Reaction mass of carbon tetrachloride and chloroform and 1,2-dichloroethane

Carcinogenicity

Description of key information

Based on the weight-of-evidence approach followed in the carbon tetrachloride dossier:

- Carcinogenicity, oral: NOAEL  = 10 mg/kg bw/day for the mouse (study not reported in this dossier)

- Carcinogenicity, inhalation: NOAEC = 32 mg/m³ air (= 5 ppm) for the rat and the mouse, 2 y (OECD TG 453); study: Nagano 2007 B), based on increase of hepatocarcinoma incidences at higher level exposures.

- Carcinogenicity, dermal: no study available

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

worst-case value used as key value in the CTC dossier

Organ:

liver

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically sound study, GLP conform, limited reporting of experimental data.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4 weeks
- Weight at study initiation: 120 g +/- 5 SD (males), 100 g +/- 3 SD (females)
- Housing: individually in stainless steel wire hanging cages (150 mm x 220 mm x 176 mm). Randomisation procedure for allocation
- Diet (e.g. ad libitum): y-irradiation-sterilized commercial pellet died, ad libidum
- Water (e.g. ad libitum): sterilized water, ad libidum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23° +/- 2 °C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 12 +/- 1 air changes/h
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

not specified

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Source and rate of air: airflow containing CTC prepared by a vaporization technique.
- System of generating particulates/aerosols: saturated vapor-air mixture generated by bubbling clean air through CTC liquid at 26°C and by
cooling it through a thermostatted cindenser at 18°C
- Temperature, humidity, pressure in air chamber: see "details on test animals and environmental conditions"

OTHER:
Four inhalation exposure chambres of 7600 L volume. Each exposure chamber accomodated 100 individual cages for 50 males and 50
females.
Chamber concentrations of CTC were monitored by GC every 15 min and mantained constant.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Two years

Frequency of treatment:

6h/day, 5 days/week, for 104 weeks

Dose / conc.:

0 ppm (analytical)

Remarks:

0 mg/m3

Dose / conc.:

5 ppm (analytical)

Remarks:

32 mg/m3

Dose / conc.:

25 ppm (analytical)

Remarks:

160 mg/m3

Dose / conc.:

125 ppm (analytical)

Remarks:

801 mg/m3

No. of animals per sex per dose:

50 males and 50 females per concentration

Control animals:

yes

Details on study design:

- Dose selection rationale: the lowest exposure concentration of 5 ppm was selected at an environmentally relevant level in consideration of the occupational exposure limit value of 5 ppm of CTC (ACGIH, 2001). The highest concentration of 125 ppm was selected not to exceed the maximum tolerated dose (MTD) based on a 13-wk inhalation study

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: one per week for the first 14 wk, and every 2 wk thereafter
- How many animals: 50 males/50 females per group (no. reduced in the higher dose groups due to deaths during the course of the study)

FOOD CONSUMPTION: Yes
- Time schedule for examinations: one per week for the first 14 wk, and every 2 wk thereafter

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after overnight fasting at the end of the 2-yr exposure period.
- Anaesthetic used for blood collection: Yes (ether)
- Study was conducted according to OECD 452, but haematologic parameters were not reported in the present publication

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after overnight fasting at the end of the 2-yr exposure period.
- How many animals: 22 males/39 females (control group), 28 males/43 females (5 ppm), 19 males/39 females (25 ppm), 3 males/1 female
(125 ppm)
- Study was conducted according to OECD 452, but only the parameters shown in table 2 were reported in the present publication


URINALYSIS: Yes
- Time schedule for collection of urine: during the last week of the 2 year testing period
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- No. of animals tested: 22 males/39 females (control group), 31 males/43 females (5 ppm), 19 males/40 females (25 ppm), 3 males/1 female
(125 ppm)
- Study was conducted according to OECD 452, but only urinary protein levels were reported in the present publication.

