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Effects on fertility

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Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
To assess the reproductive toxicity of the test chemical in rats
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALSSource: Charles River breeding laboratoriesAge at study initiation: (P) x wks; (F1) x wks : No dataWeight at study initiation: (P) Males: x-x g: 240 to 280 g Females: x-x g: 180 to 220 g.Fasting period before study: during mating periodHousing: Cages Use of restrainers for preventing ingestion (if dermal): yes/no : No Diet (e.g. ad libitum): basal diet of Purinalaboratory chow, ad libitumWater (e.g. ad libitum): Tap water, ad libitumAcclimation period: No dataENVIRONMENTAL CONDITIONSTemperature (°C): No dataHumidity (%): No dataAir changes (per hr):No dataPhotoperiod (hrs dark / hrs light):No dataIN-LIFE DATES: From: To: No data
Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
not specified
Vehicle:
other: basal diet of Purina laboratory chow
Details on exposure:
DIET PREPARATIONRate of preparation of diet (frequency): Diets were prepared twice weekly.Mixing appropriate amounts with (Type of food): The composite was mixed into the basal diet of Purina laboratory chow at concentrations of 0, 97.50 and 390 mg/kgbw
Details on mating procedure:
- M/F ratio per cage:One male was placed in a cage with two females from 4 PM to 8 AM the following day.- Length of cohabitation:from 4 PM to 8 AM. This procedure was continued until copulation was confirmed.- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancyappearance of sperm in a vaginal smear (day 0 of pregnancy- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.Males were rotated within their dietary groups at I0-day intervals until conception wasconfirmed or until each female had been mated with a maximum of two males. If oneof the two females caged with the male became pregnant, the male was consideredfertile. A female was considered infertile if she failed to become pregnant after matingwith two different males for 10 days each.- Further matings after two unsuccessful attempts: [no / yes (explain)]Males were rotated within their dietary groups at I0-day intervals until conception wasconfirmed or until each female had been mated with a maximum of two males. If one of the two females caged with the male became pregnant, the male was considered fertile. A female was considered infertile if she failed to become pregnant after mating with two different males for 10 days each.- After successful mating each pregnant female was caged (how):Individual cages- Any other deviations from standard protocol:No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
The study was divided into two parts. In Part I, the females received the basal diet from 8 wk prior to mating through the weaning of their litters. The males siring these litters were fed the test diets for 8 wk prior to mating and during the mating period. In Part II, males received the basal diet for 8 wk prior to and during mating,while the females received the test diets 8 wk prior to mating and during gestation and21 days of lactation.
Frequency of treatment:
Daily
Details on study schedule:
No data
Remarks:
Doses / Concentrations:0, 195 and 780 mg/kg bw/day Basis:no data
No. of animals per sex per dose:
six groups of 10 males and 20 females each.
Control animals:
not specified
Details on study design:
No data
Positive control:
No data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data: No dataTime schedule: No dataCage side observations checked in table [No.?] were included.: No dataDETAILED CLINICAL OBSERVATIONS: Yes / No / No data: YesTime schedule: No dataBODY WEIGHT: Yes / No / No data: YesTime schedule for examinations:No dataFOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YesFood consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data: No dataCompound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No dataWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data: No dataTime schedule for examinations: No data
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
Yes
Postmortem examinations (parental animals):
SACRIFICEMale animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]: No data Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]: One female pregnant by each male was killed by chloroform inhalation on day 13 of her pregnancy to obtain information regarding the early stages of gestation.The uterus was examined for the number and distribution of embryos, the presence of empty implantation sites, and the number of embryos undergoing resorption. Each embryo was examined under a dissecting microscope. The remaining dams were allowed to deliver normally. A necropsy was performed on all females that did not deliver a litter to determine whether pregnancy had occurred. The duration of gestationwas noted and the litters’ were examined for numbers of live and stillborn pups andgross abnormalities.
Postmortem examinations (offspring):
SACRIFICEThe F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.: F1 : The litters’ were examined for numbers of live and stillborn pups and gross abnormalities.These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:The litters were examined for numbers of live and stillborn pups and gross abnormalities. The pups were weighed at birth, and at 4 and 21 days. At 21 days all surviving pups were killed by chloroform inhalation and examined grossly for abnormalities.
Statistics:
The data were subjected to statistical analysis, using the 95% confidence level. The methods used included chi square test, analysis of variance and t test, and the Fisher exact probability testFemale Fertility Index: (No of pregnant females/No. of females mated ) as percentMale Fertility Index: (No of males siring the litter/ No. mated to fertile females) as percentGestation Indices =%age of pregnancies resulting in litters cast liveViability Indices: %age of pups cast alive that survived at 4 daysLactation Indices: %age of pups alive at 4 days that survived to weaning at 21 days
Reproductive indices:
Part I: Female fertility indices: 85,95,65 ;Male fertility indices: 90, 100, 80Gestation Indices: 100,100,100Part II: Female fertility index: 95,90,95Gestation Index: 100,100,100
Offspring viability indices:
Part I:Viability Indices: 98,99,96Lactation Indices: 98,100,98Part IIViability Indices: 92,86,92Lactation Indices: 99,98,99
Clinical signs:
no effects observed
Description (incidence and severity):
No signs of toxicity were seen throughout the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the dietary concentrations fed, there were no effects on food consumption and body weight gains of either males or females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the dietary concentrations fed, there were no effects on food consumption and body weight gains of either males or females.
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The female fertility index in the high dosage group in Part I and the average pup weight in the high dosage group in Part II were lower than the control values, but the differences were not statistically significant at the 95 % confidence level.
Dose descriptor:
NOAEL
Effect level:
616 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NO effect on body weight gains, food consumption and fertility
Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The female fertility index in the high dosage group in Part I and the average pup weight in the high dosage group in Part II were lower than the control values, but the differences were not statistically significant at the 95 % confidence level.
Histopathological findings:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
616 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on live pups , survival, pup body weights and gross pathology
Reproductive effects observed:
not specified
Conclusions:
NOAEL for F1 and F2 generation was considered to be 616 mg/kg when Sprague- Dawley CD male and female rats treated with 2-nitro-p-phenylenediamine orally in feed for 13 weeks.
Executive summary:
in a Fertility and reproductive study, Sprague- Dawley CD male and female rats were treated with 2-nitro-p-phenylenediamine in the concentration of 0, 195 and 616 mg/kg bw/day orally in feed. No significant effect food consumption and body weight gains of male and female were observed as compared to control. Similarly, no effect on male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival were observed as compared to control. Decrease in female fertility index and average pup weight was observed at 616 mg/kg bw/day as compared to control, but the differences were not statistically significant at the 95 % confidence level. In addition, no effects on abnormal pups were seen upon dissection of embryos after 13 days of gestation or upon gross examination at weaning after 21 days. Therefore, NOAEL for F1 and F2 generation was considered to be 616 mg/kg when Sprague- Dawley CD male and female rats treated with 2-nitro-p-phenylenediamine orally in feed for 13 weeks.
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
To assess the reproductive toxicity of the test chemical in rats
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
other: Charles River CD
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALSSource: Charles River CDAge at study initiation: No dataWeight at study initiation: No data Fasting period before study: No dataHousing: metal cagesDiet (e.g. ad libitum): Ralston Purina Laboratory Chow, ad libitumWater (e.g. ad libitum):Tap water, ad libitumAcclimation period: No dataENVIRONMENTAL CONDITIONSTemperature (°C): No dataHumidity (%): No dataAir changes (per hr):No dataPhotoperiod (hrs dark / hrs light):No dataIN-LIFE DATES: From: To: No data
Route of administration:
dermal
Type of inhalation exposure (if applicable):
not specified
Vehicle:
not specified
Details on exposure:
Administraion /Exposure: Day 1 to 19 of gestation.
Details on mating procedure:
- M/F ratio per cage:No data- Length of cohabitation:No data- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancyVaginal plug at day 0 - After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.No data- Further matings after two unsuccessful attempts: [no / yes (explain)]No data- After successful mating each pregnant female was caged (how):No data- Any other deviations from standard protocol:No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
Day 1 to 19 of gestation.
Frequency of treatment:
every 3 days
Details on study schedule:
No data
Remarks:
Doses / Concentrations: 2 ml/kgBasis:no data
No. of animals per sex per dose:
20 pregnant rats
Control animals:
yes
Details on study design:
Details of controls animals: 3 negative (untreated) and one positive control group
Positive control:
Positive control group received acetylsalicylic acid by gavage at a dose of 250 mg/kg on days 6 through 16 of gestation
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data: No dataTime schedule: No dataCage side observations checked in table [No.?] were included.: No dataDETAILED CLINICAL OBSERVATIONS: Yes / No / No data: YesTime schedule: No dataBODY WEIGHT: Yes / No / No data: YesTime schedule for examinations:No dataFOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YesFood consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data: No dataCompound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No dataWATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data: No dataTime schedule for examinations: No data
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
