels in residential indoor air, although available information is sufficient to indicate that such levels are higher than those in ambient air.

Therefore, point estimates of average daily intakes (on a body weight basis) were derived primarily as a basis for determining the relative contributions to total intake by the few media for which relevant data were identified (Table 1). These point estimates were based on the limited data on mean concentrations in ambient air, indoor air, and drinking-water reported in the Canadian multimedia exposure study (Conor Pacific, 1998) and reference values for body weight, inhalation volume, and amount of drinking-water consumed daily for six age groups in the general population in Canada. Although confidence in the results of the multimedia exposure study is low, due to limitations of the analytical methodology, this is one of the only studies in which exposure in residential indoor air, the likely principal medium of exposure for the general population (other than during the use of consumer products), was characterized; it is also the only investigation in which an attempt was made to characterize representative exposure of the general population of Canada. Mean concentrations in indoor air for this study, for which confidence in quantification is low, are similar to the single detected value for the only other identified investigation of a limited number of samples of residential indoor air in Italy, for which the limit of detection was not reported. Mean concentrations in the air in offices in well documented studies in other countries were lower, although maximum concentrations were often higher. The dermal uptake of 2-butoxyethanol from air can be estimated to be 0.47 µg/kg body weight per day using the following assumptions: an average K_p of 3 cm/h (Corley et al., 1997); exposure for 21 h/day (Health Canada, 1998b) to the average concentration of 2-butoxyethanol in indoor air (27.5 µg/m³; Conor Pacific, 1998); an adult average total body surface area of 19 400 cm² (Health Canada, 1998b); and an average adult body weight of 70.9 kg (Health Canada, 1998b). This dermal uptake is roughly similar to the intake by inhalation of 2-butoxyethanol from ambient air containing an average concentration of 8.4 µg/m³ (Conor Pacific, 1998) for 3 h/day (i.e., 0.2 µg/kg body weight per day for the adult age group in Table 1).

Table 1: Estimated average intake of 2-butoxyethanol by six age groups in the general population.

Since no monitoring data are available, it is not possible to determine the contribution of food to the overall intake of 2-butoxyethanol. However, 2-butoxyethanol’s volatile nature, very low log K_ow (0.83), and low bioconcentration factor mean that it is unlikely to partition to food. Indeed, based on physical/chemical properties, the principal source of 2-butoxyethanol in food is likely to be water, for which reported concentrations are very low. In addition, if intake in food were estimated on the basis of concentrations predicted in terrestrial animals and plants by fugacity modelling, these values would be more than 2 orders of magnitude less than the estimated average intake from indoor air for an average adult. Exposure to 2-butoxyethanol in soil is likely to be negligible, based on its release patterns and the relatively small quantities of soil ingested.

Based upon available data, exposure of the general population to 2-butoxyethanol is most likely via inhalation and dermal absorption during the use of a variety of consumer products containing this chemical. 2-Butoxyethanol was detected in emissions from seven consumer products, including cleaners, nail polish remover, and hair colorant (selected as those considered most likely to contain the chemical), that had been purchased in Ottawa, Canada, at rates of up to 938 mg/m³ per hour (Cao, 1999; Zhu et al., 2001).

Estimates of indoor air concentrations resulting from use of several cleaning products examined by Health Canada (Cao, 1999; Zhu et al., 2001) were derived on the basis of emission factors calculated from steady-state concentrations measured in emission chambers. Assuming a standard room volume, a conservative air exchange rate, and standard product use scenario information, estimated average concentrations of 2-butoxyethanol during the first 60 min following application range from 2.8 mg/m³ for a glass cleaner to 62 mg/m³ for an all-purpose spray cleaner (see Table 2).

Table 2: Estimates of exposure to 2-butoxyethanol through inhalation and dermal uptake from use of household cleaning products.

Estimates of daily intake of 2-butoxyethanol via inhalation and dermal absorption associated with six common household cleaning tasks involving these spray and glass cleaners are also presented in Table 2. Because these products are used primarily by adults, estimated exposures have been derived for this age group only. (The differences in intake from a given medium among age classes, as a result of age-specific differences, would be small in relation to the variation in exposure from the various sources, in any case.) It is assumed that the hands become wetted by the cleaning products during performance of the various cleaning tasks. Dermal absorption from cleaning products was estimated using five different approaches, in order to characterize the variety of estimates that could be derived using the available data.¹⁰ The estimates of dermal absorption presented in Table 2 are based on the non-steady-state approach with a K_p of 0.0014 cm/h, estimated using the Guy & Potts (1993) equation relating K_p to log K_ow and molecular mass; this approach was considered preferable in view of the fact that the lag times and/or time to steady state are not far removed from the durations for each of the tasks modelled and the limitations in the available measured data on dermal absorption of 2-butoxyethanol.¹¹ The estimated K_p was within the same order of magnitude as the measured K_p based on an in vivo study in guinea-pigs exposed dermally to 2-butoxyethanol solutions (Johanson & Fernström, 1988) and within a factor of 5 of both the estimated and measured values reported in USEPA (1992) for 2-ethoxyethanol, a structurally similar compound. The estimated dermal absorption derived by the various approaches is fairly similar in any case, differing by 9- to 32-fold across all of them, depending on the cleaning product modelled.

The estimated overall daily intakes (i.e. intakes from all tasks combined) via inhalation range from 0.074 to 0.186 mg/kg body weight per day for all-purpose spray cleaners and from 0.004 to 0.006 mg/kg body weight per day for spray glass cleaners, assuming that the user is exposed only for the task duration, average frequency of use, standard values for breathing rate consistent with "light activity," and average adult body weight. (Note that these estimates assume that aerosol generated as overspray is not inhaled by the user and that additional inhalation of background concentrations of 2-butoxyethanol in the residential air following the cleaning activities are relatively low compared with the higher intakes during active use of the products.) Dermal absorption during the performance of these tasks could contribute an additional 0.045–0.132 mg/kg body weight per day for the all-purpose cleaners and 0.007–0.013 mg/kg body weight per day for the glass cleaners, assuming contact with the palms of both hands and an estimated K_p of 0.0014 cm/h and applying a non-steady-state approach. Based on these estimated values, therefore, both inhalation and dermal absorption contribute significantly to intake of 2-butoxyethanol during use of domestic products containing the substance.

It should be noted that estimates of exposure to 2-butoxyethanol through use of consumer products were developed for only a few of the small number of products that were investigated by Health Canada and that exposure to the substance could also occur during use of a variety of other types of products. Little information was identified in the literature regarding measured human exposures from consumer products. Norbäck et al. (1995, 1996) reported that personal air samples collected in the breathing zone of Swedish house painters using water-based paints under "normal" working conditions contained a mean 2-butoxyethanol concentration of 59 µg/m³ (maximum 730 µg/m³). Concentrations were below limits of detection in personal air and area samples (i.e. <3.4 and <1.0 mg/m³, respectively) for cleaners at a school in Australia using a diluted solution of a product containing 1% 2-butoxyethanol (NICNAS, 1996). Office window cleaners in France were exposed to 2-butoxyethanol at concentrations of <1.5–3.4 mg/m³ during use of spray cleaners containing 0.9% or 9.8% of the substance (Vincent et al., 1993).

Results of animal and human studies (most of the available data are from studies conducted with rats) indicate that 2-butoxyethanol is readily absorbed following inhalation, oral, and dermal exposure (Jonsson & Steen, 1978). Absorption through the skin can be significant and may be influenced by the vehicle — water, for example, possibly facilitates percutaneous absorption (Johanson & Fernström, 1988; Wilkinson & Williams, 2002).

2-Butoxyethanol is metabolized primarily via alcohol and aldehyde dehydrogenases, with the formation of BALD and BAA, the principal metabolite (Ghanayem et al., 1987a; Medinsky et al., 1990). This is the favoured metabolic pathway for lower systemic doses of 2-butoxyethanol. Alternative pathways include O-dealkylation to ethylene glycol and conjugation to 2-butoxyethanol glucuronide and/or 2-butoxyethanol sulfate (Medinsky et al., 1990). In the study conducted by Medinsky et al. (1990), higher relative concentrations of BAA and ethylene glycol were obtained at lower vapour concentrations of 2-butoxyethanol; higher 2-butoxyethanol glucuronide levels were observed at the high exposures to 2-butoxyethanol, possibly owing to saturation of the oxidative and dealkylation pathways. In human studies, N-butoxyacetylglutamine (an amino acid conjugate of the parent compound) has been identified as a metabolite (Rettenmeier et al., 1993).