NEUROBEHAVIOURAL EXAMINATION: No data


OTHER:
Mortality:
- Time schedule: daily

Relative organ weights (liver and adrenal gland)
- How many animals: 35 males/26 females (control group), 36 males/24 females (5 ppm), 25 males/10 females (25 ppm), 1 male/1 female
(125 ppm)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all organs that are outlined in OECD 452, but only results on lung, liver and kidney were discussed in detail in the present publication
HISTOPATHOLOGY: Yes, all organs that are outlined in OECD 452, but only results on lung, liver and kidney were discussed in detail in the present publication

Statistics:

- Incidence of neoplastic lesion: Peto's test (difference from the clean air-exposed group: Fisher's exact test)
- incidence of nonneoplasitv lesions and urinary parameters: chi-square test
- survival curves: Kaplan and Meier's Method
- Difference in survival rate: Fisher's exact test
- Body weight: Dunnett's test
- Body weight: Dunnett's test
- Blood biochemical parameters: Dunnett's test

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:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Significant decreased survival rates of the 125-ppm-exposed male and female rats (observed at weeks 89 and 74 of the exposure and threreafter).
At the end of the 2-yr exposure period, the survival rates of 0-, 5-, 25-, and 125-ppm-exposed rats were 44, 58, 38, and 6% for males, and 78, 86, 78, and 2% for females, respectively.
The incidences of palpable liver masses in the abdominal cavity, which first appeared at wk 63 in a male and wk 49 in a female, were increased in the
125-ppm-exposed rats.


BODY WEIGHT AND WEIGHT GAIN (see table 1)
Growth rates at 125-ppm-exposed male and female rats significantly retarded after wk 13 and 58, respectively.
The 25 ppm-group (both sexes) exhibited a significant but marginal decrease by approx 10% in the terminal body weight


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant decrease in any CTC-exposed group of either sex as compared with the clean-air-exposed group


HAEMATOLOGY / CLINICAL CHEMISTRY ((see table 2)
Significantly increased plasma levels of AST and ALT in both the 25- and 125-ppm-exposed male rats; y-GTP significant increase only in the 125-ppm-exposed male rats. Significantly increased levels of ALT, LDH, and y-GTP in the 25-ppm-exposed female rats. Urea nitrogen increase in male and female rats exposed to 25 ppm and 125 ppm.

URINALYSIS (see table 2)
The number of animals whose urinary protein level exceeded 300 mg/dL was significantly increased in both 5- and 25-ppm-exposed rats (males and females).


ORGAN WEIGHTS (see table 1)
Relative organ weights increased in an exposure concentration-dependent manner for the liver, kidneys, and lungs of male and female rats exposed
to 25 and 125 ppm.


GROSS PATHOLOGY
Macroscopic examination: nodular surface and large circumscribed and tumor-associated masses in the liver of the 125-ppm-exposed rats of both sexes. Incidences of granular surface in the kidney were increased in an exposure concetration-manner.
Macroscopic and microscopic examinations of rats that died before the end of the 2-yr exposure to 125 ppm revealed that 28 males and 34 females were severely affected by chronic progressive nephropathy (CPN) and apparently died of CPN, while 17 males and 14 females died of various tumors including hepatocellular tumors. Hepatocellular tumors were found in 82% and 85% of the 125-ppm-exposed male and female rats, respectively, that apparently died of CPN. Therefore, the significantly decreased survival rates in the 125-ppm-exposed rats of both seyes were considered to be causally related to both various tumors including hepatocellular carcinomas and severely affected CPN.


HISTOPATHOLOGY: NON-NEOPLASTIC (see table 3)
Macroscopic and microscopic examinations of rats that died before the end of the 2-yr exposure to 125 ppm revealed that 28 males and 34 females were severely affected by chronic progressive nephropathy (CPN) and apparently died of CPN.
Incidences of hepatic altered cell foci, including clear, acidophilic, basophilic, and mixed cell loci were significantly increased in the 125-ppm-exposed females, but not in any CCl4-exposed male group except for basophilic cell foci in the 125-ppm males.
Incidences of fatty change in the liver were significantly increased in both male and female rats exposed to 25 and 125 ppm. The incidence of liver cirrhosis was significantly increased at 125 ppm-exposure, while the incidence of liver fibrosis was significantly increased in the 25-ppm-exposed rats of both sexes.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) (see table 3)
Macroscopic and microscopic examinations of rats that died before the end of the 2-yr exposure to 125 ppm revealed that 17 males and 14 females died of various tumors including hepatocellular tumors. Hepatocellular tumors were found in 82% and 85% of the 125-ppm-exposed male and female rats, respectively, that apparently died of CPN. Therefore, the significantly decreased survival rates in the 125-ppm-exposed rats of both seyes were considered to be causally related to both various tumors including hepatocellular carcinomas and severely affected CPN.
The hepatocellular carcinoma was found first at wk 73 and 50, respectively in a dead male and a dead female exposed to 125 ppm. Incidences of hepatocellular adenomas and carcinomas and their combined incidenses were increased in an exposure concentration-dependent manner. Those tumor incidences were significantly increased in the 125-ppm- exposed rates (both sexes) compared with the control group.
Multiple occurrence of hepatocellular tumors was found in the 125-ppm-exposed rats. Metastasis of hepatocellular carcinomas to the lung occured in the 125-ppm-exposed male and female rats.

