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 for cross-reference:

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Rats exposure was too short.Gravid uterus weight not dtermined. Food consumption data and results were not reported. Recording of body weights started too late.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorobenzene

EC Number:

203-628-5

EC Name:

Chlorobenzene

Cas Number:

108-90-7

Molecular formula:

C6H5Cl

IUPAC Name:

chlorobenzene

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:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, Michigan, USA.
- Age at study initiation: adult.
- Weight at study initiation: 175-220 g at breeding.
- 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 (described in Miller et al. 1980. Amer. Ind. Hyg. Assoc. J. 4, 844-846). 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 compressed air supply to the vaporization tubes was preheated (120 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: cohoused
- If co-housed:
- M/F ratio per cage: 1:1
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.

Duration of treatment / exposure:

6 h/d

Frequency of treatment:

daily from day 6 to day 15 of gestation

Duration of test:

On day 21 of gestation test animals were sacrificed

No. of animals per sex per dose:

32 /33 bred rats

Control animals:

yes

Details on study design:

Sex: female
Duration of test: Section on d 21 of gestation
Control animals were exposede 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 rats were recorded on gestation days 6, 9, 12, 16, and 21.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: uterine horns.

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 : Yes: all per litter
-sex: Yes: all per litter

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, 1956. Nonparametric statistics for the behavioral Sciences. 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 and Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York). The reported level of statistical significance was alpha=0.05. For feed and water consuption data, statistical outliers were identified by a sequential outlier test, alpha=0.02 (Grubbs FE, 1969. 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:
No maternal deaths occurred during the period of the study, and no significant effects on general appearance were observed among bred rats exposed to 0, 75, 210, or 590 ppm of monochlorobenzene on days 6 through 15 of gestation. Some evidence of maternal toxicity was observed in the 590 ppm exposure group. Monochlorobenzene exposed pregnant rats gained significantly less weight than controls on Days 6 through 8 of gestation and the absolute and relative weights of the liver on Day 21 of gestation were significantly increased but histopathological correlates were not reported. No effects on body weight gain or liver weights were observed in the 75 or 210 ppm groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEC

Effect level:

590 ppm

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:
The pregnancy rate in rats was not altered by exposure to 75, 210, 590 ppm of monochlorobenzene.No adverse effects were observed on the mean litter size or incidence of implantations which were undergoing resorption. Fetal body measurements for the monochlorobenzene exposed groups were comparable to the control values. The incidence of malformations, when considered individually or collectively, was not significantly increased for any of the exposed groups compared to the control groups.
Skeletal examinations of the fetuses reveled increased incidences of some minor variants, but all of them were not considered to be indicative of a teratogenic response.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEC

Effect level:

590 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Inhalation of monochlorobenzene was not embryotoxic or teratogenic in rats

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1

Body weights and organ weights of pregnant rats

		MCB
	Gestation day	0	75	210	590
Number of dams		27	29	27	28
Body weight	6	196±7^a	196 ±28	195±6	196±5
Body weight gain	6-8	3±2	4±3	2±3	-2±5^b
	9-11	10±4	9±3	9±4	10±4
	12 -15	13±4	14±4	15±4	15±5
	16 -20	37±8	36±11	40±7	40 ±11
Total	6 -20	63±11	63±14	67±9	64±17
Maternal liver weight	21
Absolute		9.82±1.13	9.97±0.81	10.12±0.54	10.99±0.83^b
Relative		3.79±0.28	3.85±0.28	3,86±0.21	4.25±0.42^b

^a Grams, average ± SD

^b Different from the control value by Dunnett´s test,alfa=0.05

^c Grams liver/weight/100 grams body weight, average

± SD

Table 2

Incidence of fetal alterations among litters of rats

	MCB (ppm)
	0	75	210	590
	No. fetuses (No.litters) examinated
External and skeletal examination	241 (27)	256 (29)	267 (27)	258 (28)
Soft tissue examination	128 (27)	138 (29)	141 (27)	139 (28)
	No. fetuses (No.litters) affected
External alterations
Omphalocele a	1 (1)	0	0	0
Cleft palate a	0	1 (1)	0	0
Soft tissue alterations
Liver, focal necrosis	30 (21)	25 (18)	22 (14)^b	19 (14) ^b
Renal agenesis ^a	1 (1) ^c	0	0	0
Dilated renal pelvis ^a	0	0	1 (1)	2 (2)
Right-sidedaortic arch ^a	1 (1) ^c	0	0	0
Microphthalmia ^a	2 (2) c	0	0	1(1)
Anophthalmia ^a	1 (1)	0	1 (1)	0
Skeletal alteration
Vertebrae
Delayed ossification of centra	59 (23)	91 (27) ^b	73 (23)	103 (27)^b
Bilobed centra	8 (5)	8 (7)	3 (2)	12 (11) ^b
Cervical spur(s)	25 (17)	35 (18)	22 (14)	13 (11) ^b
Sternebrae delayed ossification	75 (24)	94 (27)	86 (24)	75 (25)
Total malformed ^d	4 (4)	1 (1)	2 (2)	3 (3)

^a Considered to be a malformation.

^b Different from control incidence by a modified Wilcoxon test, alpha=0.05

^c One fetus exhibited renal agenesis (unilateral), right-sided aortic arch, and microphthalmia.

^d Total number of fetuses (litters) exhibiting one or more malformations.

Applicant's summary and conclusion

Executive summary:

The embryotoxic and teratogenic potential of inhaled chlorobenzene was evaluated in rats. This study was conducted according to OECD guideline 414 with deviations (Rats exposure was too short. Gravid uterus weight was not determined. Food consumption data and results were not reported .Recording of body weights started too late).

Pregnant female Fischer 344 rats were exposed to 0, 75, 210, or 590 ppm (0, 351, 983 or 2761 mg/m³) of chlorobenzene via inhalation for 6 hour/day during the period of major organogenesis (days 6 through 15 of gestation). No maternal deaths occurred during the period of the study, and no significant effects on general appearance were observed among rats. Some evidence of maternal toxicity was observed in the 590 ppm (2761 mg/m³) exposure group. Chlorobenzene exposed pregnant rats gained significantly less weight than controls on Days 6-8 of gestation and the absolute and relative weights of the liver were significantly increased but a histopathological correlate was not reported.

Inhalation of chlorobenzene vapors during gestation was not embryotoxic or teratogenic in rats. The pregnancy rate in rats was not altered by exposure to 75, 210, 590 ppm (351, 983 or 2761 mg/m³) of chlorobenzene. No adverse effects were observed on the mean litter size or incidence of implantations which were undergoing resorption. Fetal body measurements for the chlorobenzene exposed groups were comparable to the control values. The incidence of malformations, when considered individually or collectively, was not significantly increased for any of the exposed groups compared to the control groups. Skeletal examinations of the fetuses revealed increased incidences of some minor variants, but all of them were not considered to be indicative of a teratogenic response.

On the basis of these results the NOAEC for maternal and developmental toxicity was estimated to be 590 ppm (2761 mg/m³, John et al., 1984 ).