In general, the metabolism of 2-butoxyethanol to BAA is linearly related to exposure concentration up to levels causing mortality. In one study, after inhalation exposure in rats, 2-butoxyethanol and BAA were analysed in blood, muscle, liver, and testes. The kinetic profile of BAA tissue concentrations was similar to that of 2-butoxyethanol tissue concentrations. Sixty-four per cent of the inhaled dose of 2-butoxyethanol was eliminated in urine as BAA, and the rate of urinary excretion of BAA was dose-dependent (Johanson, 1994).

In humans exposed to 2-butoxyethanol via inhalation at 100 mg/m³ for 2 h, the concentration of 2-butoxyethanol in the blood reached a plateau of 7.4 µmol/l within 1–2 h, and the chemical could no longer be detected in the blood 2–4 h after exposure. The mean elimination half-life was 40 min. Less than 0.03% of the total uptake of 2-butoxyethanol was excreted unchanged in the urine, whereas urinary excretion as BAA ranged from 17% to 55% (Johanson et al., 1986). Similarly, after percutaneous uptake of 2-butoxyethanol, the urinary excretion of BAA peaked 3 h after exposure and subsequently declined, with an average half-life of 3.1 h. The accumulated urinary excretion of BAA corresponded to 2.5–39% of uptake (Johanson et al., 1988).

Several PBPK models of increasing sophistication have been developed for 2-butoxyethanol (Johanson et al., 1986; Johanson & Boman, 1991; Shyr et al., 1993; Corley et al., 1994; Lee et al., 1998). The Corley et al. (1994) model accurately predicted pharmacokinetics data in animals at non-toxic dose levels but overpredicted the amount of BAA excreted in the urine at haemolytic doses, presumably due to toxicity in the kidneys. The model (Corley et al., 1994) suggested that dermal intake following whole-body exposure is approximately 21% of the total, rather than the 75% suggested by Johanson & Boman (1991). An additional study further addresses dermal uptake in humans from the vapour phase but does not address direct skin contact with liquid containing 2-butoxyethanol (Corley et al., 1997).

Jones et al. (2003) determined that in human volunteers exposed to 2-butoxyethanol vapours (250 mg/m³ for 2 h), dermal absorption could account for around 11% of the total absorbed dose. This rose to as much as 39% when an industrial setting was simulated (the wearing of overalls, high temperature, and high humidity).

There are species- and sex-related variations in haematological response to 2-butoxyethanol, with rats being the most sensitive species tested to date. Alterations in haematological parameters were observed in repeated inhalation studies in both rats and mice (NTP, 2000), but the changes in mice were consistent with normocytic anaemia, compared with the macrocytic anaemia observed in rats. In both rats and mice, females are more sensitive to the haematotoxicity of 2-butoxyethanol (see section 8). The data suggest that BAA is responsible for the haematological effects. Observed species- and sex-related variations in haematotoxicity are well correlated with differences in production and clearance of BAA. Mice appear to clear BAA from the blood much more quickly than rats, and the fall-off in the rate of elimination at increased duration of exposure is less marked in mice (Dill et al., 1998). Likewise, clearance of BAA from the blood is slower in female rats than in males (Dill et al., 1998); in addition, the activity of hepatic alcohol dehydrogenase enzyme, which is involved in the metabolism of 2-butoxyethanol to BAA, is greater in females than in males (Aasmoe et al., 1998). Ghanayem et al. (1987b) also observed older rats to be more susceptible to the haemolytic effects of acute 2-butoxyethanol exposure, which is consistent with the greater rate of elimination of metabolites in the urine of younger rats.

These models do not incorporate results of in vitro investigations, in which hepatocytes from rats metabolized 2-butoxyethanol to BAA more efficiently than cells from humans; this pathway was also saturated at much lower doses in human hepatocytes (Green et al., 1996).

Green et al. (2002) compared the metabolic capacity of the rat and mouse stomach to metabolize 2-butoxyethanol. 2-Butoxyethanol was metabolized in vitro in both mouse and rat forestomach and glandular stomach fractions by alcohol dehydrogenases, forming BALD, which was rapidly converted by aldehyde dehydrogenases to BAA. There were marked species differences in alcohol dehydrogenase activity between rats and mice, with the maximum rate up to 1 order of magnitude greater in mice than in rats.

Although data are limited, the acetate derivative of 2-butoxyethanol (2-butoxyethyl acetate) appears to be rapidly hydrolysed to 2-butoxyethanol via esterases in several tissues in the body (Johanson, 1988). Where available, data on the toxicity of 2-butoxyethyl acetate have, therefore, been included in this CICAD.

Many acute toxicity studies of 2-butoxyethanol have led to the establishment of LC₅₀s or LD₅₀s in a variety of species by inhalation, oral, and dermal exposure. Inhalation LC₅₀s for 2-butoxyethanol of 2400 mg/m³ (male rats, 4 h), 2200 mg/m³ (female rats, 4 h), 3400 mg/m³ (mice, 7 h), and >3200 mg/m³ (guinea-pigs, 1 h) have been reported. Oral LD₅₀s for rats (2500 mg/kg body weight), mice (1400 mg/kg body weight), guinea-pigs (1200 mg/kg body weight), and rabbits (320 mg/kg body weight) have also been reported. Dermal LD₅₀s of 404–502 and 2000 mg/kg body weight have been reported for rabbits and guinea-pigs, respectively. Effects observed in rats, mice, and guinea-pigs exposed by inhalation to the LC₅₀ or by ingestion at the LD₅₀ include loss of coordination, ataxia, sluggishness, muscular flaccidity, enlarged kidney, blood in the bladder, haemoglobinuria, splenic lesions, and pulmonary congestion (Werner et al., 1943a; Carpenter et al., 1956; Dodd et al., 1983; Gingell et al., 1997). Inhalation exposures of female rats to 2-butoxyethanol at 299 mg/m³ for 4-h periods resulted in increased osmotic fragility of erythrocytes (Carpenter et al., 1956).

Ghanayem et al. (1987b) indicated that the haemolytic activity of 2-butoxyethanol in rats is age-dependent, with older rats being more susceptible than younger animals. In their study, 2-butoxyethanol (0, 125, or 500 mg/kg body weight) was administered orally to young (4–5 weeks old) and adult (9–13 weeks old) male F344 rats. Although no significant haematotoxic effects were observed in the younger rats administered 2-butoxyethanol at 125 mg/kg body weight, effects in older animals administered this dose included a significant decrease (P < 0.05) in the number of red blood cells, haematocrit, and haemoglobin and an increase in free haemoglobin plasma levels (P < 0.05). Histopathological evaluation of tissues collected 24 h after administration of 2-butoxyethanol to rats of various ages revealed dose- and age-dependent liver and kidney changes. These histopathological changes exhibited signs of regression when examined 48 h following exposure. Severe acute haemolytic anaemia was evidenced by a decrease in circulating red blood cells, an increase in the concentration of free haemoglobin in plasma, and the development of haemoglobinuria. Ghanayem et al. (1987b) indicated that the acute anaemia in 2-butoxyethanol-exposed rats was caused by a time- and dose-dependent decrease in the number of red blood cells, in haemoglobin concentrations, and in haematocrit, with little or no change in mean cell volume. In a follow-up study, haematological profiles revealed a time- and dose-dependent increase in haematocrit and mean cell volume. Based on these data, Ghanayem et al. (1990) concluded that 2-butoxyethanol causes spherical swelling of red blood cells followed by haemolysis.