Although other types of tumors, including testicular interstitial-cell tumors, pituitary adenomas, adrenal pheochromocytomas, monouclear cell leukemia, and mammary-gland adenomas, were observed in both CTC-exposed and clean-air-exposed rats, there was no exposure-related difference in the incidences of these tumors between any CTC-exposed group and the clean-air-exposed group.

No neoplastic lesion was induced in the respiratory tract, including the nasal cavity, pharynx, larynx, trachea and lungs. However, incidences and severities of eosinophilic globules (in the cytoplasm of both the respiratory and olfactory epithelia in the nasal cavity) tended to increase in an exposure concentration-dependent manner in male rats exposed to 25 and 125 ppm and in femal rats exposed to 5, 25, and 125 ppm.

Dose descriptor:

NOAEC

Effect level:

32 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

LOAEC

Effect level:

160 mg/m³ air

Sex:

male/female

Basis for effect level:

other: - 3 cases of hepatocellular carcinomas and increased incidences of hepatic altered cell foci were reported for the 25 -ppm-exposed females - Hepatocellular carcinomas and cirrhosis significantly occurred in the 125 -ppm-exposed rats of both sexes

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Table 1: terminal body weight and relative organ weights of rats exposed to CTC, vapor by inhalation for 2 yr

	Male				Female
Group name	Control	5 ppm	25 ppm	125 ppm	Control	5 ppm	25 ppm	125 ppm
No. of animals examinated	22	29	19	3	39	43	39	1
Body weight (g)	430 ± 38	430 ± 45	385 ± 48*	357 ± 160	331 ± 51	332 ± 42	294 ± 33**	183
Liver (%)	3.56 ± 0.38	3.47 ± 0.56	4.30 ± 0.60**	6.67 ± 0.44**	2.83 ± 0.73	2.54 ± 0.35*	5.11 ± 0.60**	8.70
Kidney (%)	0.81 ± 0.24	0.78 ± 0.20	0.88 ± 0.12*	1.09 ± 0.38	0.65 ± 0.16	0.63 ± 0.11	0.85 ± 0.18**	1.70
Lung (%)	0.35 ± 0.04	0.36 ± 0.15	0.42 ± 0.15**	0.50 ± 0.18	0.34 ± 0.11	0.33 ± 0.09	0.43 ± 0.13**	0.66

Note: values were represented as mean ± SD. Significant difference indicated by *p .05 and **p 0.01 by Dunnett’s test.

Table 2: blood biochemical and urinary parameters of rats exposed to CTC vapor by inhalation for 2 yr

	Male				Female
Group name	Control	5 ppm	25 ppm	125 ppm	Control	5 ppm	25 ppm	125 ppm
Blood biochemistry
No. of animals examined	22	28	19	3	39	43	39	1
AST (IU/L)	60 ± 23	78 ± 56	109 ± 120*	222 ± 93**	143 ± 166	144 ± 139	208 ± 318	144
ALT (IU/L)	17 ± 6	21 ± 13	28 ± 11**	92 ± 38**	33 ± 16	39 ± 24	62 ± 61**	70
LDH (IU/L)	257 ± 81	380 ± 518	350 ± 130	302 ± 41	440 ± 666	324 ± 266	823 ± 1757**	289
Γ-GTP (IU/L)	31.6 ± 7.2	35.4 ± 17.0	39.5 ± 11.5*	74.9 ± 52.9	17.9 ± 2.8	17.2 ± 3.7	29.2 ± 16.9**	60.2
Urineanalysis
No. of animals examined	22	31	19	3	39	43	40	1
Protein ^a	0	24**	15**	0	1	26**	37**	0

Note: values were represented as mean ± SD. Significant difference indicated by *p .05 and **p 0.01 by Dunnett’s test.

AST: aspartate aminotransferase, ALT: alanine aminotransferase; LDH: lactate dehydrogenase; γ-GTP: γ-glutamyl transpeptidase

^a Number of animals whose urinary protein level exceeded 300 mg/dl was counted.

