Chlorobenzene

Carcinogenicity

Description of key information

In a 2-year cancer study on male and female F344/N rats and B6C3F1 hybrid mice, groups of 50 males and 50 females were given chlorobenzene doses of 60 or 120 mg/kg/day by gavage for 103 weeks (5 days/week); male B6C3F1 mice received 30 or 60 mg/kg/day.
Increased mortality was statistically significant for male rats (p = 0.033, high dose), but not for female rats and mice. A statistically significant positive trend in the incidence of hepatocellular neoplastic nodules was observed in male rats (4/50, 2/50, 4/49, and 8/49 for untreated controls, vehicle controls, low and high dose groups, respectively). There were no increases in hepatocellular neoplastic nodules for female rats and mice, and there were no increases in hepatocellular or other site-specific tumours for either species. 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

Only 2 doses level instead of 3 were administered. Some organs were not microscopically analyzed. Food intake was not determined. No blood smear was obtained from the animals.

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI, USA)
- Age at study initiation: 7 weeks
- Weight at study initiation: 206 g (males); 110 g (females)

- Housing: five animals/polycarbonate cage
- Diet (e.g. ad libitum): Purina Laboratory Chow (pellets), Ralston Purina Co. (St.Louis, MO, USA) ad libitum
- Water (e.g. ad libitum): Edstrom automatic watering system (Waterford, WI, USA)
- Acclimation period: 17 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26 °C
- Humidity (%): 40%-70%
- Air changes (per hr): 15 changes of room air per hour were provided.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Appropriate amount of chlorobenzene and corn oil were mixed with a stirring bar of 15 minutes in a graduate cilinder to preparare highest dose. Lower dose levels prepared by sequential dilution of measured volume of high dose formulation with corn oil.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of chlorobenzene/corn oil mixtures were periodically analyzed at Betelle Columbus Laboratories. Results of these analyses ad of the refereeanalyses at Midewest Research Institute indicated that the analyzed mixtures were properly formulated.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

5 days per week

Post exposure period:

no

Remarks:

Doses / Concentrations:
0, 60, 120 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: by cage every week for the first 13 weeks and monthly thereafter

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; grossly visible lesions were performed on major tissues and organs.
HISTOPATHOLOGY: Yes; tissue masses, skin, mandibular or mesentric lymph nodes, mammary gland, salivary gland, bone marrow, sternebrae, thymus, trachea, lung and bronchi, heart, thyroid, parathyroid, esophagus, stomach, small intestine, colon, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, prostate/testes or ovaries/uterus, brain, and pituitary were microscopally examined.

Statistics:

See " Any other information on materials and methods including tables

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
The survival of high dose male rats was significantly less than that observed for the vehicle controls (p=0.033). No other significant differences in survival were observed.
No compound-related clinical sings of toxicity were observed at any time during the study.
BODY WEIGHT AND WEIGHT GAIN
Throughout the studies, mean body weights of dosed and vehicle control male rats were comparable. During the second year of the studies, mean body weights of dosed female rats were greater than those of the vehicle controls. No compound-related clinical signs of toxicicty were observed at any time during the studies.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Liver: Chlorobenzene was associated with an increased occurence of neoplastic nodules in the livers of male rats. Generally considered to be late-occurig lesions, the first neoplastic nodule of the liver was detected in a vehicle control male rat that died at week 89, and the majority in all groups were detected at study termination. The incidence of neoplastic nodules of the liver in male rats surviving for at least 89 weeks were 4/44 (9%), 2/48 (4%), 4/40 (10%) , and 8/32 (25%) in the untreated control, vehicle control, low dose, and high dose groups, respectively. Pairwise comparisons by the Fisher exact test indicated that the incidence in high dose male rats was significantly (p<0.05) increased in comparison to the vehicle controls or the combined (vehicle and untreated) controls.
Chlorobenzene was associated with an increased occurence of neoplastic nodules in the high dose (120 mg/kg/day) male rats in composite control group. The increase in the neoplastic nodules of the liver in male rats, therefore, was considered to be chlorobenzene.
Testis: Interstitial cell tumors were observed with a significant positve trend by the life table tes, and the incidence in the high dose group was significantly higher that in that vehicle controls by the life table test. Statistical significance was not indicated by either the incidental tumor of Fisher exact tests. One of the interstitial cell tumors in a vehicle control rat was malignant; none of the tumors in the dosed group were malingnant.
Urinary bladder: A transitional cell papilloma was found 1/46 (2%) low dose and 1/45 (2%) high dose male rats. This tumor type was not observed in untreated or vehicle controls.
Kidney: A tubular cell adenocarcinoma was observed in one high dose female rat. This tumor type was not observed in untreated controls, vehicle controls, or low dose female rats.
Pituitary: Adenomas in female rats, and adenomas, adenocarcinomas, or carcinomas in male rats occurred with significantl negative trends. The incidences in the high dose groups were significantly lower than those in the controls.
Uterus: Endometrial stromal polyps were observed with a significantly lower incidence in the low dose group than in controls.
HISTOPATHOLOGY: NON-NEOPLASTIC
An apparent increase in the occurrence of hepatocellular necrosis, and decreases in the occurences of of hepatocellular basophilic cytoplasmic change and granulomatous inflammation, were observed in chlorobenzene-treated male and female rats. Upon a blind review of all liver sections by an indipendent pathologist, however, the occurrence of hepatocellular necrosis in chlorobenzene-treated rats was found to be similar to that in controls. Both diagnosticians generally graded the necrotic lesions as minimal to mild severity in all groups. Therefore, the evidence for mild chlorobenzene-induced hepatocellular necrosis in these studies is considered equivocal.

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day

Sex:

male/female

Remarks on result:

other:

Remarks:

Effect type: other: systemic toxicity (migrated information)

Table 1. Analysis of liver tumors in male rats: Statistical comparisons of treated to vehicle controls

	Untreated control	Vehicle control	60 mg/kg	120 mg/kg
Neoplastic nodules
Tumor rates
Overall	4/50 (8%)	2/50 (4%)	4/49 (8%)	8/49 (16%)
Adjusted	10.4 %	4.5%	12.5%	29.3%
Terminal	2/34 (6%)	0/39 (0%)	4/32 (13%)	7/26 (27%)
Statistical tests
Life table		p=0.005	p=0.255	p=0.010
Incidental tumor test		p=0 .011	p=0.290	p=0.021
Cochran-Armitage trend test		p=0.027
Fisher Exact test			p=0.329	p=0.043
Carcinoma
Tumor rates
Overall	0/50 (0%)	2/50 (4%)	0/49 (o%9	0 /49 (0%)
Adjusted	0.0%	5.1%	0.0%	0.0%
Terminal	0/34 (0%)	2/39 (5%9	0.32 (0%)	0/26%
Statistical tests
Life table		p=0.139 N	p=0.283 N	p=0.331N
Incidental tumor test		p=0.139 N	p=0.283 N	p=0.331N
Cochran-Armitage trend test		p=0.098 N
Fisher Exact test			p=0.253 N	p=0.283 N
Neoplastic Nodule or Carcinoma
Tumor Rates
Overall	4/ 50 (8%)	4/50 (8%)	4/49 (8%)	8/49 (16%)
Adjusted	10.4 %	9.4 %	12.5 %	29.3%
Terminal	2/34 (6%)	2/39 (5%)	4/32 (13%)	7/26 (27%)
Statistical tests
Life Table		p=0.033	p=0.532	p=0.048
Incidental tumor test		p=0.054	p=0.570	p=0.083
Cochran-Armitage trend test		p=0.121
Fisher Exact test			p=0.631	p=0.168