To investigate the induction of tolerance, Ghanayem et al. (1992) assessed haematological parameters in naive or previously bled rats administered a single 2-butoxyethanol dose of 125 or 250 mg/kg body weight. The bled/recovered rats were less sensitive to 2-butoxyethanol than the naive animals. In vitro incubations with BAA revealed that red blood cells from the bled/recovered rats were less sensitive than those cells from naive animals. Ghanayem et al. (1992) concluded that young red blood cells formed during the regeneration process were less sensitive to BAA than older red blood cells. On chronic exposure to 2-butoxyethanol, some tolerance might be expected. The mechanism is probably related to the greater susceptibility of older cells to BAA; haemolysis of these cells during the initial exposure followed by their replacement with less susceptible younger cells may account for the development of tolerance.

Female rats appear to be more sensitive than males to the effects of 2-butoxyethanol exposure. The onset of haemolysis was faster and blood cell changes were observed earlier and more frequently in female F344 rats given 2-butoxyethanol at 250 mg/kg body weight by gavage than in males treated similarly (Ghanayem et al., 2000).

Toxic effects in the kidneys have been observed in rabbits exposed percutaneously to 2-butoxyethanol (Carpenter et al., 1956). Necropsy of rabbits exposed for 24 h to undiluted 2-butoxyethanol (0.48–0.64 ml/kg body weight) revealed congestion of the kidneys, haemoglobinuria, pale livers, and engorged spleens (Carpenter et al., 1956).

When 2-butoxyethanol (200, 260, 320, 375, or 500 mg/kg body weight) was applied to the shaved dorsal skin of groups of female rats, blood effects (increased mean cell volume, a lowered erythrocyte count and haemoglobin level, and haemoglobinuria) were observed at all doses except the lowest. However, there was no discernible dose–response relationship, which was attributed to the inherent biological variation in percutaneous absorption and haemolytic susceptibility and to the small number of animals (n = 3) in these dose groups (Bartnik et al., 1987).

2-Butoxyethanol is irritating to the eyes and skin. In rabbits, instillation of an unspecified amount of 2-butoxyethanol caused severe eye irritation, including conjunctival hyperaemia and oedema (von Oettingen & Jirouche, 1931), while 30% and 70% concentrations were moderately irritating (Kennah et al., 1989). When applied to the skin of rabbits for 4 h, 2-butoxyethanol caused mild irritation; extending the period of contact increased the severity of irritation (Tyler, 1984). 2-Butoxyethanol was classified as a severe skin irritant when the Draize method was used (Zissu, 1995).

2-Butoxyethanol did not induce skin sensitization in guinea-pigs (Unilever, 1989; Zissu, 1995).

Haematotoxic effects (e.g. increased osmotic fragility, decreased haemoglobin, decreased numbers of red blood cells) have been observed in rats (270–1600 mg/m³), dogs (1000–1900 mg/m³), and monkeys (1030 mg/m³) exposed repeatedly via inhalation to 2-butoxyethanol for up to approximately 30–35 days (Werner et al., 1943b; Carpenter et al., 1956). Indications of haemoglobinuria, together with histopathological changes in the kidney, were also observed in rabbits exposed for 30 days to 2-butoxyethyl acetate at 2600 mg/m³ (equivalent to a 2-butoxyethanol concentration of 2000 mg/m³) (Truhaut et al., 1979).

Dodd et al. (1983) exposed Fischer 344 rats of both sexes to 2-butoxyethanol at 0, 100, 420, or 1200 mg/m³, 6 h/day for 9 days in total (5 consecutive days of exposure, followed by 2 days of no exposure, then 4 additional consecutive days of exposure). In both sexes, exposure to 1200 mg/m³ was associated with a significant reduction in red blood cell counts (P < 0.001), haemoglobin levels (P < 0.001), and mean cell haemoglobin concentration (P < 0.01), as well as a significant increase (P < 0.001 in all cases) in mean cell volume, nucleated red blood cells, and reticulocytes. Fourteen days post-exposure, a substantial recovery of the affected erythroid parameters was observed; however, statistically significant differences from controls were still observed for the males — i.e. red blood cell count (P < 0.01), mean cell volume (P < 0.001), and mean cell haemoglobin (P < 0.001). Exposure of both sexes to 2-butoxyethanol at 420 mg/m³ was associated with a significant but less profound effect on erythroid parameters. The NOAEC in this study is 100 mg/m³.

In a study designed primarily to assess developmental effects, Tyl et al. (1984) exposed pregnant Fischer 344 rats (36 per group) and New Zealand white rabbits (24 per group) to 2-butoxyethanol (0, 120, 250, 490, or 980 mg/m³) for 6 h/day on days 6–15 of gestation for the rats and on days 6–18 of gestation for the rabbits. In rats, the blood picture was normal at 250 mg/m³; at 490 mg/m³ and above, effects included reductions in red blood cell count, mean cell haemoglobin concentration, and increases in haemoglobin, haematocrit, mean cell volume, and mean cell haemoglobin. In the rabbits, statistically significant increases in haemoglobin content and haematocrit were observed at a 2-butoxyethanol concentration of 490 mg/m³ (P < 0.01) but not at 980 mg/m³, suggesting that rabbits are less sensitive than rats. The NOAEC in this study is 250 mg/m³.

The oral administration of 2-butoxyethanol at doses of 500 or 1000 mg/kg body weight per day for 4 consecutive days to male F344 rats produced a pronounced dose-dependent effect on circulating red and white blood cells (Grant et al., 1985); however, some effects were reversible following the end of exposure. Reduced erythrocyte counts, haematocrit, haemoglobin levels, and leukocyte counts and elevated mean cell volume, reticulocyte counts, and mean cell haemoglobin concentration (P < 0.001) were observed in animals in the high-dose group. Similar, although less severe, effects were observed in the low-dose group. The LOAEL was 500 mg/kg body weight per day.

To assess the development of tolerance to the haemolytic effects of 2-butoxyethanol exposure in laboratory animals, male F344 rats were administered (by gavage) 2-butoxyethanol at 125 mg/kg body weight per day for 0, 1, 2, 3, 6, and 12 days, and haematological parameters (red blood cell counts, haemoglobin content, haematocrit) were determined after exposure (Ghanayem et al., 1987b). Administration of 2-butoxyethanol for 2 and 3 days caused significant haemolysis of red blood cells. After 3 or more days of exposure, however, there was a gradual increase in the number of red blood cells and haemoglobin content. After 12 days of exposure, red blood cells and haemoglobin approached pre-exposure levels, indicative of the development of tolerance to the haemolytic effects of 2-butoxyethanol. In a follow-up study, Ghanayem et al. (1992) assessed the haemolytic effects of 2-butoxyethanol (administered as a single dose of 0, 125, or 250 mg/kg body weight) in untreated (control) or 2-butoxyethanol-pretreated male F344 rats. The pretreated animals were administered (by gavage) 2-butoxyethanol at 125 mg/kg body weight per day for 3 days and then allowed to recover for 7 days prior to study. The pretreated animals were less sensitive than the untreated controls to the haemolytic effects of subsequent exposure to 2-butoxyethanol. In vitro incubations with BAA revealed that red blood cells from the 2-butoxyethanol-pretreated group were less sensitive than cells from the untreated controls. The investigators suggested that the development of tolerance to the haemolytic effects of 2-butoxyethanol might be due in part to the greater resilience of young erythrocytes formed during the blood regeneration process.

In mice orally administered 2-butoxyethanol at 500 or 1000 mg/kg body weight per day, 5 days/week for 5 weeks, no effect upon white blood cell counts, mean cell volume, or haemoglobin levels was observed; however, red blood cell counts were reduced at both doses (Nagano et al., 1979). The oral administration of 2-butoxyethanol to male rats at 222, 443, or 885 mg/kg body weight per day, 5 days/week for 6 weeks, principally affected red blood cells, whereas white blood cell counts were unaffected (Krasavage, 1986).

In a study in which F344/N rats and B6C3F1 mice were administered 2-butoxyethanol in drinking-water daily for 2 weeks, estimates of 2-butoxyethanol intake by rats and mice ranged from 70 to 300 mg/kg body weight per day and from 90 to 1400 mg/kg body weight per day, respectively (NTP, 1993). Survival was not affected. Decreased thymus weights were noted in male mice receiving 2-butoxyethanol at 400 or 650 mg/kg body weight per day. No haematological tests were conducted in this study.

