p-phenylenediamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 99.8 %

Test animals

Species:

rat

Strain:

other: Sprague-Dawley rats Crl: OFA (SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 to 13 weeks old
- Weight at study initiation: 209 to 292 g
- Fasting period before study: No
- Housing: Females were individually housed in plastic cages (dimensions 365 x 225 x 180 mm) with autoclaved sawdust as bedding
- Diet: ad libitum
- Water: ad libitum (filtered (0.2 μm) mains drinking water)
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2 °C (target range)
- Humidity (%): At least 40 % (target value)
- Air changes (per hr): At least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

GAVAGE
- Solvent Used: sterile water
- Preparation frequency: Formulations were performed on each treatment day.
- Preparation details: The test item was prepared as a solution in the vehicle at concentrations of 1, 2 and 4 mg/mL. Before preparation, the vehicle was degassed by sonication for at least 15 minutes and then saturated with nitrogen gas and kept under nitrogen atmosphere for approximately 15 minutes. The test substance was then immediately delivered to the animal facility at room temperature, protected from light.
- Adjusted for purity: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method of Analysis: HPLC
Conducted on all dose levels: yes
Results: Actual concentrations of the test substance formulated at 1, 2 and 4 mg/mL were in agreement with the nominal values since the deviations obtained were within -0.1 % to 4.0 %, the requirement of the testing facility being within ± 15% of the nominal value.

Details on mating procedure:

- Impregnation procedure: 100 time-mated females. The females were mated at the supplier with a documented day of mating. They were received at the testing facility on day 0 of gestation (G0).

Duration of treatment / exposure:

days 6-19 of gestation (G6-19)

Frequency of treatment:

Once daily

Duration of test:

to day G20

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a published embryo-fetal developmental toxicity study (Re TA, Loehr RF, Rodwell DE, D'Aleo CJ, and Burnett CM (1981). The absence of teratogenic hazard potential of the test substance in Sprague-Dawley rats. Fundam. Appl. Toxicol., 1:421-425). The test substance was administered by gavage to pregnant Sprague-Dawley rats at dose levels of 5, 10, 15, 20 and 30 mg/kg/day. Maternal toxicity was demonstrated at the two highest dose levels (2/21 pregnant females died at 30 mg/kg/day; lower body weight gains and lower food consumption were noted in the groups given 20 and 30 mg/kg/day). There was no evidence of teratogenic or embryotoxic effect of the test substance in this study. Furthermore, no adverse effect level in a 13-week oral toxicity study in the Sprague-Dawley rat was 4 mg/kg/day (Toxicol Laboratories study LRL/44/94, April 1995).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for clinical signs. During the treatment period, the animals were observed once before and at least once after dosing to detect any clinical signs or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed individually on days 0, 6, 9, 12, 15, 18 and 20 of gestation.

FOOD CONSUMPTION: Yes
- Individual food consumption was measured for the periods (days) 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 during gestation.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The ovaries and uterus of each female were removed and examined. The placentae were also examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: individual fetal weights, fetal sex.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The data were checked for homogeneity of variance across groups using Bartlett’s test. Homogenous data were then analysed by parametric methods, i.e. ANOVA followed by Dunnett's test if ANOVA is significant. Non-homogenous data were analysed by non-parametric methods, i.e. Kruskal-Wallis test followed by Dunn’s test if the Kruskal-Wallis is significant. The numbers of resorptions, number of dead fetuses and all litter-based percentages were analysed using the above non-parametric methods. Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There were no unscheduled deaths and no signs of general toxicity in any group. A slightly transient lower mean gestation body weight gain was noted in the intermediate (10 mg/kg/day) and high (20 mg/kg/day) dose groups during the first three days of treatment. The low dose (5 mg/kg/day) group was not affected. No treatment-related effects on maternal food consumption or macroscopic findings were observed.

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (nominal)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At least 23 females were pregnant in each group. One control and one high dose female had no viable foetuses. There were no differences in pre-or post-implantation data between the treated and control groups, except for an equivocal increased incidence of early resorptions at the high dose level. Mean live litter sizes were comparable between the control and treated groups. Mean foetal weight and the mean gravid uterus weight were slightly lower in the high dose females than in the other groups (not statistically significant). The foetal sex ratio was comparable between groups. There were no malformed foetuses in any group. The incidences of foetuses with morphological anomalies or variations did not suggest any adverse effects of the test item.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Developmental toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). The test substance was non-embryo-fetotoxic. The maternal NOEL was 5 mg/kg/day and the developmental NOAEL was 10 mg/kg/day.

Executive summary:

Three groups of 25 mated female Sprague-Dawley rats were given the test substance orally (by gavage) from days 6 to 19 of gestation inclusive at dose levels of 5, 10, and 20 mg/kg/day. A control group of 25 mated rats was given the vehicle (sterile water) over the same period. Clinical condition, body weights and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on day 20 of gestation and litter parameters were recorded.

 

There were no unscheduled deaths and no signs of general toxicity in any group. A slightly transient lower mean gestation body weight gain was noted in the intermediate (10 mg/kg/day) and high (20 mg/kg/day) dose groups during the first three days of treatment. The low dose (5 mg/kg/day) group was not affected. There was no effect of treatment on maternal food consumption in any group. There were no treatment-related macroscopic findings at the terminal necropsy examination of the adult females.

At least 23 females were pregnant in each group. One control and one high dose female had no viable foetuses. There were no differences in pre-or post-implantation data between the treated and control groups, except for an equivocal increased incidence of early resorptions at the high dose level. Mean live litter sizes were comparable between the control and treated groups. Mean foetal weight and the mean gravid uterus weight were slightly lower in the high dose females than in the other groups (not statistically significant). The foetal sex ratio was comparable between groups. There were no malformed foetuses in any group. The incidences of foetuses with morphological anomalies or variations did not suggest any adverse effects of the test item. 

 

It was concluded that the test substance was non-embryo-fetotoxic, and that the maternal NOEL was 5 mg/kg/day whereas the developmental NOAEL was 10 mg/kg/day.