No data
Postmortem examinations (parental animals):
SACRIFICEMale animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]: No data Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]: Twenty pregnant rats from each group were sacrificed on day 20 of gestation by chloroform anesthesia, and Cesarean sections were performed.The uteri were examined, corpora lutea of pregnancy counted, and the number, distribution, and location of live, dead, and resorbed fetuses recorded.
Postmortem examinations (offspring):
SACRIFICEThe F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.: F1 : All fetuses were examined for gross anomalies, sexed, and weighed. These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:Approximately one-third the fetuses from each litter were fixed in Bouin's solution and subsequently examined for visceral anomalies by razor blade sectioning. The remaining fetuses in each litter were fixed in 95% ethyl alcohol, eviscerated, cleared, stainedwith KOH-alizarin red S, and examined for skeletal anomalies
Statistics:
All statistical analyses compared the treatment groups with the control groups. The number of females exhibiting resorption sites, number of females exhibiting two or more resorptions, number of dead or resorbed fetuses, and the number of fetuses with soft-tissue or skeletal anomalies and accessory ribs was compared using chi-square test criterion with Yates' correction on 2 X 2 contingency tables as described by Steel and Torrie or Fisher's exact probability test as appropriate to judge the significance of difference.The mean number of corpora lutea, implantation sites, live fetuses, andresorption sites was compared by analysis of variance (one-way classification) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge the significance of differences.The live fetal weights were compared by analysis of variance (hierarchal classification) as described by Steel and Torrie using Dunnett's multiple comparison tables to judge the significance of differences.
Reproductive indices:
No data
Offspring viability indices:
No data
Clinical signs:
no effects observed
Description (incidence and severity):
No signs of toxicity were seen throughout the study.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
Body weight and food consumption:Changes in female body weights were similar for rats in the untreated controls and all dye-treated groups. A marked reduction in maternal weight gain through gestation was observed in the rats receiving acetylsalicylic acid as compared with either the untreated control rats or dye-treated rat. Mean food consumption for all groups throughout gestation was similar except for rats in the acetylsalicylic acid group; these rats showed a moderate decrease in food consumption from days 7 to .13 of gestation. This decrease was not seen from days 13 to 20 of gestation.Gross pathology:The dye formulations produced no significant differences in the mean number of corpora lutea, implantation sites and live fetuses, and the sexratio when compared with the untreated control groups. No differences between groups were seen regarding the number of females exhibiting resorption sites or mean resorptions per pregnancy. No significant changes were observed regarding soft-tissue anomalies between the dye-treated groups and the untreated control groups. Normally occurring skeletal variations were present in all groups; the most frequent variation noted was accessory ribs.
Dose descriptor:
NOAEL
Effect level:
0.004 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effect on clinical sign, body weight, food consumption and reproduction
Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Normally occurring skeletal variations were present in all groups; the most frequent variation noted was accessory ribs. There were no biologically significant soft tissues or skeletal changes in the fetuses.
Histopathological findings:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.004 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on live fetuses, sex ratio and soft-tissue anomalies in foetuses
Reproductive effects observed:
not specified
Conclusions:
NOAEL for F1 and F2 generation was considered to be 0.0035519 mg/kg when mated Charles River CD female rats treated with 2-nitro-p-phenylenediamine topically for 7 days.
Executive summary:
In a Teratogenicity study, mated Charles River CD female rats (presence of sperm in the vagina considered day 0 of gestation) treated with 2-nitro-p-phenylenediamine in the concentration of 0.0035519 mg/kg bw/day (1.1 % in 2 ml ) topically for 7 days. Acetylsalicylic acid 250 mg/kg served as a positive control. No effect on clinical sign and body weight were observed in treated female rats as compared to control. Except for the changes in the color of the skin and hair at the site of dye application, no irritation or other changes in appearance were seen. Similarly, no effect on food consumption and no repro-developmental effect such as mean number of corpora lutea, implantation sites and live fetuses, and the sex ratio, resorption sites or mean resorptions per pregnancy were observed as compared to control. In addition, No significant changes were observed regarding soft-tissue anomalies in foetuses of treated female rats. Therefore, NOAEL for F1 and F2 generation was considered to be 0.0035519 mg/kg when mated Charles River CD female rats treated with 2-nitro-p-phenylenediamine topically for 7 days.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
616 ng/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Dat is of Klimish 2 and from peer reviewed Journal
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
0.004 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is of Klimish 2 and from peer reviewed Journal
Additional information