Female B6C3F1 mice were given oral doses (0, 50, 150, or 500 mg/kg body weight) of 2-butoxyethanol or BAA daily for 10 days. Marked hyperkeratosis in the forestomach was observed, with BAA being more toxic than 2-butoxyethanol. The NOEL for the former was 50 mg/kg body weight, whereas that for the latter was 150 mg/kg body weight (Green et al., 2002).

Oral administration of neat 2-butoxyethanol to male and female B6C3F1 mice (400, 800, or 1200 mg/kg body weight per day for 2 days, reduced to 200, 400, and 600 mg/kg body weight per day for a further 2 days because of unexpected mortality) caused dose-related forestomach lesions (epithelial hyperplasia and inflammation) (Poet et al., 2003), similar to those reported in long-term inhalation studies in mice (NTP, 2000). Forestomach lesions were also observed in mice administered 400 mg/kg body weight per day for 4 days by intraperitoneal or subcutaneous injection (Poet et al., 2003).

In an inhalation study in F344/N rats exposed at 150–2500 mg/m³ for 14 weeks (NTP, 2000), there were changes in haematological parameters characteristic of macrocytic, normochromic, responsive anaemia (i.e. increased mean cell volume, lack of change in mean cell haemoglobin values, and increased reticulocyte count). Females were more sensitive than males (LOECs of 150 mg/m³ and 610 mg/m³, respectively); the NOEC in males was considered to be 310 mg/m³. Severity of these effects increased with concentration, and there was no evidence of amelioration over time. Females at the higher concentrations had an increased incidence of thrombosis in the blood vessels of several tissues as well as bone infarction, which was hypothesized to have resulted from severe acute haemolysis or anoxic damage to endothelial cells, causing compromised blood flow. Other effects consistent with regenerative anaemia observed in both male and female rats included excessive haematopoietic cell proliferation in the spleen, haemosiderin pigmentation in the hepatic Kupffer cells and renal cortical tubules, and bone marrow hyperplasia. Inflammation and/or hyperplasia of the forestomach also occurred in rats of both sexes exposed to the higher concentrations of 1200 and 2500 mg/m³, while changes in relative kidney and liver weights were noted at 310 mg/m³ and above in females and 1200 mg/m³ and above in males.

Dodd et al. (1983) exposed Fischer 344 rats of both sexes (16 per group) to 2-butoxyethanol at 0, 25, 120, or 380 mg/m³ by inhalation, 6 h/day, 5 days/week, for 13 weeks. After 6 weeks, animals exposed to 380 mg/m³ had a slight but statistically significant decrease in red blood cell counts (P < 0.01) and haemoglobin level (statistics not reported), accompanied by an 11% increase in mean cell haemoglobin concentration (P < 0.001). At the end of the study, these effects had either lessened or returned to the ranges of control values (contrary to the observations in this strain of rats by the NTP [2000]). The only significant haemolytic effect for male rats in the 380 mg/m³ exposure group was a 5% decrease in red blood cell count after 66 exposures to 2-butoxyethanol (statistics not provided). The NOAEC in this study is 120 mg/m³.

Alterations in haematological parameters indicative of haemolytic anaemia (haemoglobin, haematocrit, and erythrocyte counts) were also the most sensitive end-points observed in B6C3F1 mice exposed for 14 weeks (NTP, 2000). However, the anaemia in mice was considered to be normocytic, normochromic, and responsive (compared with the macrocytic anaemia noted in rats), as 2-butoxyethanol did not induce any changes in mean cell volume. In addition, based on the magnitude of the changes, the anaemia was less severe in mice than in rats, although females were again more sensitive than males (LOECs in females and males were 150 mg/m³ and 610 mg/m³, respectively). As in rats, effects consistent with regenerative anaemia (haemosiderin pigmentation and increased haematopoiesis in the spleen) were also observed. The incidence of hyperplasia of the forestomach was increased in female mice exposed to 610 mg/m³ or more and in males at the highest concentration, 2500 mg/m³; various lesions also appeared in other tissues in females at 2500 mg/m³ (a concentration that was also associated with increased mortality in both sexes).

In older studies, haematotoxic effects (e.g. increased osmotic fragility, decreased haemoglobin, decreased red blood cell numbers) have been observed in mice (490–2000 mg/m³), dogs (2000 mg/m³), and monkeys (490 mg/m³) exposed repeatedly by inhalation to 2-butoxyethanol for up to approximately 90 days (Werner et al., 1943c; Carpenter et al., 1956).

Groups of F344/N rats and B6C3F1 mice (10 per sex per concentration) were administered 2-butoxyethanol in drinking-water (0, 750, 1500, 3000, 4500, or 6000 mg/l) daily for 13 weeks; estimated intakes ranged from 70 to 500 mg/kg body weight per day in rats and from 100 to 1300 mg/kg body weight per day in mice (NTP, 1993). Effects observed in both species included decreased body weight gain and water consumption. In rats, reduced red blood cell counts and histopathological lesions in the liver, spleen, and bone marrow were observed in males and females (at concentrations of 3000–6000 mg/l and 750–6000 mg/l, respectively). Reduced thymus weights (at concentrations of 4500 and 6000 mg/l in males and females, respectively), diminished uterine size (at 4500 and 6000 mg/l in females), and diminished sperm concentration (at 750–6000 mg/l in males) were also noted. A NOAEL could not be identified in rats owing to a mild to moderate anaemia present in most dose groups. In mice, the only effect observed was reduced body weight gain in males and females at concentrations of 3000–6000 mg/l, although haematological parameters may not have been examined in mice.

Siesky et al. (2002) investigated the hepatic effects of 2-butoxyethanol in rodents. Male B6C3F1 mice and male F344 rats were given 2-butoxyethanol by gavage at 225, 450, or 900 mg/kg body weight per day (mice) or 225 or 450 mg/kg body weight per day (rats) for up to 90 days. DNA synthesis, oxidative damage, haematocrit, and iron deposition in the liver were assessed. There was an increase in haemolysis (as indicated by a decrease in haematocrit and an increase in relative spleen weight) in both rats and mice. The percentage of iron-stained Kupffer cells was increased following treatment with 450 or 900 mg/kg body weight per day in mice and in all treated rats. In the mouse liver, a biphasic increase in oxidative damage (elevated 8-hydroxydeoxyguanosine and malondialdehyde) was observed after 7 and 90 days of treatment, whereas no such effect was seen in treated rats. Vitamin E levels were reduced in both mouse and rat liver following treatment with 2-butoxyethanol, although (importantly) the basal level of vitamin E was approximately 2.5-fold higher in the rat than in the mouse. There was also a biphasic induction of DNA synthesis following 2-butoxyethanol treatment in the mouse; increased DNA synthesis was observed in hepatocytes at 90 days and in endothelial cells at 7 and 14 days at all doses. No change in DNA synthesis was seen in 2-butoxyethanol-treated rat liver.

No overt signs of toxicity and no effects on the weight or microscopic appearance of unspecified organs or on haematology (including osmotic fragility tests) were observed in rabbits administered daily dermal 2-butoxyethanol applications (covered) of up to 150 mg/kg body weight per day for 13 weeks (CMA, 1983).

Results are available for inhalation bioassays in which F344/N rats and B6C3F1 mice were exposed (whole body) for 6 h/day, 5 days/week, for up to 2 years to 2-butoxyethanol concentrations of 0, 153, 308, or 614 mg/m³ (rats) and 0, 308, 614, or 1230 mg/m³ (mice) (NTP, 2000). In rats, chronic exposure to 2-butoxyethanol at 153 mg/m³ (the lowest concentration tested) or greater resulted in haemolytic anaemia (characterized as macrocytic, normochromic anaemia, based on decreases in haematocrit, haemoglobin concentrations, and erythrocyte counts, increases in mean cell volume and mean cell haemoglobin, and the lack of effect on mean cell haemoglobin concentration). Consistent with results observed in earlier studies and toxicokinetic data that indicate slower clearance of the active metabolite (i.e. BAA) and greater activity of the relevant isoenzyme in females, the severity of haematological effects was, in general, greater in females than in males; alterations in multiple parameters were observed at the lowest concentration tested in female rats (i.e. 153 mg/m³, considered to be the LOEC), while only mean cell volume was affected in males at this concentration. The severity of these effects increased with exposure level, and the effects were persistent throughout the 12 months during which haematological parameters were monitored; there was no indication of amelioration over time in males, while in females, there were slight decreases in the magnitude of the changes in some parameters at 12 months. The anaemia was considered to be responsive, based on the observation of increased reticulocyte and nucleated erythrocyte counts and a decrease in the myeloid to erythroid ratios.