Table 3: incidences of selected histopathological lesions in the rats exposed to CTC vapor by inhalation for 2 yr

	Male				Female
Group name	Control	5 ppm	25 ppm	125 ppm	Control	5 ppm	25 ppm	125 ppm
No. of animals examinated	50	50	50	50	50	50	50	50
Neoplastic lesions – liver
Hepatocellular adenoma	0	1	1	21**	0	0	0	40**
Hepatocellular carcinoma	1	0	0	32**	0	0	3	15**
(Metastasis to lung)	(0)	-	-	(4)	-	-	(0)	(1)
Hepatocellular tumors ^a	1	1	1	40**	0	0	3	44**
Pre-neoplastic lesions – liver
Altered cell foci	14	12	13	16	3	4	24^##	ND
Clear cell foci	10	9	7	3	2	2	14^##	ND
Acidophilic cell foci	2	1	5	3	0	0	16^##	ND
Basophilic cell foci	1	3	2	12^##	0	1	6^#	ND
Mixed cell foci	2	3	1	2	1	1	4	ND
Non-neoplastic lesions – liver
Fatty change	4	7	39^##	49^##	6	7	49^##	46^##
Fibrosis	0	0	43^##	2	0	0	45^##	0
Cirrhosis	0	0	1	40^##	0	0	2	50^##
Non-neoplastic lesions – kidney
Chronic progressive nephropathy	49 (1.8)	49 (2.0)	50^# (2.2)	49^## (2.5)	44 (1.3)	45 (1.2)	49^## (1.7)	50^## (2.7)
Non-neoplastic lesions – lung
Uremic pneumonia	1	5	2	19^##	0	0	1	4^#
Non-neoplastic lesions – nasal cavity
Eosinophilic globules	43 (1.0)	47 (1.0)	50^## (1.5)	47^## (1.7)	39 (1.0)	49^## (1.3)	50^## (1.5)	50^## (1.9)

Note. Values indicate number of animals bearing lesion. The values in parentheses indicate the average of severity grade index of the lesion.  Grade: 1 slight, 2 moderate, 3 severe.

Significant difference indicated by *p .05 and **p 0.01 by Fisher exact test; ^#p .05 and ^##p 0.01 by chi-square test.

ND: altered cell foci were not detected, since almost all area of the liver was occupied by hepatocellular tumors.

^a Hepatocellular tumors include hepatocellular adenoma and hepatocellular carcinoma.

Conclusions:

Under the test conditions, the NOAEC for carcinogenicity in rats was 32 mg/m3.

Executive summary:

Carcinogenicity and chronic toxicity of CTC were examinated by inhalation exposure of 50 F344 rats of both sexes at 0, 5, 25, and 125 ppm (v/v) (= 0, 32, 160, 800 mg/m³) for 6h/day, 5days/wk, for 104 wk (OECD 453, GLP study).

Incidence of hepatocellular adenomas and carcinomas in rats of both sexes were significantly increased dose-dependently. Hepatocellular carcinomas and cirrhosis significantly occured in the 125 -ppm-exposed rats of both sexes. Hepatocellular carcinomas metastasized to the lung. Survival rates were decreased in the 125 -ppm-exposued rats of both sexes in association with decreased body weights. The decrased survival rates were considered to be causally related to both various tumors including hepatocellular carcinomas and severe chronic progressive nephropathy in rats.

In the 25 ppm exposed groups no statistically significant rise in carcinogenic incidences were noted, though 3 cases of hepatocellular carcinomas and increased incidences of hepatic altered cell foci were reported for the 25 -ppm-exposed females.

In the 5 ppm groups none of the carcinogenic parameters were significantly raised.

The study provided clear evidence of carcinogenicity for CTC in the high dose group. A cytotoxic-proliferative mode of action for CTC -induced hepatogenesis was suggested.

Under these experimental conditions, the NOAEC for CTC carcinogenicity in rats was 32 mg/m3.