Table 9. Comparative incidenced of lung lesions in male and female rats

	Male (a)				Female (b)
Group	Untreated control	VehicleControl	60 mg/kg	120mg/kg	UntreatedControl	Vehiclecontrol	60mg/kg	120 mg/kg
No. of lungs Examinated	50	50	50	50	47	49	49	49
LESION
Aspiration foreign body	0 (0) (b)	4 (3)	15 (10)	10 (3)	0(0)	0(0)	5 (1)	9 (4)
Inflammation acute/ chronic	7	2	9	4	2	1	7	11
Inflammation,focal granulomatous	0	11	4	1	0	14	8	1

(a) Number of animals with specified lesion

(b) The numbers in parentheses include only those animals that were not diasognes as having died from gavage.

Table 3. Analysis of pituitary tumors in rats

	UntreatedControl	Vehicle Control	60 mg/kg	120mg/kg
MALES
Adenoma
Tumor Rates
Overall	20/49 (41%)	10/50 (20%)	9/42 (21%)	3/47 (6%)
Adjusted	47.5%	24.2%	27.4%	10.6%
Terminal	12/33 (36%)	8/39 (21%)	7/30 (23%)	2/25 (8%)
Statistical Tests
Life table		p=0.172N	p=0.477	p=0.162N
Incidental tumor Test		p=0.109N	p=0.532	p=0.101N
Cohran-Armitage Trend Test		p=0.047N
Fisher Exact Test			p=0.534	p=0.046N
Adenoma, Adenocarcinoma or Carcinoma
Tumor Rates
Overall	20/49 (41%)	12/50 (24%)	9/42 (21%)	3/47 (6%)
Adjusted	47.5%	28.3%	27.4 %	10.6%
Terminal	12/33 (36%)	9/39 (23%)	7/30 (23%)	2/25 (8%)
Statistical Tests
Life table		p=0.084N	p=0.541N	p=0.086N
Incidental tumor Test		p=0.044N	p=0.462N	p00.044N
Cohran-Armitage Trend Test		p=0.016N
Fisher Exact Test			p=0.484N	p=0.015N
FEMALES
Adenoma
Overall	27/48 (56%)	23/46 (50%)	18/46 (39%)	13/43 (30%)
Adjusted	63.6%	67.0%	56.1%	41.6%
Terminal	20/35 (57%)	16/27 (59%)	15/29 (52%)	9/26 (35%)
Statistical Tests
Life table		p=0.027N	p=0.146N	p=0.039M
Incidental tumor Test		p=0.016N	p=0.252N	p=0.021N
Cohran-Armitage Trend Test		p=0.036N
Fisher Exact Test			p=0.201N	p=0.046N

         

Table 11. Analysis of endometrial stromal polyps of the uterus in female rats

	UntreatedControl	VehicleControl	60 mg/kg	120mg/kg
Tumor Rates
Overall	9/49 (18%)	16/50 (32%)	4/49 (8%)	10/50 (20%)
Adjusted	23.2%	51.3%	13.3%	29.3%
Terminal	7/36 (19%)	14/29 (48%)	4/30 (13%)	8/31 (26%)
Statistical Tests
Life Table		p=0.060N	p=0.002N	p=0.090N
Incidental Tumor Test		p=0.059N	p=0.002N	p=0.088N
Cochran-Armitage Trend Test		p=0.085N
Fisher Exact Test			p=0.003N	p=0.127N

                                                                                   

Executive summary:

NTP, 1985.