There was a marginal increase in the incidence of pheochromocytomas (primarily benign, with one malignant tumour) of the adrenal gland in female rats at the highest concentration (614 mg/m³), which, while not statistically significantly elevated compared with concurrent controls, was greater than the incidence of this lesion observed in historical controls at the NTP. (Incidences of benign or malignant pheochromocytomas combined were 3/50, 4/50, 1/49, and 8/49 in the 0, 153, 308, and 614 mg/m³ exposure groups.) There was also a non-statistically significant increase in the incidence of hyperplasia of the adrenal medulla of females at 614 mg/m³. No such increases were observed in males. Other exposure-related histopathological changes observed in rats included increased incidences of minimal hyaline degeneration of the olfactory epithelium (which was considered to be adaptive/protective rather than adverse), increased incidences of Kupffer cell pigmentation in the liver of both sexes at the two highest concentrations, and an increase in splenic fibrosis in males at 308 mg/m³ and above. Based on the results of this study, the NTP concluded that there was no evidence of carcinogenic activity in male F344/N rats and equivocal evidence of carcinogenic activity in female rats of this strain, since the slight increase in pheochromocytomas could not be attributed with certainty to exposure to 2-butoxyethanol.

As in shorter-term studies, B6C3F1 mice were less sensitive than rats to the haematological effects associated with exposure to 2-butoxyethanol. Anaemia, characterized by decreases in haematocrit, haemoglobin concentrations, and erythrocyte count, was present in mice exposed to the two higher concentrations (614 and 1230 mg/m³), and there was some evidence of anaemia in females at 308 mg/m³, but only at one time point. In general, based on the lack of consistent changes in mean cell volume and mean cell haemoglobin concentrations, the effects were consistent with normocytic, normochromic anaemia. Although the anaemia was considered responsive, based on the increased reticulocyte counts, this response ameliorated over time. In addition, contrary to the observations in rats, there were no decreases in myeloid to erythroid ratios. Thrombocytosis (increase in platelet counts) was present at all concentrations. Females were again more sensitive than males, with alterations in haematological parameters generally occurring earlier and at lower exposure levels in female mice.

There were increased incidences of papillomas or carcinomas (combined) of the forestomach in both sexes, which were statistically significant in females exposed to 1230 mg/m³ compared with concurrent and historical controls and in males at 614 and 1230 mg/m³ compared with historical controls (but not study controls). (Incidences of squamous cell papilloma were 1/50, 1/50, 2/49, and 2/49 in male mice and 0/50, 1/50, 2/50, and 5/50 in female mice at concentrations of 0, 308, 614, and 1230 mg/m³, respectively. Incidences of squamous cell papilloma or carcinoma combined in female mice were 0/50, 1/50, 2/50, and 6/50 at concentrations of 0, 308, 614, and 1230 mg/m³, respectively. In addition, the incidence of hyperplasia of the epithelium of the forestomach was significantly increased in a concentration-related manner in all exposed groups, which was accompanied by a concentration-related trend in the incidence of ulcers of the forestomach in female mice. The severity of the epithelial hyperplasia in females also increased with exposure level, as mean severity scores in animals with lesions were 1.8, 2.0, 2.4, and 2.9 at 0, 308, 614, and 1230 mg/m³, respectively.

There was also a concentration-related increase in the incidence of haemangiosarcomas of the liver in male mice (significant at 1230 mg/m³). (Incidences were 0/50, 1/50, 2/49, and 4/49 in mice exposed to 0, 308, 614, and 1230 mg/m³, respectively.) Haemangiosarcomas were also detected in the bone marrow of two mice exposed to 1230 mg/m³ (one of which also had a haemangiosarcoma in the spleen, while the other had a haemangiosarcoma in the heart) and in one mouse exposed to 308 mg/m³. A significant increase in the incidence of hepatocellular carcinomas was also observed in males at the highest concentration, although the incidence was within the range observed in historical controls. In addition, the incidences of hepatocellular adenomas were lower in exposed mice than in controls, and there was no indication of an association between exposure and induction of a related preneoplastic lesion. In spite of these facts, a potential role of 2-butoxyethanol in the development of malignant liver tumours could not be ruled out, and it was concluded that they may be exposure-related. Haemosiderin pigmentation of the Kupffer cells of minimal severity was also noted in the liver of exposed mice.

Based on the increased incidence of hemangiosarcoma of the liver (males) and squamous cell papillomas or carcinomas of the forestomach (females), it was concluded by the investigators that there was some evidence of carcinogenic activity of 2-butoxyethanol in male and female B6C3F1 mice, and the LOAEC for non-neoplastic effects (haematotoxicity and forestomach lesions) was 308 mg/m³ in both sexes (NTP, 2000).

2-Butoxyethanol has been tested for genotoxicity in a range of in vitro and in vivo assays.

In vivo mutagenicity tests have yielded uniformly negative results for 2-butoxyethanol. These assays have included three bone marrow micronucleus tests utilizing intraperitoneal injection in rats and mice (Elias et al., 1996; Elliott & Ashby, 1997; NTP, 2000); a [³²P]post-labelling assay for DNA adducts in the brain, kidney, liver, spleen, and testes of orally dosed rats (Keith et al., 1996); an assay for DNA methylation in the brain, kidney, liver, spleen, and testes of rats and in FVB/N transgenic mice carrying the v-Ha-ras oncogene (Keith et al., 1996); as well as a test for tumour formation in FVB/N transgenic mice (Keith et al., 1996). Although the results of in vitro tests for mutagenicity of 2-butoxyethanol are inconsistent, the negative results from in vivo studies suggest that 2-butoxyethanol does not have significant in vivo genotoxic potential.

In standard tests in bacteria, 2-butoxyethanol was not mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA97, TA98, TA100, and TA102 (Zeiger et al., 1992; Hoflack et al., 1995; Gollapudi et al., 1996). However, the results for strain TA97a were inconsistent, with one report of mutagenicity observed in both the presence and absence of metabolic activation (Hoflack et al., 1995) and another report of no mutagenicity (Gollapudi et al., 1996).

2-Butoxyethanol was not mutagenic at the HPRT locus in Chinese hamster ovary cells in either the presence or absence of metabolic activation (McGregor, 1984; Chiewchanwit & Au, 1995). However, there was evidence that it caused gene mutations at the HPRT locus in Chinese hamster lung (V79) cells (Elias et al., 1996). An in vitro assay for unscheduled DNA synthesis in rat hepatocytes yielded equivocal results (Elliott & Ashby, 1997). 2-Butoxyethanol produced sister chromatid exchanges in human peripheral lymphocytes but not in Chinese hamster lung (V79) or ovary cells. In vitro cytogenetic assays conducted with human lymphocytes, Chinese hamster lung (V79) cells, and Chinese hamster ovary cells revealed no induction of chromosomal aberrations. An in vitro micronucleus assay in Chinese hamster lung (V79) cells, which incorporated a test for aneuploidy, yielded equivocal results (Elliott & Ashby, 1997). 2-Butoxyethanol failed to produce cell transformation in Syrian hamster embryo cells tested at concentrations up to 20 mmol/l with 7-day exposures (Park et al., 2002).

Mutagenicity studies have also been performed on two metabolites of 2-butoxyethanol: BAA and BALD. BAA was not mutagenic in a series of in vitro assays, in addition to an in vivo micronucleus assay in mice administered the chemical by intraperitoneal injection (Hoflack et al., 1995; Elias et al., 1996; Elliott & Ashby, 1997). BAA (tested at up to 20 mmol/l for 7 days) failed to produce cell transformation in Syrian hamster embryo cells (Park et al., 2002). BALD exhibited genotoxic potential in several in vitro studies (including tests for HPRT gene mutation, chromosomal aberrations, micronuclei, aneuploidy, and sister chromatid exchange); however, in the absence of data from in vivo studies, it is not possible to reach a firm conclusion concerning the possible mutagenic hazard of this metabolite (Chiewchanwit & Au, 1995; Hoflack et al., 1995; Elias et al., 1996; Elliott & Ashby, 1997).