Reference 2

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically sound study, GLP conform, limited reporting of experimental data

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Species:

mouse

Strain:

other: Crj:BDF1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4 weeks
- Weight at study initiation: 23.4 +/- 0.8 g (males), 19.8 +/- 0.7 g (females)
- Housing: individually in stainless steel wire hanging cages (100 mm x 120 mm x 120 mm). Randomisation procedure for allocation
- Diet (e.g. ad libitum): y-irradiation-sterilized commercial pellet died, ad libidum
- Water (e.g. ad libitum): sterilized water, ad libidum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23° +/- 2 °C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 12 +/- 1 air changes/h
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

not specified

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Source and rate of air: airflow containing CTC prepared by a vaporization technique.
- System of generating particulates/aerosols: saturated vapor-air mixture generated by bubbling clean air through CTC liquid at 26°C and by
cooling it through a thermostatted cindenser at 18°C
- Temperature, humidity, pressure in air chamber: see "details on test animals and environmental conditions"

OTHER:
Four inhalation exposure chambres of 3700 L volume. Each exposure chamber accomodated 100 individual cages for 50 males and 50
females.
Chamber concentrations of CTC were monitored by GC every 15 min and mantained constant.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Two years

Frequency of treatment:

6h/day, 5 days/week, for 104 weeks

Dose / conc.:

0 ppm (analytical)

Remarks:

0 mg/m³

Dose / conc.:

5 ppm (analytical)

Remarks:

32 mg/m³

Dose / conc.:

25 ppm (analytical)

Remarks:

160 mg/m³

Dose / conc.:

125 ppm (analytical)

Remarks:

801 mg/m³

No. of animals per sex per dose:

50 males and 50 females per concentration

Control animals:

yes

Details on study design:

- Dose selection rationale: the lowest exposure concentration of 5 ppm was selected at an environmentally relevant level in consideration of the occupational exposure limit value of 5 ppm of CTC (ACGIH, 2001). The highest concentration of 125 ppm was selected not to exceed the maximum tolerated dose (MTD) based on a 13-wk inhalation study

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: one per week for the first 14 wk, and every 2 wk thereafter
- How many animals: 50 males/50 females per group (no. reduced in the higher dose groups due to deaths during the course of the study)

FOOD CONSUMPTION: Yes
- Time schedule for examinations: one per week for the first 14 wk, and every 2 wk thereafter

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


OPHTHALMOSCOPIC EXAMINATION: No data


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after overnight fasting at the end of the 2-yr exposure period.
- Anaesthetic used for blood collection: Yes (ether)
- Study was conducted according to OECD 452, but haematologic parameters were not reported in the present publication

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after overnight fasting at the end of the 2-yr exposure period.
- How many animals: 35 males/26 females (control group), 36 males/24 females (5 ppm), 25 males/10 females (25 ppm), 1 male/1 female
(125 ppm)
- Study was conducted according to OECD 452, but only the parameters shown in table 2 were reported in the present publication


URINALYSIS: Yes
- Time schedule for collection of urine: during the last week of the 2 year testing period
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- No. of animals tested: unclear
- Study was conducted according to OECD 452, but no urinary parameters were reported in the present publication.

NEUROBEHAVIOURAL EXAMINATION: No data


OTHER:
Mortality:
- Time schedule: daily

Relative organ weights (liver and adrenal gland)
- How many animals: 35 males/26 females (control group), 36 males/24 females (5 ppm), 25 males/10 females (25 ppm), 1 male/1 female
(125 ppm)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all organs that are outlined in OECD 452, but only results on lung, liver, kidney and adrenal glands were discussed in detail in the present publication
HISTOPATHOLOGY: Yes, all organs that are outlined in OECD 452, but only results on lung, liver, kidney and adrenal glands were discussed in detail in the present publication

Statistics:

- Incidence of neoplastic lesion: Peto's test (difference from the clean air-exposed group: Fisher's exact test)
- incidence of nonneoplasitv lesions and urinary parameters: chi-square test
- survival curves: Kaplan and Meier's Method
- Difference in survival rate: Fisher's exact test
- Body weight: Dunnett's test
- Body weight: Dunnett's test
- Blood biochemical parameters: Dunnett's test

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:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival rates of 125-ppm-exoposed male mice and both 25- and 125-ppm-exposed female mice were significantly decreased as compared with those of the clean air-exposed groups. At the end of the 2-yr exposure period, the survival rates of 0-, 5-, 25-, and 125-ppm exposure mice were 70, 72, 50 and 2% in males and 52, 48, 20 and 2% in females.
The incidences of palpable liver masses, which first appeared at wk 43 in a male and wk 41 in a female, were increased in 125-ppm-exposured mice.

