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Toxicological information

Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.
EC Number:
EC Name:
1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.
Cas Number:
Molecular formula:
Reaction mass of N-phenyl,N'-o-tolyl-phenylene diamine, N,N'-diphenyl-p-phenylene diamine and N,N'-di-o-tolyl-phenylene diamine

Test animals

Details on test animals or test system and environmental conditions:
Caesarean-originated albino rats were supplied by Charles River Labs, North Carolina. Females were ~10 week of age (218 - 273 g) on gestation day 0. One hundred male rats of same strain & origen were obtained from same supplier. Animals were quarantined for 7 days prior to mating at which time, females were individually housed with a male rat. Females were examined for vaginal sperm and/or copulation plugs for evidence of insemination on successive days until insemination was confirmed. Sufficient parings were conducted to ensure 4 groups of 25 sperm-positive females were available for the test project. Diet & drinking water were provided ad libitum. No fasting of animals was employed in this study.

Housing of animals was conducted in a 12/12 hr light/dark cycle with temperature range 68-75 degrees C, and relative humidity of 55 +/- 2.1 %. The in-life study period was conducted in November-December 1994.

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
Rats were maintained on pelleted Purina Rodent Chow.

Dosing solutions were prepared by dissolving test chemical in corn oil at target concentration of 4.0, 14.0, and 40.0 mg/L. Dosing volume to test animals was 5 ml/kg which delivered 20, 70, and 200 mg/kg-day to the three exposed test groups. A negative control group received corn oil only at the same volume level. Corn oil was commonly used as vehicle by the lab and found acceptable for developmental studies, and was shown to dissolve the test chemical. The highest dose for study (200 mg/kg-day) was chosen due to effects observed in range-finding gavage test in which 600 mg/kg-day caused maternal mortality whereas a lower dose 200 mg/kg-day produced non-lethal but demonstrable maternal & developmental effects. 20 & 70 mg/kg-day were without effects in the range finding study.

The test solutions were analyzed and confirmed to be within 10% of target concentrations.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Dosing solutions were prepared by adding test chemical to corn oil. These mixtures were then heated to accelarate dissolution of test chemical (~90 degrees C). One preparation per dose level was used throughout the gavage period. Stabilities of the test solutions were confirmed analytically.

Each corn oil/chemical sample sent to Goodyear for analysis by gas chromatography (nitrogen/phosphorus dection) was diluted with up to 100 volumes of toluene prior to analysis. Duplicate samples were assayed to a limit of detection of 1 ug/sample. Test samples used in the developmental study ranged from 95.0 - 108.5% of targeted concentrations.
Details on mating procedure:
Cohabitation of one female/one male continued until females were confirmed to be sperm positive or 100 sperm-positive females were available for division into 4 test groups of 25 each.
Duration of treatment / exposure:
Daily gavage dosing of sperm-positive females began on gestation 6, and continued through gestation Day 15.
Duration of test:
Test females were sacrificed on gestation day 20, two days prior to expected parturition.
No. of animals per sex per dose:
25 sperm-positive females per dose group including negative controls.


Maternal examinations:
Cage side clinical observations were recorded at least once daily up to sacrifice. Body weights were established for maternal animals on gestations days 0, 6, 9, 12, 15, 18, and 20. Feed consumptions were measured for periods from Day 0 to 6, 6 to 9, 9-12, 12 - 15, 15 - 18, & 18 - 20, and expressed as g/day-maternal rat. Water consumption was not monitored.

Maternal rats were sacrificed on gestation Day 20 by carbon dioxide anesthesia. All were subjected to thoracic and abdominal cavity gross examinations as were all major organs. Gravid uteri and livers were weighed at sacrifice of test animals.
Ovaries and uterine content:
Uteri exams confirmed pregnancy status of all females. Where no implantation sites were visible, uteri were stained with ammonium sulfide to visualize sites for possible early resorptions. Dead & live fetus plus late resorptions were counted as were numbers of corpora lutea.
Fetal examinations:
All fetuses were examined for sex, external morphological, and visceral changes. One half of all fetuses were decapitated prior to dissection, and heads fixed with Bouin's solution for free-hand sectioning and exams. All fetal carcasses were eviscerated and stained with alcian blude/alizarin red S, and half with intact fetluses were examined for skeletal malformations & variations.
Test units for comparisons were pregnant females or the litters.
Corpora lutea, implantation sites per litter, resorption sites, fetal deaths, non-live implants, fetuses examined, external malformations, visceral malformations, skeletal malformations & variations,
Historical control data:
Historical data were available in test report for this lab and the rat strain used in this study.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weight gain was depressed by 14 % in the high dose relative to the control.

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Body weight gain was depressed by 14% in the high dose females relative to controls.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
ca. 200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
ca. 70 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
Effect level:
ca. 200 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 effects

Details on embryotoxic / teratogenic effects:
Although there were slight body weight deficits seen for both male (3.5%) & female (5.2%) fetuses in the high dose groups, these were not statistically significant nor were considered an important adverse effect. No other compound-related adverse effects were seen in the study.

Effect levels (fetuses)

Dose descriptor:
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in sex ratio
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

A NOEL of 70 mg/kg-day was observed in maternal animals for the study while the NOAEL for fetuses was 200 mg/kg-day.
Executive summary:

A developmental study was conducted in rats employing oral gavage dosing to test chemical in corn oil. Dose levels were 20, 70 & 200 mg/kg-day. Very minimal effects were observed in maternal animals at the highest dose level (body weight gain deficit). There were no compound related adverse changes seen in fetuses at any exposure level in this study.