2-NITROPROPANE
Explanation
This extraction solvent has been previously considered by the
Joint FAO/WHO Expert Committee on Food Additives in 1979 and 1981 (see
Annex 1, Ref. 50, 56). A toxicological monograph was issued in 1982
(see Annex 1, Ref. 57). Since the previous evaluation, additional data
have become available and are summarized and discussed in the
following monograph. The previously published monograph has been
expanded and reproduced in its entirety below:
BIOLOGICAL DATA
Biochemical Aspects
Ingestion of either 2-nitropropane or 1-nitropropane by rabbits
resulted in the formation of nitrite (Scott, 1943). Metabolism to
acetone and nitrite is effected in the liver (Ulrich et al., 1978).
Groups of 8-10 Wistar rats were administered 2-nitropropane by
intraperitoneal injection or inhalation. Injection of 1.7 g/kg caused
death in two hours and 89% blood methemoglobin. Quantities of nitrite
1-2.5 mg/100 g tissue) were found in the heart, lungs, kidney, spleen
and liver. 2-nitropropane was found in the liver at 1.34 mg/100 g of
tissue. There was no trace in the other organs and pulmonary excretion
amounted to 76% of the injected dose. Injection of 0.11 g/kg/day for
15 days followed by killing the rats 36 hours after the last injection
produced no methemoglobin, but nitrite was found in all organs
examined except the liver. 2-nitropropane was found in the liver and
lungs at concentrations of 18.7 and 360 mg/100 g tissue, respectively.
Urinary excretion of nitrite was also noted. When groups of rats
inhaled 2-nitropropane in air at 80 ppm (0.008%) for eight hours per
day and were killed the day after the fifth exposure, methemoglobin
was not detected, but nitrite was found in all organs examined except
the liver. No nitrite was detected in the urine during the whole
exposure period and no trace of 2-nitropropane was found in the organs
(Dequidt et al., 1972).
Male Sprague-Dawley rats (200-300 g) were administered 2-(14C)
nitropropane (2-(14C)NP) by inhalation for 6 hours at concentrations
of either 20 or 150 ppm, and the disposition of 14C in these animals
was followed for 48 hr. Over 40% of the amount inhaled was absorbed.
The absorbed 2-(14C)NP was rapidly metabolised and eliminated. About
50% was excreted as 14CO2 at both exposure levels after 48 hr. The
fraction of unchanged 2-NP excreted was approximately 4% and 25% at
the low and high exposure levels. Urine and feces represented minor
routes of excretion.
The distribution of 14C in the tissues of the rats was inversely
proportional to the dose, and was primarily present in the excretory
organs (liver, kidneys, and lung). Only a small amount (less than 5%)
of the 14C was incorporated into liver macromolecules. Concentration
of 14C in the blood at 0 and 48 hours, and pulmonary elimination of
unchanged 2-14C-NP at the two exposure concentrations indicate that
at the 150 ppm exposure concentration, the kinetics of 2-14C-NP
metabolism were non-linear (Nolan, et al., 1982).
In vitro studies with microsomal preparations from the induced
livers of rats show that in the presence of NADPH, nitropropane
degrades the heme moiety of hepatic microsomal P-450 (Ivantich et al.,
1978; Sakurai, 1980).
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity
A group of 125 male and 125 female Sprague-Dawley rats were
exposed to 2-nitropropane by inhalation at a concentration of 25 ppm
(0.0025%) for seven hours per day, five days per week for 22 months.
The control group also consisted of 250 rats evenly divided between
sex. The technical grade 2-nitropropane used in the study was 95.6%
pure; the remainder being other lower nitroparaffins. Representative
groups of animals were killed after 2, 3, 6, or 12 months of exposure.
All rats remaining alive after 22 months were killed for necropsy. No
exposure-related effects were found upon periodic examination of serum
and blood chemistry. At necropsy final brain, liver, kidney and body
weights were compared. Only a slight increase in relative liver
weights was apparent. This increase was significant at the P 0.05
level for the 6-month exposed animals and at the P 0.01 level for
the 22-month exposure group. Detailed microscopic examination was
performed only on liver tissue. Liver congestion was present in 1
of 125 control males versus 8 of 125 exposed. In females, the
corresponding incidence was 0 versus 7. Focal areas of hepatocellular
nodules were present in 2 of 125 control males versus 10 of 125
exposed, and 1 of 125 control females versus 3 of 124 exposed. Focal
vacuolization of hepatocyte cytoplasm was observed in 46% of exposed
males versus 18% in controls. One liver angioma was observed in a
control male and one liver adenoma in an exposed female. The authors
concluded that no significant pathologic changes or malignancies were
attributable to exposure of rats to 25 ppm (0.0025%), seven hours per
day, five days per week, for 22 months (Griffin et al., 1980). A
complete histologic study of tissues and organs of the rats from this
study showed that apart from the 2-NP related changes in the liver,
the distribution of tumors and lesions was similar in control and
exposed groups (Griffin et al., 1981).