BODY WEIGHT AND WEIGHT GAIN:
Growth rates at 25- and 125-ppm-exposed male and female mice were significantly retarded along the time course of the 2-yr exposure period as compared with those of both clean air-exposed and 5-ppm-exposed mice of both sexes.
Terminal body weight was significantly decreased by 32% and 22% in the 25-ppm-exposed males and females, and by 39% and 31% in the 125-ppm-exposed mice.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant decrease in any CTC-exposed group of either sex as compared with the clean-air-exposed group


HAEMATOLOGY / CLINICAL CHEMISTRY:
Plasma levels of AST, ALT, LDH, and urea nitrogen were significantly increased in the 25-ppm-exposed mice, but did not attain statistical significance because of the insufficient number of animals.



ORGAN WEIGHTS
An exposure concentration-dependent increase in relative organ weights was observed in the liver and adrenal gland of male and female mice exposed to 25 and 125 ppm (although the number of 125-ppm-exposed mice was insufficient for the statistical comparison).


GROSS PATHOLOGY
Macroscopic examination: the numbers of mice bearing tumor-associated masses in the liver were increased in an exposure concentration-dependent manner, as in the abdominal masses that were observed as a clinical sign during the 2-yr exposure period.


HISTOPATHOLOGY: NON-NEOPLASTIC
Hepatocellular tumors were found to occupy the abdominal cavity and to compress other organs in many male and female mice exposed to 125 ppm. Enlargment of the adrenal gland was observed in 8 and 10 cases out of the 125-ppm-exposed male and female mice, respectively.
Incidences of hepatic altered cell foci were not increased in any CTC-exposed group of either sex. Incidences of deposition of ceroid-like yellow pigment, bile-duct proliferation, and hydropic change in centrilobular hepatocytes were significantly increased in both male and female mice exposed to 25 and 125 ppm.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Macroscopic and microscopic examinations of mice that died before the end of the 2-yr exposure period showed that 44 males and 39 females died of hepatocellular tumors in the 125-ppm-exposed group. 18 males and 14 females also died of hepatocellular tumors in the 25-ppm-exposed mice.Therefore, the decreased survival rates in both the 25- and 125-ppm- exposed mice of both sexes were considered to be causally related to the significantly increased number of mice that died of hepatocellular tumors.
Incidences of hepatocellular adenomas and carcinomas and their cobined incidences were increased in an exposure concentration-dependent manner. The incidence of hepatocellular tumors were significantly increased in the 25- and 125-ppm-exposed mice. The incidence of hepatocellular adenomas in the 5-ppm-exposed females (16.3%) was significantly increased. In addition, the tumor incidence exceeded the upper range of the JBRC historical control data. The combined incidence of hepatocellular adenomas and carcinomas in the 5-ppm-exposed females (18.4%) also exceeded the upper range of the JBRC historical control data. However, the incidence of hepatocellular carcinomas in the 5-ppm-exposed female mice was not significantly increased.
Multiple occurence of hepatoellular tumors was found in the 25- and 125-ppm-exposed mice of both sexes, and the tumor tissues occupied almost all areas of the entire liver tissue in the 125-ppm group.
The incidence of metastasis of hepatocellular carcinomas to the lung were increased in an exposure concentration-dependent manner. The hepatocellular tumors in the 25 and 125-ppm-exposed groups (males and females) occurred earlier than those in the clean air-exposed group
Pheochromocytomas in the adrenal gland occured in an exposuire conc-dependent manner, and those tumor incidences were significantly increased in the 25- and 125-ppm-exposed male mice and in the 125-ppm-exposed female mice.
The adrenal tumor was benign and characterized by m,assive proliferation of adreno-medullary cells compressing the adjacent adreno-cortical tissue.

Although other types of tumors, including malignant lymphoma, uterine histiocytic sarcomas, and pituitary adenomas, were observed in both CTC-exposed and clean-air-exposed rats, there was no exposure-related difference in the incidences of these tumors between any CTC-exposed group and the clean-air-exposed group.

No neoplastic or nonneoplastic lesion were observed in the respiratory tract (including the nasal cavity, pharynx, larynx, trachea and lungs) at the end of the 2-yr exposure period.