8.7.1 Effects on fertility

Heindel et al. (1990) used a continuous breeding protocol (Heindel et al., 1989) to assess the reproductive toxicity of 2-butoxyethanol. Male and female Swiss CD-1 mice were administered 2-butoxyethanol in drinking-water (0, 0.5, 1, or 2%; equivalent to 0, 700, 1300, and 2100 mg/kg body weight per day) 7 days prior to and during a 98-day cohabitation period (20 pairs of mice per dose). Exposure to 1% or 2% 2-butoxyethanol in drinking-water was associated with increased mortality in the females and a significant reduction (P < 0.05) in the number of live pups per litter, the proportion of pups born alive, and the live pup weights (both absolute and adjusted). Other signs of maternal toxicity included decreased body weight, decreased water consumption, and increased kidney weight. Necropsy revealed that testes and epididymis weights were normal, as were sperm number and motility. The reproductive toxicity of 2-butoxyethanol was evident only in female mice, at doses that also elicited general toxicity (Heindel et al., 1990). (It has been hypothesized that fetal deaths may have been due to hydrops foetalis, associated with severe anaemia induced by 2-butoxyethanol or its metabolite, BAA, transported across the placenta [Atkins, 1999]; however, no description of the possible cause of fetal death was presented in the report of this study.) The NOAEL was 700 mg/kg body weight per day.

Effects on male and female reproductive organs (including reduced weight or histopathological changes in the epididymis or testes, decreased sperm concentration, altered sperm morphology, or uterine atrophy) were noted in F344 rats and B6C3F1 mice exposed to 2-butoxyethanol in the subchronic studies conducted by the NTP (1993, 2000), although some of these effects were not considered to be of biological significance and occurred only at doses or concentrations that also induced haematological and other effects.

Effects on the testes were not observed in studies in which Alpk/Ap (Wistar-derived) rats were exposed by inhalation to 2-butoxyethanol at 4000 mg/m³ for 3 h (Doe, 1984), JCL-ICR mice were orally administered 2-butoxyethanol at doses ranging from 500 to 2000 mg/kg body weight per day, 5 days/week, for 5 weeks (Nagano et al., 1979), or rats were administered 2-butoxyethanol (by gavage) at doses ranging from 222 to 885 mg/kg body weight per day, 5 days/week, for 6 weeks (Krasavage, 1986). Testicular damage was not observed in groups of Alpk/Ap (Wistar-derived) rats administered a single oral dose of BAA at 174, 434, or 868 mg/kg body weight (Foster et al., 1987).

8.7.2 Developmental toxicity

No adverse effects were observed in either the dams or pups (number of resorptions, fetal weights, and incidence of malformations) in a study in which Sprague-Dawley rats were exposed by inhalation for 7 h/day on days 7–15 of gestation to 2-butoxyethanol at 740 or 980 mg/m³. Adults died at 1200 or 2500 mg/m³ (Nelson et al., 1984).

Tyl et al. (1984) exposed Fischer 344 rats (36 per group) and New Zealand white rabbits (24 per group) to 2-butoxyethanol at 0, 120, 250, 490, or 980 mg/m³ for 6 h/day on days 6–15 of gestation for the rats and on days 6–18 of gestation for the rabbits. No adverse reproductive or developmental effects were observed in rats or rabbits exposed to 2-butoxyethanol at concentrations up to 250 mg/m³. At 490 mg/m³ and above, signs of delayed ossification were seen in the rats, and maternal toxicity (decreased weight gain and increased resorptions) was evident at 980 mg/m³. In rabbits, maternal toxicity (reduced weight gain) and delayed ossification were seen at 980 mg/m³.

Maternal deaths and a reduction in the number of viable litters were observed when CD-1 mice were orally administered 2-butoxyethanol at 4000 mg/kg body weight per day on days 7–14 of gestation (Schuler et al., 1984).

No maternal, embryotoxic, fetotoxic, or teratogenic effects were detected when 2-butoxyethanol (106 mg, about 1.6 g/kg body weight per day) was applied to the shaved interscapular skin of female Sprague-Dawley rats, 4 times daily on days 7–14 of gestation (Hardin et al., 1984).

Effects on the immune system have been examined in rats. In one study, Sprague-Dawley rats were administered 2-butoxyethanol at 0, 2000, or 6000 mg/l (males) or 0, 1600, or 4800 mg/l (females) in drinking-water for 21 consecutive days. Treatment had no effect on antibody production, delayed-type hypersensitivity reactions, and interferon or interleukin-2 production. However, natural killer cell cytotoxicity responses were enhanced (P < 0.05) in rats receiving the lowest concentrations of 2-butoxyethanol (Exon et al., 1991). In a second study, male Fischer rats were administered (by gavage) 2-butoxyethanol at 0, 50, 100, 200, or 400 mg/kg body weight per day for 2 consecutive days, following immunization with trinitrophenyl-lipopolysaccharide. A reduction (P < 0.05) in the serum haemagglutination titre was observed 3 days later in rats administered 2-butoxyethanol at 200 mg/kg body weight per day. All animals in the highest dose group died (Smialowicz et al., 1992).

An abstract reports that statistically significant effects on indicators of immune function were observed in BALB/c mice administered repeated oral 2-butoxyethanol doses of 50 mg/kg body weight per day or more for 10 days. There was an increase in the mixed lymphocyte reaction and concanavalin A mitogenic stimulation of splenocytes at all tested doses. Increased cytotoxic T lymphocyte activity and elevated lipopolysaccharide stimulation of splenocytes were reported at the higher dose levels (Morris et al., 1996). Effects on immune function were also observed in a study in which BALB/c mice were given dermal doses of 100, 500, 1000, or 1500 mg/kg body weight per day for 4 days. Reduced splenic T cell proliferative response to concanavalin A and mixed lymphocyte response to allogeneic antigen were observed at 500 mg/kg body weight per day, whereas there was an increase in spleen cellularity and spleen to body weight ratio at the top dose (Singh et al., 2001). Dermal exposure also resulted in a decrease in oxazolone-induced contact hypersensitivity response in female BALB/c mice. Application of 4 mg of 2-butoxyethanol in 4:1 acetone and olive oil vehicle at the time of induction of sensitization, challenge, or both decreased the contact hypersensitivity response (evaluated by measuring ear thickness before and after challenge) by 18%, 18%, and 22%, respectively (Singh et al., 2002).

Reduced weights or histopathological changes were observed in the thymus or spleen of both mice and rats exposed to 2-butoxyethanol by repeated inhalation; however, these effects were considered likely to be secondary to haemolysis and decreased body weight (NTP, 1993, 2000).

No specific investigations on potential neurological effects associated with exposure to 2-butoxyethanol were identified. However, adverse effects on the central nervous system associated with exposure to high doses or concentrations of 2-butoxyethanol have been observed in short-term studies. These included loss of coordination, sluggishness, narcosis, muscular flaccidity, and ataxia (Carpenter et al., 1956; Dodd et al., 1983; Hardin et al., 1984; Krasavage, 1986).

Bartnik et al. (1987) examined the effects of 2-butoxyethanol and BAA on human (from healthy males) and rat (four male Wistar) erythrocytes in vitro. The lowest concentration of BAA administered (1.25 mmol/l) resulted in 25% haemolysis of rat erythrocytes after 180 min, whereas 3.75 mmol/l caused complete lysis. In contrast, BAA at 15 mmol/l did not produce measurable haemolysis in human erythrocytes over the same time. Complete lysis of rat and human cells occurred at 2-butoxyethanol concentrations of 200 mmol/l and 175 mmol/l, respectively. These results indicate that rats may be more susceptible than humans to the haemolytic effects of 2-butoxyethanol and its metabolite BAA (Bartnik et al., 1987).

