Chlorobenzene

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: Comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Inhalation teratology study on monochlorobenzene in rats and rabbits

Author:

John JA, Hayes WC, Hanley TR, Johnson KA, Gushow TS & Rao KS

Year:

1984

Bibliographic source:

Toxicol. Appl. Pharmacol. 76:365 - 373

Materials and methods

Test guideline

Qualifier:

equivalent or similar to

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Body weights of the animals were recorded too late. Exposure of the rabbits was too short. Gravid uterus weight was not determined. Results of food consuption was not reported.

GLP compliance:

not specified

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Name of test material (as cited in study report): monochlorobenzene.
- Analytical purity: 99.982%
- Impurities (identity and concentrations): Incidental impurities (percentage by weight) consisted of benzene, < 0.005%; bromobenzene, 0.018%; and water, 0.0077%.
- Other: The sample of monochlorobenzene was obtained from the Monsanto Company, St.Louis, Missouri, USA

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Langshaw Rabbitry, Augusta Michigan
- Age at study initiation: adult
- Weight at study initiation: 3.5- 4.5 kg
- Housing: Animals were housed in wire-bottom cages
- Diet (e.g. ad libitum): Feed (Certified Laboratory Animal Chow, Ralston Purin Co., St. Louis, Mo.) were available ad libitum.
- Water (e.g. ad libitum): tap water was available ad libitum.
- Acclimation period: at least 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 22°C
- Humidity (%): approx. 50%
- Photoperiod (hrs dark / hrs light): 12hrs dark / 12hrs light

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: compressed air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposures were conducted in 4.3 m³ Rochester-type stainless-steel and glass chambers under dynamic airflow conditions
- Source and rate of air: dynamic airflow conditions (circa 800 liters air/min).
- System of generating particulates/aerosols: exposure concentrations of monochlorobenzene were generated by metering the liquid test material at controlled rates into vaporization tubes as described by Miller et al. 1980. Vapors from the tubes were swept into the chamber inlet ducts with compressed air where they were mixed and diluted with incoming air by turbulence. The compresses air supply to the vaporization tubes was preheated (120°C to 135 °C) to facilitate complete vaporization of the liquid test material.
- Temperature, humidity, pressure in air chamber: temperature was approximately of 21°C, and the humidity of approximately 50%.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Monochlorobenzene concentrations in the chambers were analyzed one to two times per hour throughout the exposure periods by infrared spectrophotometry using a Miran IA-CVF analyzer equipped with a variable pathlength gas cell.

Details on mating procedure:

- Impregnation procedure: artificial insemination

Duration of treatment / exposure:

6 h/d

Frequency of treatment:

daily on d 6 - 18 of gestation

Duration of test:

On day 29 of gestation the rabbits were sacrificed

Doses / concentrations

Remarks:

Doses / Concentrations:
Study 1: 75, 210 or 590 ppm (351, 983 or 2761 mg/m3). Study 2: 0, 10, 30, 75 or 590 ppm (47, 140, 351 or 2761 mg/m3)
Basis:
nominal conc.

No. of animals per sex per dose:

30 rabbits were employed in the study 1 and 32 in study 2

Control animals:

other: control animals were exposed to filtered air

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of rabbits were measured on Days 6, 9, 12, 15, 19, and 29 of gestation.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Uterine horns


OTHER: In addition, maternal liver weights of rabbits were obained at the time of cesarean section.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and position of fetuses in utero, number of live and dead fetuses.

Fetal examinations:

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

Statistics:

Statistical evaluation of the frequency of alterations and of resorptions among litters and the fetal population was conducted by the Wilcoxon test as modified by Haseman & Hoel, 1974. Statistical analysis of the percentage of pregnancy and other incidence data were conducted by the Fisher exact probability test (Siegel S, 1996. Nonparametric statistics for the behaviorallsciences. McGraw-Hill, New York). Analyses of the other data were made by parametric or non parametric analysis of variance followed by either Dunnett test or the Wilcoxon test as appropriate (Steel RG and Torrie HH, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York). The reported level of statistical significance was alpha=0.05. For feed and water consumption data, statistical outliers were identified by a sequential outlier test, alpha=0.02 (Grubbs FE, 1969. Procedures for detecting outlying observations in samples. Technometrics 11, 1-21), and were deleted from the calculation of mean values.