Dose descriptor:

NOAEC

Effect level:

32 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: Based on the absence of malignant tumours.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Table 1: terminal body weight and relative organ weights of mice exposed to CTC, vapor by inhalation for 2 yr

	Male				Female
Group name	Control	5 ppm	25 ppm	125 ppm	Control	5 ppm	25 ppm	125 ppm
No. of animals examinated	35	36	25	1	26	24	10	1
Body weight (g)	44.6 ± 8.0	47.1 ± 7.8	30.5 ± 4.0^**	27.0	35.9 ± 4.5	39.3 ± 5.6^*	28.0 ± 2.7^**	24.8
Liver (%)	4.56 ± 1.82	3.78 ± 0.93	17.97 ± 5.54^**	18.71	5.01 ± 1.88	4.32 ± 0.92	14.46 ± 5.82^**	17.52
Adrenal gland (%)	0.025 ± 0.007	0.026 ± 0.020	0.048 ± 0.017^**	0.052	0.039 ± 0.01	0.037 ± 0.008	0.048 ± 0.008^*	0.097

Note: values were represented as mean ± SD. Significant difference indicated by *p .05 and **p 0.01 by Dunnett’s test.

Table 2: blood biochemical and urinary parameters of mice exposed to CTC vapor by inhalation for 2 yr

	Male				Female
Group name	Control	5 ppm	25 ppm	125 ppm	Control	5 ppm	25 ppm	125 ppm
Blood biochemistry
No. of animals examined	22	28	19	3	39	43	39	1
AST (IU/L)	60 ± 23	78 ± 56	109 ± 120*	222 ± 93**	143 ± 166	144 ± 139	208 ± 318	144
ALT (IU/L)	17 ± 6	21 ± 13	28 ± 11**	92 ± 38**	33 ± 16	39 ± 24	62 ± 61**	70
LDH (IU/L)	257 ± 81	380 ± 518	350 ± 130	302 ± 41	440 ± 666	324 ± 266	823 ± 1757**	289
γ-GTP (IU/L)	31.6 ± 7.2	35.4 ± 17.0	39.5 ± 11.5*	74.9 ± 52.9	17.9 ± 2.8	17.2 ± 3.7	29.2 ± 16.9**	60.2
Urea nitrogen	31.5 ± 24.1	23.6 ± 4.2	47.6 ± 26**	40.1	16.3 ± 2.6	16.5 ± 2.9	27.7 ± 11.3**	70.2

Note: values were represented as mean ± SD. Significant difference indicated by *p .05 and **p 0.01 by Dunnett’s test.

AST: aspartate aminotransferase, ALT: alanine aminotransferase; LDH: lactate dehydrogenase; γ-GTP: γ-glutamyl transpeptidase

Table 3: incidences of selected histopathological lesions in the mice exposed to CTC vapor by inhalation for 2 yr

	Male				Female
Group name	Control	5 ppm	25 ppm	125 ppm	Control	5 ppm	25 ppm	125 ppm
No. of animals examinated	50	50	50	50	50	49^a	50	49^a
Neoplastic lesions – liver
Hepatocellular adenoma	9	10	27^**	16	2	8*	17^**	5
Hepatocellular carcinoma	17	12	44^**	47^**	2	1	33^**	48^**
(Metastasis to lung)	(3)	(3)	(10)	(14)	(1)	(0)	(4)	(8)
Hepatocellular tumors ^b	24	20	49^**	49^**	4	9	44^**	48^**
Neoplastic lesions – adrenal gland
Pheochromocytoma: benign	0	0	16^**	31^**	0	0	0	22^**
Pre-neoplastic lesions – liver
Altered cell foci	4	7	1	1	1	0	0	2
Clear cell foci	2	6	0	1	1	0	0	0
Acidophilic cell foci	1	1	1	0	0	0	0	1
Basophilic cell foci	1	1	0	0	0	0	0	1
Non-neoplastic lesions – liver
Deposit of ceroid	2	1	36^##	36^##	0	0	28^##	35^##
Proliferation: bile duct	0	0	19^##	22^##	0	0	5^#	9^##
Hydropic change: centribular	1	0	8^#	9^##	1	0	13^##	12^##

Note. Values indicate number of animals bearing lesion. Significant difference indicated by *p .05 and **p 0.01 by Fisher exact test; ^#p .05 and ^##p 0.01 by chi-square test.

^a Number of mice examined was 49 instead of 50, because one mouse accidentally died.

^b Hepatocellular tumors include hepatocellular adenoma and hepatocellular carcinoma.

Conclusions:

Under the test conditions, the NOAEC for carcinogenicity in mice was 32 mg/m3.

