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. REFERENCES BRUSICK, D.J. (1977) Mutagenic evaluation of P-135766459T, final report, Litton Bionetics, Inc., 5516 Nicholson Lane, Kensington, Maryland 20795. COULSTON, F. (1982) Influence of glutathione and diethyle maleate on the induction of hepatocellular carcinomas in rats exposed to 200 ppm of 2-nitropropane. Unpublished data, submitted to WHO, by Durkee Foods Division, SCM Corp., USA. DEQUIDT, J., VASSEUR, P., & POTENCIER, J. (1972) Etude toxicologue expérimentale de quelques nitroparafinnes, Bull. Soc. Pharm. Lille, 83-87: Experiemntal toxicologic study of some nitroparaffins, translation by International Minerals & Chemical Corporation, 666 Garland Place, des Plaines, Illinois 60016. GRIFFIN, T.B., et al. (1978) Chronic inhalation toxicity of 2-nitropropane in rats. Pharmacologist, 20: 145. GRIFFIN, T.B., COULSTON, F., & STEIN, A.A. (1980) Chronic inhalation exposure of rats to vapors of 2-nitropropane at 25 ppm. Ecotoxicol. & Environ. Safety, 4: 267-281. GRIFFIN, T.B., STEIN, A.A., & COULSTON, F. (1981) Histologic study of tissues and organs from rats exposed to vapors of 2-nitropropane at 25 ppm. Ecotoxicol. & Environ. Safety, 5: 194-201. 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See Also: Toxicological Abbreviations Nitropropane, 2- (EHC 138, 1992) Nitropropane, 2- (WHO Food Additives Series 16) Nitropropane, 2- (WHO Food Additives Series 26) Nitropropane, 2- (IARC Summary & Evaluation, Volume 71, 1999)