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Effects on fertility

Additional information

There are no specific studies on toxicity to reproduction using inhalative exposure, but there is a 13 week inhalation study which also evaluated the reproductive organs and can therefore be taken into account for this exposure route.

Male and female F344/N rats were exposed to 0, 1.1, 2.3, 4.5, 9, 18 ppm (0, 7, 14.7, 28.8, 57.6, 115.2 mg/m3), 6 hours per day, 5 days per week over a period of 13 weeks (NTP, 1993). At the end of the study sperm morphology and vaginal cytology evaluations were performed of animals in the 0, 4.5, 9 and 18 ppm groups (reproductive organs of animals of the two lower exposure groups were not evaluated).

There were no clear clinical signs of toxicity. All rats survived till the end of the study. Concentration-related increase in methaemoglobinaemia and oxidative damage to red blood cells occurred from the first days of exposure and resulted in a regenerative anaemia; target organs were kidneys, spleen, liver, erythrocytes and nasal cavity respiratory epithelium. Males of the 18 ppm group showed decreases in cauda epididymis weights and in the spermatid count and spermatid heads/testis (NOAEL reproductive organs = 9 ppm). Female reproductive system was not affected by treatment (NOAEL reproductive organs = 18 ppm).

Male and female B6C3F1 mice were exposed to 0, 1.1, 2.3, 4.5, 9, 18 ppm (0, 7, 14.7, 28.8, 57.6, 115.2 mg/m3), 6 hours per day, 5 days per week over a period of 13 weeks (NTP 1993). At the end of the study sperm morphology and vaginal cytology evaluations were performed of animals in the 0, 4.5, 9 and 18 ppm group (reproductive organs of animals of the two lower exposure groups were not evaluated): There were no clinical signs of toxicity. 2/10 male mice exposed to 18 ppm died; target organs were kidneys, spleen and liver. Male mice in all evaluated dose groups demonstrated a decrease in sperm motility (a NOAEL reproductive organs for male mice was not determined); in females no effects were observed (NOAEL reproductive organs = 18 ppm).

In a 5 week feeding study 12 B6C3F1 mice/sex/dose received 0, 50, 500 or 5000ppm 1-chloro-2-nitrobenzene. Males of the highest dose group showed decreased testis weight without histopathological changes (Bayer 1991, 1993).

There is a carefully performed study on toxicity to reproduction in mice using oral treatment (NTP 1992): Male and female Swiss CD-1 mice were exposed to 1-chloro-2-nitrobenzene dissolved in corn oil by gavage to assess reproduction and fertility using the NTP continuous breeding protocol: Groups of 20 breeding pairs received 40, 80 or 160 mg/kg bw per day 2-chloronitrotoluene for 7 days prior to cohousing and for 98 days of continuous breeding. 40 breeding pairs received the corn oil vehicle only. The last litter born during the holding period following the continuous breeding phase from control and high dose groups was reared by the dam until weaning, after which time treatment of the F1 animals was initiated by the same route and at the same concentration as the F0 animals. These F1 animals were used for the assessment of second generation fertility.

Data from a 2-week dose-range-finding study were used to set exposure concentration. The highest dose used in the reproduction study was one-half of that caused mortality in the dose-range-finding study.

In the F0-generation mortality occurred in 2, 2, 2 and 3 mice in the control to the high dose groups, respectively, which was suggested not to be treatment related. There was a slight increase in male and post partum dam terminal weights. 3 females in the high dose group appeared cyanotic. No other clinical signs were observed. Necropsy of the high dose mice showed increased spleen weights by 50-100 % and 4-6 fold increased methaemoglobin level. No other necropsy data were collected.

Reproductive performance and function of the F0-mice was not affected by treatment: number of litters, pup weight, and viability were all unchanged; live pups per litter and proportion of pups born alive were increased (15% resp. 10%) in the high dose group.

In the final litter of the holding period following the continuous breeding phase, pup weight gain during suckling was lower in the treated groups. At weaning, pups of the high dose group weighed 12% less than control. None of the pups showed clinical signs of toxicity.

Mating of the adult F1 mice (only control and high dose group) revealed no difference between the groups in terms of proportion of mated pairs, number of litters per group, number of live pups per litter and pup weight or viability. Treated F1 male and female mice had 3-fold increased methaemoglobin level compared to the control and were approximately 7 and 5 % heavier than their control counterparts. At necropsy, liver and spleen weights were increased by 40 to 60 %. In male mice, abs. right epididymis and kidney/adrenals weights were increased, seminal vesicle-to-body weight was reduced compared to controls. Sperm measured were unaffected by 1-chloro-2-nitrobenzene exposure (epididymal sperm motility, sperm count, percentage of abnormal sperm). In females, oestrous cycle were unaffected by 1-chloro-2-nitrobenzene exposure. Thus, NOAEL for fertility is 160 mg/kg bw/day.