Executive summary:

Carcinogenicity and chronic toxicity of CTC were examinated by inhalation exposure of 50 BDF1 mice of both sexes at 0, 5, 25, and 125 ppm (v/v) (= 0, 32, 160, 800 mg/m³) for 6h/day, 5days/wk, for 104 wk. Incidence of hepatocellular adenomas and carcinomas and adrenal pheochromocytomas in both sexes were significantly increased dose-dependently. Hepatocellular carcinomas were induced in mice of both sexes at 25 and 125 ppm, and hepatocellular adenomas occured in females at 5 ppm without any degenerative or necrotic change in hepatocytes. Hepatocellular carcinomas metastasized to the lung. Survival rates were decreased in the 125 -ppm-exposued rats of both sexes and in the 25 -ppm-exposed females, in association with decreased body weights. The decreased survival rates were considered to be causally related to various tumors including hepatocellular carcinomas in rats and mice.

In the 5 ppm groups no significant increase in carcinomas was found. In the female group at 5 ppm an increased number of hepatocellular adenomas was reported.

The study provided clear evidence of carcinogenicity for CTC. A cytotoxic-proliferative and genotoxic mode of action for CTC -induced hepatogenesis was suggested.

Under these experimental conditions, the NOAEC for carcinogenicity in mice was 32 mg/m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

32 mg/m³

Study duration:

chronic

Species:

other: rats and mice

Organ:

liver

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification

The constituent carbon tetrachloride has an harmonized classification in Category 2 for carcinogenicity (H351) according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available data, no additional classification is proposed for the constituent carbon tetrachloride.

There were no data regarding dermal route for this constituent.

Additional information

For this endpoint, several studies are available by the oral and inhalation route in the carbon tetrachloride dossier. Since the most relevant route of exposure for human is inhalation, the key parameter was assessed mainly for inhalation. Nevertheless, the studies by oral exposure support the results found by inhalation: the liver was the main target organ for both routes of exposure. Cytotoxicity seems to be an important factor in the generation of pre-neoplastic lesions. Taking into account that no genotoxicity has been observed with CTC in vivo, a threshold for carcinogenicity is postulated for CTC.

 

Carcinogenicity, inhalation:

In a combined 2-year combined carcinogenicity/repeated dose study in the rat and the mouse by Nagano et al (2007 B), animals were exposed for 6 h/d, 5 d/week during 104 weeks. The study was performed in compliance with OECD TG 453 and GLP. In both species, at 160 and 800 mg/m³ (25 and 125 ppm), a marked to severe liver toxicity and an increase in incidences for liver carcinomas were determined. At 32 mg/m³ (5 ppm) the number of carcinomas in both species was not raised and only minor toxic effects were apparent. In the female mice group at 32 mg/m³ (5 ppm), an increased number of hepatocellular adenomas was reported at 32 mg/m³ (5 ppm) but only with a low statistical significance (p ≤ .05); in the medium and high dose groups, hepatocellular carcinomas were observed (both in mice and in rats). Therefore a NOAEL of 5 ppm (v/v) (= 32 mg/m³) in both species after chronic exposure to CTC via the inhalation route can be derived from this study. This NOAEL is in accordance with the opinion of the SCOEL in the "Recommendation of the Scientific Expert Group on Occupational Exposure Limits for Carbon Tetrachloride" (SEG/SUM/31, 2009). In conclusion the NOAEC of 32 mg/m³ (= 5 ppm) in rats for the carcinogenic potential of CTC and the corresponding LOAEC of 64 mg/m³ (10 ppm) is considered applicable for the risk assessment.

 

Carcinogenicity, oral: (not reported in this dossier)

Tumorigenic responses of rats, mice and hamsters have been observed after oral administration of CTC. The liver was the main target for appearance of tumours. In the Eschenbrenner study (1946), the NOAEL was 10 mg/kg bw /day in mice (non-guideline protocol). In another study by oral route in rats, the LOAEL was 25 mg/kg bw/d, based on a dose-dependent increase of carcinogenic effects at the double dose (78 weeks exposure). In Syrian hamsters, the LOAEL was found to be 25 mg/kg bw/d after 78 weeks exposure by oral route (Della Porta, 1960).

 

Carcinogenicity, dermal:

No study is available for this route.

 

General conclusion on carcinogenicity

Studies in humans were inadequate to show an association between exposure to carbon tetrachloride and carcinogenicity. None of the human epidemiology studies reported associations to cancer of the liver, which is the main site of carcinogenicity in animal studies. In conclusion, as reported by IARC, there is inadequate evidence in humans for the carcinogenicity of carbon tetrachloride, but there is sufficient evidence in experimental animals for the carcinogenicity of carbon tetrachloride.

For additional information on carbon tetrachloride, its dossier is available on the ECHA website.