The addition of 2-butoxyethanol at concentrations of 5 or 10 mmol/l in rat blood had no effect on haematocrit, whereas a concentration of 20 mmol/l caused significant haemolysis (P < 0.05). The addition of BAA to rat erythrocytes to concentrations of 0.5 or 1 mmol/l caused a time- and concentration-dependent increase in haematocrit followed by haemolysis. Incubation with BAA at 2 mmol/l caused a faster time-dependent increase in haematocrit, with the haematocrit reaching a maximum after 2 h, followed by nearly complete haemolysis after 4 h. Also examined was the effect of BAA (0.5, 1, 2, 4, or 8 mmol/l) on human blood obtained from healthy young male and female volunteers (Ghanayem, 1989). No significant changes in haematocrit or haemolysis were observed at BAA concentrations of 4 mmol/l or less; at 8 mmol/l, there was a slight but significant increase in haematocrit (P < 0.05), followed by a slight but significant haemolysis (P < 0.05) of erythrocytes (Ghanayem, 1989).

In a subsequent study, Ghanayem & Sullivan (1993) assessed the haemolytic activity of BAA (1 or 2 mmol/l) in blood collected from rats, mice, hamsters, baboons, rabbits, pigs, guinea-pigs, dogs, cats, and humans. BAA caused a time- and concentration-dependent increase in mean cell volume and haematocrit of blood from rats, rabbits, hamsters, mice, and baboons. However, no or minimal effects were observed on blood from humans, guinea-pigs, dogs, cats, and pigs (Ghanayem & Sullivan, 1993), demonstrating the sensitivity of rat erythrocytes and the relative insensitivity of human erythrocytes to the haemolytic effects of BAA.

Udden (2000) compared the morphological appearance of rat erythrocytes with that of erythrocytes derived from humans when each was exposed in vitro to BAA. Stomatocytes (cup-shaped cells) and spherocytes were the principal morphological features of cells from rats, whereas none of these was observed in human red blood cells incubated with BAA at up to 2.0 mmol/l. In a subsequent study, Udden (2002) reported that human erythrocytes required exposure to a concentration (up to 10 mmol/l) of BAA that was 100-fold greater than that required by rat erythrocytes in order to develop sub-haemolytic effects (loss of deformability, increased osmotic fragility, and increased erythrocyte sodium); the investigators suggested that such a high concentration is not likely to occur under normal human use of 2-butoxyethanol-containing products. The results give further support to the role of BAA in the haemolytic effect of 2-butoxyethanol in the rat and the higher sensitivity of rats compared with humans.

The effect of BAA on red blood cells from healthy young and older individuals (Udden & Patton, 1994) and individuals with a possible susceptibility to 2-butoxyethanol-induced haemolysis (i.e. sickle cell and spherocytosis patients) (Udden, 1994, 1996) has also been examined. Along with haemolysis, BAA at 0.2 and 2 mmol/l caused decreased red blood cell deformability and increased mean cellular volume in rat red blood cells (Udden & Patton, 1994). However, none of the human blood samples exhibited prehaemolytic changes (i.e. decreased deformability and increased mean cellular volume) or haemolysis after BAA treatment at 2 mmol/l for up to 4 h (Udden, 1994, 1996; Udden & Patton, 1994). The results of these in vitro studies provide further evidence that rat erythrocytes are more susceptible than human erythrocytes to BAA-induced haemolysis.

8.10.1 Forestomach papillomas and carcinomas in female mice

Mechanisms by which 2-butoxyethanol may induce tumours in the forestomach have been assessed (USEPA, 2005). The first stage in the proposed sequence of steps is the deposition of 2-butoxyethanol/BAA in the stomach and forestomach via consumption or reingestion of 2-butoxyethanol-laden mucus, salivary excretions, or fur material. 2-Butoxyethanol/BAA may be retained in food particles in the forestomach long after being cleared from other organs. 2-Butoxyethanol is metabolized to BALD, which is then rapidly converted to BAA systemically and in the forestomach. Irritation of the target cells leads to hyperplasia and ulceration, with continued injury and degeneration resulting in high cell proliferation and turnover. The final stage on the path to tumour formation is the high cell proliferation and turnover leading to clonal growth of spontaneously initiated forestomach cells (USEPA, 2005).

The weak positive effects induced by 2-butoxyethanol at high concentrations in some in vitro DNA repair, sister chromatid exchange, and cell transformation assays make it difficult to completely exclude the potential for contribution from direct interaction of a 2-butoxyethanol metabolite with DNA. Although these positive findings may be due to study design artefacts such as changes in pH or osmolarity associated with high 2-butoxyethanol concentrations, they may also be due to the short-lived metabolite BALD, which has caused clastogenic changes in Chinese hamster lung and human lymphocyte cells. Available evidence from PBPK modelling, modified to include kinetics for the metabolism of BALD, suggests that the conditions in the in vitro assays for genotoxicity (no metabolic activation; high cytotoxic concentrations of BALD) have little relevance to the expected target organ environment (high metabolic activity; low concentrations of BALD). Additional research to verify the PBPK modelling and explore further the relevance of genotoxic activity would enable a more definitive determination regarding the possible role of BALD in the formation of forestomach tumours in female mice.

8.10.2 Liver tumours in male mice

A mode of action for the development of haemangiosarcomas of the liver and hepatocellular carcinomas in male mice has been proposed (USEPA, 2005).¹² The first stage in the proposed sequence of steps is haemolysis of red blood cells by the 2-butoxyethanol metabolite BAA. This haemolysis leads to the accumulation of haemosiderin (iron) in phagocytic cells of the liver of both rats and mice. Oxidative damage and increased synthesis of endothelial hepatocyte DNA are initiated by the generation of reactive oxygen species from iron within Kupffer cells and perhaps from within hepatocytes and sinusoidal endothelial cells and/or by the activation of Kupffer cells to produce cytokines/growth factors that suppress apoptosis and promote cell proliferation. Recent research indicates that the mouse is more or uniquely sensitive to these effects. It is hypothesized that these events can contribute to the transformation of the endothelial cells to haemangiosarcomas and of hepatocytes to hepatocellular carcinomas in male mice.

As described above (see section 8.10.1) for forestomach tumours observed in female mice, the weak positive effects induced by 2-butoxyethanol at high concentrations in some in vitro genotoxicity assays and the reported clastogenicity of the 2-butoxyethanol metabolite BALD make it difficult to completely exclude the potential for contribution from direct interaction of a 2-butoxyethanol metabolite with DNA. Again, PBPK modelling suggests that the conditions in the in vitro genotoxicity assays (no metabolic activation; high cytotoxic concentrations of BALD) are of little relevance to the expected target organ environment (high metabolic activity; low concentrations of BALD). As in the case for the forestomach tumours, additional research to verify the PBPK modelling and explore further the relevance of genotoxic activity would enable a more definitive determination regarding the role of BALD in the formation of liver tumours in male mice.

Information on human health effects associated with exposure to 2-butoxyethanol are limited to a few case reports, a clinical investigation, and a cross-sectional survey. The principal human health effects attributed to 2-butoxyethanol exposure have involved the central nervous system, the blood, and the kidneys (ATSDR, 1998).

In a cross-sectional survey, slight, but statistically significant, changes in some haematological parameters (haematocrit and mean corpuscular haemoglobin concentration) were observed in a group of 31 men occupationally exposed to average concentrations of 2-butoxyethanol of 3.64 or 2.20 mg/m³ compared with unexposed workers. However, there was no correlation with levels of BAA in the urine, and information on exposure was limited to personal monitoring samples taken during only one workshift (Haufroid et al., 1997).

In one report involving a number of small studies, the exposure of two males to 2-butoxyethanol at 560 mg/m³ for 4 h produced nose and eye irritation as well as disturbed taste, but there was no evidence of haemolytic effects. Similar effects were observed in a second study in which two males and one female were exposed to 2-butoxyethanol at 960 mg/m³ for two 4-h periods, separated by a 30-min period of no exposure. When two males and two females were exposed to 2-butoxyethanol at 490 mg/m³ for 8 h, the effects included vomiting and headache. No clinical signs of haemolysis were observed in any of the subjects (Carpenter et al., 1956).