Indices:

no data

Historical control data:

yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
In the first study, evidence of slight maternal toxicity was observed among rabbits exposed to 210 or 590 ppm; the absolute and relative weights of the liver were significantly increased in these groups. No statistically identified or consistent effects on body weight or body weight gain were observed in any of the exposure groups of rabbits. The pregnancy rate in rabbits was not altered by exposure to monochlorobenzene. At the same time any adverse effects on the mean litter size or the incidence of implementations which were undegoing resorption were observed.
In the second study, exposure to 590 ppm produced an increase in the liver weight of maternal animals. A significant increase in the percentage of implantations undergoing resorption was observed in the 590 ppm group. In this group, 61% of the litters (12% of fetuses) contained resorptions compared to 41% of the litters for the controls (6% of the fetuses).The resorption rate was not signifcantly altered at the lower exposure concentations.

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

> 590 ppm

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
A significant increase in the mean fetal crown-rump lenght was observed in litters from the 210 ppm group, but this increase was not considered to be of toxicological importance. Mean fetal body weights of litters from the exposed groups were not significantly altered. Exposure of rabbits of 0, 75, 210 or 590 ppm of monochlorobenzene resulted in a variety of malformations in all groups, at an incidence shlightly higher than historical controls. Forelimb flexure, the malformation most often observed, occurred more often among controls than among any of the treatment groups, as malformations of the skeletal system.
The latter induced hemivertebrae, missing or fused vertebrae, crokked long bones, and a variety of rib malformations. However there were several cases of external and visceral malformations which were scattered among exposed groups. For instance spina bifida (single case) and low incidence of heart anomalies were observed in the 210 and 590 ppm groups, whereas these malformations were not observed among the concurrent control group.
In the second study, fetal body measurements were not adversely affected by exposure by monochlorobenzene. The incidence of malformations from the exposed groups, when considered individually compared to the control group. Fetuses with external, soft tissue, and skeletal malformations were observed among all groups including controls.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

> 590 ppm

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

The embryotoxic and teratogenic potential of inhaled chlorobenzene was evaluated in rabbits.

This study was conducted accordingly with OECD guideline 414 with deviations (Body weights of the animals were recorded too late. Exposure of the rabbits was too short. Gravid uterus weight was not determined. Results on food consumption were not reported).

First 30 artificially inseminated (per sex/dose groups) New Zealand White rabbits were exposed to 0, 75, 210, or 590 ppm (0, 351, 983 or 2761 mg/m³) of chlorobenzene via inhalation for 6 hr/day during the period of major organogenesis. To further evaluate the effects of chlorobenzene in rabbits, additional groups were exposed to 0, 10, 30, 75, or 590 ppm (0, 47, 140, 351 or 2761 mg/m³) using the same experimental conditions..

In the first study, evidence of slight maternal toxicity was observed among rabbits exposed to 210 or 590 ppm (983 or 2761 mg/m³); the absolute and relative weights of the liver were significantly increased in these groups. No statistically identified or consistent effects on body weight or body weight gain were observed in any of the exposure groups of rabbits. The pregnancy rate in rabbits was not altered by exposure to chlorobenzene. At the same time any adverse effects on the mean litter size or the incidence of implementations which were undergoing resorption were observed.

In the second study, exposure to 590 ppm (2761 mg/m³) produced an increase in the liver weight of maternal animals. A significant increase in the percentage of implantations undergoing resorption was observed in the 590 ppm group (2761 mg/m³). In this group, 61% of the litters (12% of fetuses) contained resorptions compared to 41% of the litters for the controls (6% of the fetuses). The resorption rate was not significantly altered at the lower exposure concentrations. A significant increase in the mean fetal crown-rump lenght was observed in litters from the 210 ppm (983 mg/m³) group, but this increase was not considered to be of toxicological importance. Mean fetal body weights of litters from the exposed groups were not significantly altered. In the first study, exposure of rabbits of 0, 75, 210 or 590 ppm (0, 351, 983 or 2761 mg/m³) of chlorobenzene resulted in a variety of malformations in all groups, at an incidence slightly higher than historical controls. Forelimb flexure, the malformation most often observed, occurred more often among controls than among any of the treatment groups, as malformations of the skeletal system. The latter induced hemivertebrae, missing or fused vertebrae, crooked long bones, and a variety of rib malformations. However there were several cases of external and visceral malformations which were scattered among exposed groups. For instance spina bifida (single case) and low incidence of heart anomalies were observed in the 210 and 590 ppm (983 or 2761 mg/m³) groups, whereas these malformations were not observed among the concurrent control group. In the second study, fetal body measurements were not adversely affected by exposure by chlorobenzene. The incidence of malformations from the exposed groups, when considered individually or collectively, was not significantly increased compared to the control group. Fetuses with external, soft tissue, and skeletal malformations were observed among all groups including controls.

On the basis of the data of the both studies the NOAEC for maternal toxicity (derived by the notifier), and the NOAEC for the teratogenicity (derived by the notifier) were > 590 ppm (2761 mg/m³, John et al 1984).