Short description of key information:
Following inhalational exposure of F344/N rats and B6C3F1 mice for 13 weeks, only in males 1-chloro-2-nitrobenzene affects the reproductive organs. Performance of a specific study on toxicity to reproduction (NTP Continuous Breeding Protocol) reveals that 1-chloro-2-nitrobenzene was without reproductive toxicity in a different mice strain following oral treatment by gavage despite of significant changes in liver and spleen weights and despite elevated methaemoglobin levels. The NOAEL fertility in Swiss CD-1 mice after oral application is 160 mg/kg bw/day whereas the dams showed general toxicity effects at this concentration.
Because 1-chloro-2-nitrobenzene affected the reproductive organs in systemic toxic doses in male rats and in males of one strain of mice after subchronic inhalation there is a concern for a reproductive toxicity potential, even if an impairment of reproduction after oral administration in males of a second strain of mice could not be detected.

Effects on developmental toxicity

Description of key information
Developmental toxicity was examined by two studies with Sprague Dawley rats which both have methodological deficiencies. In one study, due to high mortality rate at the highest dose level, only two doses could be evaluated: NOAEL for maternal toxicity is 25 mg/kg bw/day, a NOAEL for developmental toxicity could not be conclusively derived since there was an increase in the number of litters exhibiting specific skeletal variations. In the second study only one dose was applied: NOAEL for developmental toxicity is 100 mg/kg bw/day, a NOAEL for maternal toxicity could not be derived. Based on the available studies the overall conclusion is, that there is no indication of developmental toxicity, although there are some limitations within the studies.
Additional information

25 female Sprague-Dawley rats per group received 0, 25, 75 or 150 mg/kg bw/day 1-chloro-2-nitrobenzene dissolved in corn oil by gavage from day 6 to day 15 of gestation. Due to severe toxicity and high mortality rate of the dams in the 150 mg/kg bw/day group, all females of the 150 mg-group were terminated prior to scheduled sacrifice. One year later, in another laboratory, a third dose group (100 mg/kg bw/day) was examined together with a concurrent control group (see below).

No evidence of maternal toxicity was exhibited at the 25 mg/kg level. For gestation days 6-10 a slight, but not significant reduction in maternal body weight gain at the 75 mg/kg bw/day level, urinary staining and alopecia were noted in some dams when compared to the respective control groups. The difference in maternal body weight gain was accompanied by reductions in food consumption for days 6-10. The reductions noted at 75 mg/kg bw/day were recovered later in gestation.

Maternal reproductive parameters and fetal body weight in the treatment groups were similar to the respective control groups except for the mean number of early resorptions and postimplantation loss at the 75 mg/kg level. However, postimplantation loss in the respective control group was very low compared to the historical control value.

No differences in the number of the litters exhibiting malformations were evident in the treatment groups compared to the control group. Increased incidences of variations were seen in the 25 and 75 mg/kg group: cervical #7 rib (significant at 75 mg/kg); and 13 full pairs of ribs with lumbar #1 rudimentary rib; in the 25 mg/kg group: 12 full pair ribs with #13 unilateral full rib and/or rudimentary rib(s). No historical control data were given. Thus, NOAELmaternal toxicty is 25 mg/kg bw/day, a NOAELdevelopmental toxicity could not be conclusively derived (Monsanto 1990).

In an additional study which was performed in a different laboratory one year later and which was intended to clarify the observation of the first study, mated female rats received 0, or 100 mg 1-chloro-2-nitrobenzene/kg bw in corn oil by gavage from day 6 to day 15 of gestation. For gestation day 6-10 slight reduction in maternal body weight loss accompanied by reduction in food consumption for days 6-16 was noted. Maternal reproductive parameters and fetal body weights in the treatment group was comparable to the respective control group. No teratogenic effect nor statistically significant increase of skeletal variations like in the first experiment were observed (IRDC 1984).

Justification for classification or non-classification

As no adverse effects on fertility were observed at the highest tested dose in the continious breeding study with rats, classification of 1 -chloro-2 -nitrobenzene for reproductive toxicity is not warranted in accordance with EU Directive 67/548 and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

In the study of Monsanto, increased incidences of variations were noted at 25 and 75 mg/kg bw/day in rats administered the test substance at days 6 -15 of gestation. The NOAEL for developmental toxicity could not be conclusively derived. In the study of IRDC, 1984, performed in order to clarify the results of the Monsanto study, no teratogenic effects were noted at 100 mg/kg bw/day in the presence of maternal toxicity. Based on these results, classification of 1 -chloro-2 -nitrobenzene for developmental toxicity is not warranted in accordance with EU Directive 67/548 and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.