Haemoglobinuria, erythropenia, hypotension, metabolic acidosis, shock, non-cardiogenic pulmonary oedema, albuminuria, hepatic laboratory abnormalities, haematuria, and mental status depression have been reported in case-studies of individuals who had attempted suicide by ingesting 2-butoxyethanol-containing cleaning solutions (involving an estimated ingestion of 25–60 g 2-butoxyethanol) (Rambourg-Schepens et al., 1988; Gijsenbergh et al., 1989; Bauer et al., 1992; Gualtieri et al., 1995, 2003; McKinney et al., 2000). In several of the cases, haemodialysis was employed, and all patients recovered fully with appropriate treatment. A survey of paediatric poisonings identified 24 children who had ingested 5–300 ml of glass cleaners containing 2-butoxyethanol (Dean & Krenzelok, 1992). The two children with the highest intake exhibited no evidence of haemolytic effects. Other effects characteristic of poisoning with ethylene glycol (a metabolite of 2-butoxyethanol in humans), such as coma, metabolic acidosis, and renal effects, as well as changes in levels of hepatic enzymes (of uncertain biological significance), have been reported in several cases or cross-sectional studies (e.g. Rambourg-Schepens et al., 1988; Collinot et al., 1996; Haufroid et al., 1997; Nisse et al., 1998). 2-Butoxyethanol is reportedly not a skin sensitizer in humans (Greenspan et al., 1995).

10.1.1 Hazard identification and dose–response assessment

Few data were identified on the potential effects of 2-butoxyethanol in humans. Although effects on the blood have been observed in exposed workers and in several cases of incidental exposure (Rambourg-Schepens et al., 1988; Gijsenbergh et al., 1989; Bauer et al., 1992; Haufroid et al., 1997), limitations of these studies mean that characterization of health hazards associated with 2-butoxyethanol is based primarily on studies in laboratory animals.

10.1.1.1 Haematological effects

The majority of toxicological investigations with 2-butoxyethanol have been conducted in rats, in which the most sensitive target tissue is the blood. Alterations in haematological parameters characteristic of haemolytic anaemia have been observed in this species following single, short-term, medium-term, and long-term exposure to 2-butoxyethanol via inhalation, ingestion, and dermal application (Carpenter et al., 1956; Dodd et al., 1983; Bartnik et al., 1987; Ghanayem et al., 1987b; NTP, 1989). In some of these studies, the effects appeared to be reversible after cessation of exposure, as the severity of the haematological changes decreased with increasing time since exposure (Grant et al., 1985; Krasavage, 1986; NTP, 1989; Ghanayem et al., 1992). Similarly, tolerance, or autoprotection, was suggested by the results of two studies in which the haematological effects were less severe in rats that had been exposed to 2-butoxyethanol prior to administration than in rats receiving only the subsequent doses, although the protective effect declined with increasing time between exposures. In addition, bleeding rats prior to acute exposure to 2-butoxyethanol reduced the severity of the haematotoxicity (Ghanayem et al., 1992; Sivarao & Mehendale, 1995). These data suggest that older red blood cells are more susceptible to 2-butoxyethanol-induced effects (which has also been demonstrated in in vitro studies); as they are replaced by more resilient younger cells, the severity of the haematotoxic response declines.

However, such reversibility or autoprotection is likely limited, since it was not observed in rats repeatedly exposed to 2-butoxyethanol for longer durations. In rats exposed to 2-butoxyethanol in the drinking-water for 13 weeks, symptoms of regenerative haemolytic anaemia were still present at the end of the study in females at all doses tested (NTP, 1993). Similar effects were also noted in the same strain of rats exposed via inhalation for 14 weeks to 2-butoxyethanol at all concentrations tested, based on results of studies conducted by the NTP (2000). The anaemia was considered to be macrocytic, normochromic, and responsive. Other indicators of regenerative anaemia, including increased haematopoiesis, haemosiderin accumulation in the liver and kidney, and bone marrow hyperplasia, were also observed in both sexes; thrombosis, likely associated with severe acute haemolysis, was also noted in females at the higher exposure levels. In addition, in the chronic study conducted by the NTP (2000), in which rats were exposed to 2-butoxyethanol by inhalation for up to 2 years, haemolytic anaemia was evident in animals monitored at regular intervals up to 12 months. The anaemia, which was again characterized as macrocytic, normochromic, and responsive, was observed at all concentrations; the severity of the effects increased with exposure level and did not ameliorate significantly over time.

2-Butoxyethanol-induced haematological effects have also been observed in other species of laboratory animals. In mice, a decrease in red blood cell count was noted following short-term oral exposure (Nagano et al., 1979, 1984), while alterations in haematological parameters were reported in subchronic and chronic inhalation studies (NTP, 2000), although, as discussed above, mice appear to be less sensitive than rats to 2-butoxyethanol-induced haematotoxicity. Haematological effects were also noted in limited short-term studies in rabbits, dogs, and monkeys (Carpenter et al., 1956; Truhaut et al., 1979; Union Carbide, 1980; Tyl et al., 1984), although available data are inadequate to allow the evaluation of differences in species sensitivity. In general, these effects were observed only at doses greater than those that induced similar, more severe effects in rats.

There is also some evidence that the blood is a sensitive target tissue in the developing young following in utero exposure to 2-butoxyethanol in both rats and mice. Haematological effects were reported in the fetuses of rats exposed to doses of 2-butoxyethanol that were also haematotoxic in the dams (NTP, 1989), while an increase in fetal mortality in mice (Heindel et al., 1990) has been hypothesized to be due to hydrops foetalis, associated with severe anaemia induced by 2-butoxyethanol or its metabolite, BAA, transported across the placenta (Atkins, 1999); however, no description of the possible cause of fetal death was presented in the report of this study.

In view of the extensive database that indicates that 2-butoxyethanol is haematotoxic in multiple laboratory species and the limited evidence of changes in haematological parameters in occupationally and incidentally exposed humans, 2-butoxyethanol is considered likely to be haematotoxic in humans.

Although limited, available data from toxicokinetic studies and comparative in vitro investigations suggest that humans may be less sensitive than rats to 2-butoxyethanol-induced haematotoxicity (although few data were identified on interindividual sensitivity in humans); rats appear to be the most sensitive of the animal species investigated.

10.1.1.2 Other non-neoplastic effects

Other target organs of 2-butoxyethanol-induced effects in various species (rats, mice, rabbits, or guinea-pigs) following single, short-term, or long-term exposure (Carpenter et al., 1956; Truhaut et al., 1979; Krasavage, 1986; Bartnik et al., 1987; Ghanayem et al., 1987b; NTP, 1993, 2000) include the liver, kidney, spleen, and bone marrow. Many of the observed effects, such as accumulation of haemosiderin pigment, increased haematopoiesis and cellularity, and haemoglobinuria, are considered to be secondary or in response to haemolytic anaemia (NTP, 2000), while other effects, such as alterations in relative organ weights and some histopathological changes, occurred only at doses that were also haemolytic. Although cytoplasmic changes were noted in the liver of male rats exposed over the medium term to oral doses lower than those that induced alterations in blood parameters (although these doses were haematotoxic in females), these effects may have been related to the induction of enzymes involved in the metabolism of 2-butoxyethanol (NTP, 1993).

The forestomach was also a critical target for 2-butoxyethanol-induced toxicity in mice. In the chronic inhalation study in mice, increased incidences of inflammation, epithelial hyperplasia, and/or ulceration were noted. Females were more sensitive than males, and there was some evidence of concentration-related trends in incidence and severity of forestomach lesions. These effects were also observed in both mice and rats exposed over the medium term to higher concentrations that also induced haematological changes (NTP, 2000). These effects on the forestomach are also in concordance with neoplastic lesions at this site in mice. Forestomach lesions have also been observed in mice administered 2-butoxyethanol by intraperitoneal or subcutaneous injection.

In investigations of the potential developmental toxicity of 2-butoxyethanol in rats, mice, or rabbits, embryotoxic or fetotoxic effects or malformations have generally been observed only at or above doses that are also maternally toxic (Hardin et al., 1984; Schuler et al., 1984; Wier et al., 1987; NTP, 1989; Heindel et al., 1990). Similarly, effects on female reproductive ability or on male and female reproductive organs (some of which were not considered to be of biological significance) were observed only at doses or concentrations t