2-nitroaniline

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study report, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Principles of method if other than guideline:

Mated female Sprague-Dawley rats received 100, 300 or 600 mg/kg bw/day of orthonitroaniline, orally by gavage, on days 6-15 of gestation.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-nitroaniline

EC Number:

201-855-4

EC Name:

2-nitroaniline

Cas Number:

88-74-4

Molecular formula:

C6H6N2O2

IUPAC Name:

2-nitroaniline

Details on test material:

- Name of test material (as cited in study report): ortho-nitroaniline
- Analytical purity: no data
- Lot/batch No.: 1769152

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, N.Y.
- Strain: (Crl:CD(SD (BR))
- Age at study initiation: females - 12 weeks; males - 9 weeks
- Weight at study initiation: females - 204-276 g; males - >=197g
- Housing: individually
- Diet (ad libitum): Purina Certified Rodent Chow 5002
- Water (ad libitum): tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Appropriate amounts of test material were mixed with corn oil and heated to approximately 110 °C to produce test mixture concentrations of 10, 30 and 60 mg/mL orthonitroaniline for the low-, intermediate- and high-dose groups, respectively. Test mixtures were prepared once and used throughout the entire treatment period.

Three treatment groups, each consfstinq of 25 mated females, received 100, 300 or 600 mg/kg bw/day of orthonitroaniline at a constant volume of 10 mL/kg bw. A concurrent control group of equal size receivad an equivalent volume of the corn oil vehicle only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analyses of the test material in the vehicle were performed on each test mixture preparation used for dose administration. Stability was determined at 1, 2, and 4 weeks, prior to study initiation, using the low- and high-dose test mixture concentrations from the range-finding study.
Analyses confirmed that test mixtures were stable over a four-week period and concentrations were within acceptable limits.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug day 0 of pregnancy

Duration of treatment / exposure:

6-15 of gestation

Frequency of treatment:

once daily

Duration of test:

Until day 21 of gestation

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, and 600 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25 mated female rats/dose

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each female an gestation day 0, daily from days 6 through 20, and on day 21 prior to sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual female body weights were recorded on gestation days 0, 6, 10, 13, 16 and 21.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Time schedule for examinations: Individual feeder weights were recorded on gestation days 0, 6, 10, 13, 16 and 21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Immediately following sacrifice, the uterus was opened and the fetuses removed. The number and relative placement of live fetuses, dead fetuses, early resorptions and late resorptions were recorded. The number of corpora lutea per ovary was also determined.

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

Fetal examinations:

All live fetuaes were subjected to a gross external examination, sexed, weighed and tagged for identification. Approximately one-half of each litter was placcd in Bouin's fixative for subsequent serial sectioning and visceral examination. The remaining fetuses were skinned, eviscerated and fixed in
alcohol for subsequent staining with Alizarin Red S and skeletal examination. Abnormal findings were classified as malformations or developmental variations.


- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litte]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

Statistics:

Maternal body weight means, mean maternal body weight change, maternal food consumption means and fetal body weight means were compared using Dunnett's test. Percent pre- and postimplantation loss and the number of live fetuses, dead fetuses, early resorptions, late resorptions, nidations and Corpora lutea were compared using the Mann-Whitney U-test. Pregnancy rate and the percent of litters with malformed fetuses were compared using Fisher's Exact test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
All females survived to their scheduled day of sacrifice with the exception of one animal in the 300 mg/kg bw group, which died on gestation day 15. At necropsy, a perforation in the esophagus was noted, indicating that its death was due to a dosing error.
Yellow to orange staining of the mouth, nose, urogenital area and tail, and yellow to orange colored urine, were frequently noted in all treatment groups but were not noted in the control group. Piloerection was also observed in the 300 and 600 mg/kg groups.
Postmortem findings were comparable for all groups.
Mean maternal body weight was reduced in all treatment groups on gestation days 10, 13, 16 and 21 when compared to the control group. However, reduced mean weights were also evident in the 100 and 600 mg/kg groups on gestation day 6, prior to treatment. Reductions were statistically
significant only in the 600 mg/kg group on gestation days 10, 13, 16, and 21.
A statistically significant mean body weight loss was noted in the 300 and 600 mg/kg groups for days 6-10 of gestation. A reduced weight gain was noted in the 100 mg/kg group for days 6-10 and 10-13, and in all treatment groups for days 13-16, 16-21, and 6-21. Reductions for days 6-21 appeared to occur in a dose-response pattern.
Mean maternal food consumption was reduced in all treatment groups for days 6-10, 10-13 and 16-21 of gestation when comparad to the control group. In addition, reductions were noted in the 100 and 600 mg/kg groups for days 13-16. However, reduced mean food consumption was also evident in the 100 and 600 mg/kg groups for days 0-6 prior to treatment. Reductions were statistically significant in the 300 and 600 mg/kg groups for days 6-10 and also in the 600 mg/kg group for days 13-16. A statistically significant reduction in mean food consumption was evident for days 6-21 in all treatment groups. These reductions occurred in a dose-response pattern.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean fetal body weight appeared to be slightly reduced in the 600 mg/kg group when compared to the control group. However, this reduction was not statistically significant.
The number of female fetuses was greater than the number of male fetuses in the 100 and 300 mg/kg groups. However, equal numbers of each sex were present in the 600 mg/kg group. Therefore, the differences in the 100 and 300 mg/kg group are not considered to be biologically meaningful.
The total number of litters and fetuses exhibiting malformations was comparable for all groups. Multiple malformations were exhibited by one fetus in each of the control, 100 and 300 mg/kg groups, and by one fetus in each of two litters in the 600 mg/kg group. The major malformations observed in these fetuses included: control group - short body and spinal cord mispositioned; 100 mg/kg group - exencephaly and spina bifida; 300 mg/kg group - bone malformations of the jaws and hindlimbs; 600 mg/kg group - situs inversus of the stomach with heart malformations. Several fetuses with single malformations were also observed in the control and 600 mg/kg groups.
The incidence of litters exhibiting developmental variations was considered to be comparable for all groups. A slight increase in the number of litters exhibiting asymmetry of sternocostal articulations, sternebral dislocation, separation of the subcutaneous muscle from skin and retarded ossification of the skull was noted in some treatment groups when compared to the control group. These findings are not considered to be biologically meaningful, as they were also exhibited by the control group, and dose-response patterns were not evident.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Discussion

 

Piloerection and reductions in maternal body weight gain and food consumption indicate that orthonitroaniline is toxic to pregnant rats at levels of 300 mg/kg bw and greater, though treatment-related mortality was not observed. The slight reduction in body weight and parallel reduction in food consumption at the 100 mg/kg bw level may not be entirely due to treatment, as mean maternal body weight and food consumption in this group were also reduced prior to initiation of treatment. The yellow to orange colored urine and staining of the fur noted in the treatment groups may be due to staining properties of the test material rather than abnormal physiological processes.

Pregnancy rate and the mean number of live and dead fetuses, early and late resorptions, total nidations and Corpora lutea were comparable for all groups. Orthonitroaniline was not embryotoxic at the levels tested.

The slight reduction in fetal body weight at the 600 mg/kg bw level may be indicative of fetotoxicity. However, the reduction was very slight and was not evident at lower levels.

No meaningful difference in fetal sex distribution was evident.

No meaningful difference in the total number of litters or fetuses exhibiting malformations was evident. However, one fetus in each of two litters at the 600 mg/kg bw level exhibited partial situs inversus and similar heart malformations. Though malformations were not exhibited by other fetuses in these two affected litters, and similar malformations were not exhibited at lower levels, the material-related effect does exist.

In conclusion, definitive evidence of maternal toxicity was noted at the 300 and 600 mg/kg bw levels. However, treatment with orthonitroaniline did not result in a teratogenic response at levels of 300 mg/kg bw/day or less.