Groups of 50 male rats and 15 male rabbits were exposed to either
27 or 207 ppm (0.0025% or 0.0207%) of 2-nitropropane seven hours per
day, five days per week for periods up to 24 weeks. Groups of equal
size were expsoed to filtered air and served as controls. Ten rats
from each group were killed after 2 days, 10 days, 1 month, 3 months,
and 6 months. Five rabbits from each group were killed after 1, 3,
and 6 months. Body weight gains for both rats and rabbits at either
exposure concentration were similar to the control groups. No
discernable exposure-related effect was seen in haematological
evaluations. The liver weights were significantly elevated in the rats
exposed to 207 ppm (0.0207%) for 2, 3, and 6 months, however, those of
rabbits did not show any consistent exposure-related weight gain. No
gross or microscopic changes were apparent in rat or rabbit tissues
from the low exposure groups. Nor were any seen in rabbit tissues from
the high exposure group. However, multiple hepatocellular carcinomas
and neoplastic nodules were present in the livers from all 10 rats in
the high exposure groups after six months. Numerous focal areas of
hepatocellular hypertrophy, hyperplasia, and necrosis were seen in the
livers of the high exposure group of rats after three months. There
was also some incidence of haemorrhagic lesions in the lungs of the
high exposure group of rats. The lungs of three of five rabbits in the
high exposure group showed microscopic alterations. During the six
months of the experiment very few classical signs of toxicity were
observed in any exposure group. It should be noted that LC50 for a
six hour exposure to 2-nitropropane in male rats was found to be
approximately 400 ppm (0.04%). No females died after an exposure to
580 ppm (0.058%), whereas all male rats died at this concentration
(Lewis et al., 1979).
A group of male and female rats were exposed to 2-nitropropane at
a concentration of 200 ppm (0.02%), seven hours per day, five days per
week for up to six months. Groups were killed after 10 days, 1 month,
3 months, and 6 months. One group was held for an additional 6-month
post exposure period. Morphological changes occurred more extensively
in males and included hepatocellular nodules, hyperplasia, necroses,
and multivacuolated fatty metamorphosis. The livers of six of 10 rats
had pre-neoplastic foci and in nice out of 10 rats that were held six
months postexposure metastasizing tumours were apparent. A similar,
though not as severe, pathology was encountered after exposing rats to
2-nitropropane at a concentration of 100 ppm (0.01%) for 18 months.
Hepatocellular carninoma occurred in males after 12 months of exposure
and in females after 18 months (Griffin et al., 1978, 1980).
Special studies on mutagenicity
The mutagenic activity of 2-nitropropane was studied in the
Salmonella typhimurium (Ames) test with and without microsomal
activation (Hite & Skeggs, 1979). A dose-related increase in
revertants was found in all four tester strains. The increase in
revertants was significant in all four strains tested and was enhanced
in the presence of microsomal preparations. However, negative results
were obtained in the mouse micronucleus test.
In another study, mutagenic activity with microsomal activation
of 2-nitropropane was shown in S. typhimurium strains TS-98 and
TA-100. Repeat tests in TA-98 using higher concentrations
(10-20 ul/plate) confirmed the mutagenic effect (Brusick, 1977).
2-nitropropane (2-NP) was subjected to the following battery of
mutagenic tests.
An unscheduled DNA synthesis (UDS) asssy was carried out in human
diploid fibroblasts with exposures of 3 hr duration and concentrations
up to 5.000 ug/ml of culture medium. There was no increase in UDS in
cells treated with 2-NP.
A dominant lethal test was carried out in male rats with exposure
to atmospheres containing 25 ppm or 200 ppm 2-NP for 7 hr/day for 5
consecutive days. There were no large effects attributable to 2-NP in
the dominant lethal test on pregnancy frequency, with numbers of
corpora lutea or implantations, or the frequency of early deaths.
Reductions to 75% were seen, however, in pregnancy frequencies in
weeks 1 and 5 of the 200 ppm atmosphere group and in the frequencies
of live implantations and late deaths in week 2 of the 200 ppm
atmosphere group (P 0.05).