Carcinogenesis studies of chlorobenzene were conducted by administering the test chemical in corn oil by gavage to groups of 50 male and 50 female F344/N rats at doses of 60 or 120 mg/kg, according to OECD guideline 451 with restrictions (Only 2 doses level instead of 3 were administered. Some organs were not microscopically analyzed. Food intake was not determanined. Blood smears were not obtained from the animals). Chlorobenzene was administered five times per week for 103 weeks. Groups controls, of 50 rats of each sex received corn oil by gavage on the same schedule and served as vehicle controls , and additional groups of 50 rats of each sex served as untreated controls,. Mean body weights of dosed rats were essentially the same or greater than those of the controls during the 2-year studies. Survivals of low dose male and female rats were not adversely affected by administration of chlorobenzene. Survival of high dose male rats in the 2 -year study was signifcantly (p=0.033) lower than that of the vehicle controls. No chlorobenzene-induced toxic lesions responsable for this reduction in survival were observed. Male rats dosed with chlorobenzene exhibited a signifcant (p< 0.05) increase in the incidence of animals with neoplastic nodules of the liver (overall incidences: untreated control, 4/50 ( 8%); vehicle control, 2/50 (4%); low dose, 4/49; high dose 8/49 (16%)). Increased incidences of hepatocellular carcinomas in male rats or of neoplastic nodules or hepatocellular carcinomas in female rats were not observed. No increased tumor incidences were observed in female rats. Under the conditions of these studies, chlorobenzene administration increased the occurence of neoplastic nodules of the liver in high dose (120 mg/kg/day) male F344/N rats, providing some but not clear evidence of carcinogenicity. Carcinogenic effects of chlorobenzene were not observed in female F344/N rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP study similar to guideline

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification is required

Additional information

In a 2-year cancer study on male and female F344/N rats and B6C3F1 hybrid mice, groups of 50 males and 50 females were given chlorobenzene doses of 60 or 120 mg/kg/day by gavage for 103 weeks (5 days/week); male B6C3F1 mice received 30 or 60 mg/kg/day. Due to the fact that neither in rats nor in mice chlorobenzene-related toxic effects were observed, the NOAEL for non-neoplastic effects was considered to be the highest doses tested120 mg/kg/day and 60 mg/kg/day in rats and mice, respectively, leading to the overall NOAEL. to be 60 mg/kg bw/day. Despite the relative closeness of the doses in the subchronic and chronic studies, non-neoplastic lesions clearly attributed to chlorobenzene were not observed in the chronic study. The failure of chlorobenzene to elicit toxic responses in the kidney, liver or haematopoetic system in the chronic study indicates at the most a slight potential for progressive toxicity with continued chlorobenzene administraton beyond 13 weeks.

Regarding the neoplastic effects, the NTP concluded that "under the conditions of these studies, chlorobenzene administration increased the occurrence of neoplastic nodules of the liver in high-dose male F344/N rats, providing some, but not clear evidence of carcinogenicity of chlorobenzene in male rats. Carcinogenic effects of chlorobenzene were not observed in female F344/N rats or in male or female B6C3F1 mice (NTP 1985). In an additional evaluation of this carcinogenicity study extensive statistical calculations led to the conclusion that chlorobenzene cannot be assessed as a carcinogen on the basis of the data determined (Roe, 1987) This was also confirmed by the authors of the study (Kluwe 1987).

In the NTP study (NTP, 1985), the only type of tumour found to occur at a statistically significantly increased incidence in mono- chlorobenzene-treated animals was benign neoplastic liver nodule in male rats of the high-dose group (120 mg/kg/day). The increased incidence was significant by dose-related trend tests, and pair-wise comparisons between the vehicle (corn oil) controls (2/50) but not the untreated controls (4/50) and the high-dose animals (8/49). The only hepatocellular carcinomas found, occurred in two untreated male controls. The committee did not consider these findings indicative of carcinogenic potential of monochlorobenzene (Scientific Committee on Occupational Exposure Limits for Monochlorobenzene - SCOEL/SUM/42, January 2003).

Based on inadequate/equivocal evidence of carcinogenicity in experimental animal data, in combination with predominantly negative genetic toxicity data and the absence of epidemiological data, the EPA has rated chlorobenzene in Group D: inadequate evidence of human carcinogenicity.

Justification for selection of carcinogenicity via oral route endpoint:
key study(rats) is used

Carcinogenicity: via oral route (target organ): digestive: liver