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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
publication
Title:
Chronic Toxicity, Teratologic, and Reproduction Studies with Hair Dyes
Author:
THEODORE WERNICK, BEN MARR LANMAN AND JEAN LOUIS FIUUX
Year:
1975
Bibliographic source:
TOXICOLOGY AND APPLIED PHARMACOLOGY 32,450-460 (1975)

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Developmental toxicity prformed on Sprague Dawley CD male and female Rats
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):2 nitro p phenylenediamine- Substance type:Organic- Physical state:Solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALSSource: Charles River breeding laboratoriesAge at study initiation: No dataWeight at study initiation: (P) Males: 240 to 280 g Females: 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 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

Administration / exposure

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
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
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 was confirmed 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 considered fertile. 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 was confirmed 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 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
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:
No data
Duration of test:
Daily
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

Examinations

Maternal examinations:
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.
Ovaries and uterine content:
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 and gross abnormalities.
Fetal examinations:
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
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
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yesDetails on maternal toxic effects:In both studies, no dose-related significant differences were observed in male and female fertility, length of gestation, number of females with absorption sites, live pups per litter, pup body weights, and pup survival. 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.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
351 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects: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.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
554 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Generation F0 and F1

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
A semi-permanent hair-coloring composite containing 0.24 percent 2NPPD and 0.16 percent 4NOPD was administered to groups of 10 male and 20 female rats in their diets in concentrations of (In Part I study) 0, 86, 351 mg/kg/day (average) and (In Part II study) 0, 124, 554 mg/kg/day (average). Sixty male and 120 female Sprague-Dawley CD strain rats were divided into six groups of 10 males and 20 females each. The body weights of the male rats ranged from 240 to 280 g while those of the females ranged from 180 to 220 g. The composite was mixed into the basal diet of Purina laboratory chow. Diets were prepared twice weekly. 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 and 21 days of lactation.One male was placed in a cage with two females from 4 PM to 8 AM the following day. Food was withheld during this time interval to prevent access to the inappropriate diet. Water was available at all times. This procedure was continued until copulation was confirmed by the appearance of sperm in a vaginal smear (day 0 of pregnancy). Males were rotated within their dietary groups at IO-day intervals until conception was confirmed 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. Pregnant females then were placed in individual cages. 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.In both studies, no dose-related significant differences were observed in male and female fertility, length of gestation, number of females with absorption sites, live pups per litter, pup body weights, and pup survival. There were no abnormal pups. No effects on feed consumption or body weight gains of either sex were found.The NOAEL for the F0 and F1 generation: Part I study: 351mg/kg/day; Part II study: 554mg/kg/day.
Executive summary:

A semi-permanent hair-coloring composite containing 0.24 percent 2NPPD and 0.16 percent 4NOPD was administered to groups of 10 male and 20 female rats in their diets in concentrations of (In Part I study) 0, 86, 351 mg/kg/day (average) and (In Part II study) 0, 124, 554 mg/kg/day (average).

Sixty male and 120 female Sprague-Dawley CD strain rats were divided into six groups of 10 males and 20 females each. The body weights of the male rats ranged from 240 to 280 g while those of the females ranged from 180 to 220 g. The composite was mixed into the basal diet of Purina laboratory chow. Diets were prepared twice weekly. 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 and 21 days of lactation.

One male was placed in a cage with two females from 4 PM to 8 AM the following day. Food was withheld during this time interval to prevent access to the inappropriate diet. Water was available at all times. This procedure was continued until copulation was confirmed by the appearance of sperm in a vaginal smear (day 0 of pregnancy). Males were rotated within their dietary groups at IO-day intervals until conception was confirmed 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 considered fertile. A female was considered infertile if she failed to become pregnant after mating with two different males for 10 days each. Pregnant females then were placed in individual cages. 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.

In both studies, no dose-related significant differences were observed in male and female fertility, length of gestation, number of females with absorption sites, live pups per litter, pup body weights, and pup survival. There were no abnormal pups. No effects on feed consumption or body weight gains of either sex were found.

The NOAEL for the F0 and F1 generation: Part I study: 351mg/kg/day; Part II study: 554mg/kg/day.