A sperm abnormality test was carried out in male mice with
exposure to atmospheres containing 25 ppm or 200 ppm 2-NP for 7 hr/day
for 5 consecutive days. Sperm abnormality frequency was not increased
by 2-NP treatment, but neither was the frequency increased in EMS
treatment. These results must be considered inconclusive.
A cytogenetic test was carried out in male and female rat bone
marrow cells from rats exposed to atmospheres containing 25 ppm or
200 ppm, 2-NP for 7 hr/day for 5 consecutive days. A single exposure
of 7 hr duration followed by sampling after 6 hr, 24 hr and 48 hr.
There were no increases in the frequencies of chromosomal aberrations
in male or female rats.
A sex-linked recessive lethal (SLRL) test was carried out in
Drosophila melanogaster with exposure to atmospheres of 700 ppm 2-NP
for 4.5 hr. Sex-linked recessive lethal mutation frequency was
increased except in mature spermatozoa in one stock of flies. Since
the increase was not reproduced, its significance is doubtful
(McGregor, 1981).
Special studies on reproduction
A group of adult female Sprague-Dawley rats were injected i.p.
with 170 mg/kg b.w. of 2-nitropropane on days 1-15 of gestation.
Litters were examined one day prior to parturation. A 1-2 day
retardation of heart development was observed in pups from nine out of
10 litters (Harris et al., 1979).
Acute toxicity
Animal Route Lethal dose Reference
Rat oral LD50 725 mg/kg IMC, 1977
inhl LCL0 1513 ppm (0.1513%)/ Treon & Dutra, 1972
4.5 h
inhl LC50 3712 ppm (0.3712%)/ IMC, 1979
1 h
(males) inhl LC50 400 ppm (0.04%)/ Lewis et al., 1979
6 h
Guinea-pig inhl LCL0 4622 ppm (0.4622%)/ Treon & Dutra, 1972
5.5 h
Rabbit oral LCL0 500 mg/kg Machle et al., 1940
inhl LCL0 2381 ppm (0.2381%)/ Treon & Dutra, 1972
4.5 h
Cat inhl LCL0 714 ppm (0.0714%)/ Treon & Dutra, 1972
4.5 h
Short-term studies
Rabbits and guinea-pigs were administered 2-nitropropane by the
oral (stomach tube) and inhalation routes. Progressive weakness,
ataxia, and collapse were noted as well as twitching and convulsions.
General visceral and cerebral congestion as well as some degree of
liver damage was present in all animals dying from exposure. Oedema,
cloudy swelling, fatty infiltration, and necrosis were seen in the
liver. Changes in the kidney, myocardium and other organs and tissues
were marked by oedema, pallor and cloudy swelling (Machle et al.,
1940).
Five species of laboratory animals were exposed by inhalation to
2-nitropropane at concentrations ranging from 83 to several thousand
ppm for up to seven hours per day until acute toxic effects were
observed. Toxic effects after acute exposure decreased in the
following order: cat, rat, rabbit, guinea-pig. Signs of toxicity
included, weakness, dyspnoea, cyanosis, prostration, convulsions, and
coma. Pathologic changes included general vascular endothelial
damage/pulmonary oedema and haemorrage, selective disintegration of
brain neurones, and hepatocellular damage. Formation of methemoglobin
and Heinz bodies was related to the severity of the exposure. No
pathologic changes occurred after exposure to air concentrations of
328 ppm (0.0328%) or 83 ppm (0.0083%) in the tissues of rats, rabbits,
guinea-pigs, or monkeys. In the cat, 328 ppm (0.0328%) caused severe
liver damage and slight to moderate toxic degeneration of the heart
and kidneys (Treon & Dutra, 1972).
Subsequent examination of tissue sections from this study has
revealed the presence of clear cell foci in the livers of rats exposed
to air containing 328 ppm (0.0328%) of 2-nitropropane for 17 exposure
periods of seven hours each (NIOSH, 1977). These cell foci are
commonly believed to be "cytologically similar to the cellular
elements of neoplastic nodules". The proliferative nodules are known
to be induced by carcinogens and "at the least, they indicate an
increased probability for the development of hepatocellular carcinoma"
(Squire & Levitt, 1975).