Reproduction by oral route

In a study conducted by Wernicket al.(1975), Reproduction was evaluated in Sprague- Dawley CD male and female rats by using 2-nitro-p-phenylenediamine in the concentration of 0, 195 and 616 mg/kg bw/day orally in feed. No significant effect food consumption and body weight gains of male and female were observed as compared to control. Similarly, no effect on male and female fertility, length of gestation, numbers of females with resorption sites, live pups per litter, pup body weights, and pup survival were observed as compared to control. Decrease in female fertility index and average pup weight was observed at 780 mg/kg bw/day as compared to control, but the differences were not statistically significant at the 95 % confidence level. In addition, no effects on abnormal pups were seen upon dissection of embryos after 13 days of gestation or upon gross examination at weaning after 21 days. Therefore, NOAEL for F1 and F2 generation was considered to be 616 mg/kg when Sprague- Dawley CD male and female rats treated with 2-nitro-p-phenylenediamine orally in feed for 13 weeks.

In a study conducted by Wernicket al.(1975), Teratogenicity was evaluated in CFE-S female rats by using 2-nitro-p-phenylenediamine in the concentration of 0, 195 and 616 mg/kg bw/day orally in feed. No significant effect on average number of implantation sites, live pups, or early and late resorptions per litter, or in the number of females with one or more resorption sites. Similarly, No grossly abnormal pups were observed in 195 and 616 mg/kg bw/day treated female rats. Therefore, NOAEL for F1 and F2 generation was considered to be 616 mg/kg bw/day when mated Charles River CD female rats treated with 2-nitro-p-phenylenediamine orally for 10 days.

In a study conducted by Wernicket al.(1975), Teratogenicity was evaluated in New Zealand White Rabbits by using 2-nitro-p-phenylenediamine in the concentration of 0, 19.5 or 97.5 mg/kg/day orally by gavage. No effect on clinical sign of treated female rabbits was observed as compared to control. Similarly, no effect on fetal survival was observed as compared to control. In addition, variations in the degree of ossification and in the number of ribs were observed but, distribution of these changes showed no relationship to the treatment. Therefore, NOAEL for F1 and F2 generation was considered to be 97.5 mg/kg bw/day when New Zealand White female Rabbits treated with 2-nitro-p-phenylenediamine orally for 13 days.

Reproduction by dermal route

In a study conducted by Burnettet al.(1976), Teratogenicity was evaluated in mated Charles River CD female rats (presence of sperm in the vagina considered day 0 of gestation) by using 2-nitro-p-phenylenediamine in the concentration of 0.0035519 mg/kg bw/day (1.1 % in 2 ml ) topically for 7 days. Acetylsalicylic acid 250 mg/kg served as a positive control. No effect on clinical sign and body weight were observed in treated female rats as compared to control. Except for the changes in the color of the skin and hair at the site of dye application, no irritation or other changes in appearance were seen. Similarly, no effect on food consumption and no repro-developmental effect such as mean number of corpora lutea, implantation sites and live fetuses, and the sex ratio, resorption sites or mean resorptions per pregnancy were observed as compared to control. In addition, No significant changes were observed regarding soft-tissue anomalies in foetuses of treated female rats. Therefore, NOAEL for F1 and F2 generation was considered to be 0.0035519 mg/kg when mated Charles River CD female rats treated with 2-nitro-p-phenylenediamine topically for 7 days.

Thus, from the above available studies, the substance 2-nitro-p-phenylenediamine (CAS no 5307-14-2) as per the CLP classification likely to be non hazards for reproduction in rats and rabbit by oral and dermal route.

Short description of key information:
NOAEL for F1 and F2 generation was considered to be 616 mg/kg when Sprague- Dawley CD male and female rats treated with 2-nitro-p-phenylenediamine orally in feed for 13 weeks.

Justification for selection of Effect on fertility via oral route:
NOAEL for F1 and F2 generation was considered to be 616 mg/kg when Sprague- Dawley CD male and female rats treated with 2-nitro-p-phenylenediamine orally in feed for 13 weeks.

Justification for selection of Effect on fertility via dermal route:
NOAEL for F1 and F2 generation was considered to be 0.0035519 mg/kg when mated Charles River CD female rats treated with 2-nitro-p-phenylenediamine topically for 7 days

Justification for classification or non-classification

2-nitro-p-phenylenediamine (CAS no 5307-14-2) as per the CLP classification likely to be non hazards for reproduction in rats and rabbit by oral and dermal route.