Special studies
Effect of glutathione and diethylmaleate on the hepatoxicity of 2-NP
The study was carried out on groups each of 25 male Sprague-
Dawley rats. The control groups consisted of Group I, untreated Group
II was administered 100 mg/day glutathione by gavage and Group III was
administered diethyl maleate (0.6 ml/kg) i.p. The test groups
consisted of similar groups exposed to 200 ppm, 2-NP by inhalation,
7 hr/day, 5 day/week. The glutathione treated animals in the test
group were treated 5 day/week prior to exposure, to 2-NP, and the
diethyl maleate group, 5 day/week and later 3 day/week prior to
exposure to 2-NP. Interim sacrifices (5 rat/group) were carried out at
3 months and 6 months, and the remaining animals were sacrificed at
7 months. Glutathione did not have any effect on the progressive
development of nodular hyperplasia and hepatocellular carcinoma.
Diethyl maleate, which causes a marked reduction in blood glutathione
levels, delayed the appearance of hepatic cell injury and no
hepatocellular carcinomas were observed at terminal sacrifice in this
group (Coulston, 1982).
OBSERVATIONS IN MAN
Five of six people exposed daily in an industrial setting
to 2-nitropropane at concentrations ranging from 20-45 ppm
(0.002-0.0045%) complained of daily episodes of anorexia, nausea,
diarrhoea, vomiting, and occipital headaches. Two workers in another
plant, where the concentration of 2-nitropropane in their breathing
zone varied between 10 and 25 ppm (0.001 and 0.0025%) were apparently
symptom free (Skinner, 1947). 25 ppm (0.0025%) is the current United
States occupational exposure standard (OSHA, 1975).
An epidemiological study on 1481 employees of a Sterlington,
Louisiana production facility was completed in 1979. The study covered
the years between January 1955 and July 1977. Depending on which
department the employees worked in, classification was made into three
cohorts (direct, indirect, or no exposure). Prior to 1977, there was
no formalized monitoring system. Between 1955 and 1962 corrective
action was taken to reduce exposure based upon informal subjective
odour threshold evaluation. Between 1962 and 1977 measured
concentrations were made above 25 ppm (0.0025%). These excursions
above 25 ppm (0.0025%) were at times accompanied by symptoms of
headache and nausea. For a six-month period in 1977, 98% of 144 air
samples were below 10 ppm (0.001%) (time-weighted average). Causes of
death were coded from death certificates and compared with those
expected using age-time-cause specific mortality rates. It was
concluded that the data does not suggest any unusual cancer or disease
mortality. It was further noted: "However, both because the cohort is
small and because the period of latency (the time between first
exposure and observation) is for most relatively short, one cannot
conclude from these data that 2-NP is non-carcinogenic in humans".
Three unusual findings were also pointed out: (1) there were
4 lymphatic cancers where 0.9 was expected in the "no exposure" male
population; (2) there were 4 deaths from all cancers deaths from
"sarcomatous" cancer in the "no exposure" population (Miller & Temple,
1979).
An occupational health examination programme was carried out in
workers exposed to 2-NP, generally at levels less than 25 ppm, during
an 8-hour workday. The period of exposure ranged from less than 1 year
to 40 years. Of the 28 exposed workers examined (out of a total
workforce of 46 examined), no excess abnormalities of the skin, blood,
renal, liver, pulmonary and cardiac system were noted that could be
associated with exposure to 2-NP (Tabershaw, 1980).
Comments
Most of the available data relates to exposure of animals to
2-nitropropane by inhalation. There is very limited acute oral data
which indicates that under these conditions of exposure, the toxic
effects are similar to those observed by the inhalation route under
acute conditions. Inhalation data clearly indicate that 2-nitropropane
is hepatoxic to rodents and at high levels of exposure there is
evidence of a carcinogenic effect.
Information on the metabolism of 2(14C) NP suggest that the
dose-dependant changes in the metabolism of 2-NP may account for the
marked increase in toxicity observed in rats exposed to high
concentrations of 2-NP. Although the toxicity is decreased at lower
dose levels, the available information suggests that even at these
dose levels, there is some indication of a possible pre-neoplastic
response.
2-nitropropane has been shown to be mutagenic in the Ames assay,
but gave inconclusive results in mammalian systems. The limited human
data shows that 2-nitropropane can cause headaches, anorexia, nausea,
diarrhoea, and vomiting at air concentrations as low as 20 ppm
(0.002%). An epidemiological study on a population of industrially
exposed workers proved to be inconclusive in establishing
carcinogenicity in man.
EVALUATION
2-nitropropane is carcinogenic to rats at relatively high
concentration of exposure by inhalation (between 25 and 200 ppm).
Estimate of temporary acceptable daily intake for man
For use as a fractionating solvent for fats and oils with
residues levels, the lowest technologically possible (below the
current limit of detection - 10 ug/kg in the fractionated oil).
FURTHER WORK
Required by 1989
Carcinogenicity studies using per os dosing.
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