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

Endpoint summary

Description of key information

Based on the weight of evidence approach followed in the REACH dossier of CTC :

- Repeated dose toxicity, oral:  NOAEL = 1 mg/kg bw/day for the rat based on minimal to moderate hepatic effects in repeated dose toxicity studies at higher level exposures (increase of hepatic enzymes, histological changes)

- Repeated dose toxicity, inhalation: NOAEC = 32 mg/m³  (= 5 ppm) for the rat, based on liver fatty changes at higher levels in the chronic toxicity study.

- Repeated dose toxicity, dermal: no study available

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically sound study, but no individual data, limited biochemical and histological investigations.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- rats (15 - 16 animals per dose, 4 doses) were treated with orally via gavage.
- dosing regime: 5 days a week
- blood samples were taken from 5 rats of each group at 2, 4, 6, 8, 10 and 12 weeks after study initiation, every rat was selected as blood donor 2 times with an interval of 6 weeks

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TIMCO Breeding Laboratories (Houston)
- Weight at study initiation: 200-250 g (used for second experiment)
- Housing: in group of 5 or 6 animals in stainless-steel cages
- Diet (e.g. ad libitum): Ralston Purina Formulab chow, ad libidum
- Water (e.g. ad libitum): tap water, ad libidum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- Dose preparation: concentrations were adjusted so that required doses were applied at 1 mL/animal
- Vehicle: of Mazola corn oil from Best Foods CPC international (Engleufood Cliffs, N.J., U.S.A.)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

- 12 weeks

Frequency of treatment:

- 5 days a week

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

33 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15-16, males only

Control animals:

yes, concurrent vehicle

Details on study design:

- blood samples were taken from 5 rats of each group at 2, 4, 6, 8, 10 and 12 weeks after study initiation, every rat was selected as blood donor 2 times with an interval of 6 weeks (see remark1 for details)
- 7 to 9 animals were killed after 12 weeks of treatment, the rest of the respective group (6-9 animals) were kept for additional 13 days recovery period (no treatment with substance or vehicle)
- liver and kidneys weighted at final necropsy
- histopathology of liver and kidney samples after final sacrifice (see remark 2)

Positive control:

- no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
Time schedule for examinations: twice weekly



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


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


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: No


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after sacrifice
- Animals fasted: No
- How many animals: 5 per group
- Parameters checked in table 1 were examined.


URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:


OTHER: relative weight of organ/bodyweight determined for liver and kidneys

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes, liver and kidney slices

Statistics:

- Students paired and unpaired t-test
- one way ANOVA

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- body weight development was reported only graphically: significant decrease only in the high dose group (becoming significant from day 30)

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:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: - biochemistry: moderate increase in SDH levels - histological findings: mild centrilobular vacuolization

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: - absence of significant effects

Critical effects observed:

not specified

- table 2: Clinical Chemistry Data from Subchronic Study

Week of evaluation after study initiation	Daily dose of CCl4 (mg/kg)	Total dose of CCl4 (mg/kg)	OCT activity (mmol CO2/ml)	SDH activity (IU/ml)	GPT activity (IU/ml)	BUN (mg/dl)
2	0	0	43 ± 17	2.9 ± 0.7	23 ± 1	ND
	1	10	44 ± 13	8.3 ± 4.0	21 ± 1	ND
	10	100	31 ± 10	7.3 ± 1.5	25 ± 2	ND
	33	330	240 ± 56 ^d	111.5 ± 17.0 ^d	117 ± 28 ^d	ND
4	0	0	21 ± 6	2.6 ± 0.1	22 ± 1	15.6 ± 1.5
	1	20	18 ± 4	ND	23 ± 2	17.8 ± 1.2
	10	200	26 ± 3	2.9 ± 0.3	22 ± 1	18.5 ± 1.2
	33	660	140 ± 29 ^d	90.2 ± 14.7 ^d	75 ± 21 ^d	18.4 ± 0.5
6	0	0	55 ± 13	2.5 ± 0.5	10 ± 1	16.2 ± 0.2
	1	30	44 ± 5	2.4 ± 0.0	14 ± 2	15.4 ± 0.8
	10	300	40 ± 10	8.8 ± 0.8 d	16 ± 2	15.4 ±1.3
	33	990	251 ± 58 ^d	119.3 ± 16.9 ^d	82 ± 20 ^d	15.0± 0.8
8	0	0	18 ± 3	2.2 ± 0.2	21 ± 1	ND
	1	40	34 ± 9	1.6 ± 0.3	21 ± 1	ND
	10	400	30 ± 4	4.2 ± 1.5	19 ± 2	ND
	33	1320	151 ± 21 ^d	110.6 ± 10.0 ^d	130 ± 41 ^d	ND
10	0	0	28 ± 8	3.5 ± 0.4	18 ± 1	14.8 ± 0.5
	1	50	23 ± 3	2.3 ± 0.6	20 ± 1	16.6 ± 1.0
	10	500	55 ± 10	7.6 ± 2.5 d	23 ± 1	17.6 ± 0.5
	33	1650	148 ± 48 ^d	134.8 ± 15.0 ^d	617 ± 334 ^d	18.2 ± 2.5
12	0	0	45 ± 4	3.2 ± 0.4	20 ± 0.3	16.6 ± 0.7
	1	60	61 ± 12 d	1.9 ± 0.1	19 ± 1	17.1 ± 0.6
	10	600	69 ± 16	8.7 ± 2.0 d	27 ± 2 d	15.3 ± 1.2
	33	1980	247 ± 31 ^d	145.7 ± 57.9 ^d	502 ± 135 ^d	16.2 ± 0.8
14 ( 2weeks recovery phase)	0	0	169 ± 33	4.7 ± 0.5	20 ± 2	15.1 ± 0.5
	1	60	111 ± 25	3.4 ± 0.5	20 ± 1	16.8 ± 0.3
	10	600	169 ± 42	4.3± 0.8d	19 ± 1	16.7 ± 0.4
	33	1980	174 ± 24	6.0 ± 0.8 ^d	40 ± 7 ^d	14.6 ± 2.0

ND: not determined

d: significantly different from control at p0.05, paired I test

e: significant increase with increasing dose at p0.05, analysis of variance

- table 3: Severity of Liver Lesions in the Subchronic Studies

Time point of sacrifice after study initiation	Daily dose of CCl4 (mg/kg)	Lipid vacuolization	Nuclear and cellular polymorphism	Bile duct hyperplasia	Periportal fibrosis
12 weeks	0	0.0 + 0.0	0.0 + 0.0	0.0 + 0.0	0.0 + 0.0
	1	0.0 + 0.0	0.0 + 0.0	0.0 + 0.0	0.0 + 0.0
	10	3.7 + 0.7	3.5 + 0.3	0.0 + 0.0	0.0 + 0.0
	33	4.0 + 0.4	5.7 + 0.7	4.0 + 0.6	3.7 + 0.7
14 weeks	0	0.0 + 0.0	0.0 + 0.0	0.0 + 0.0	0.0 + 0.0
	1	0.0 + 0.0	0.0 + 0.0	0.3 + 0.3	0.0 + 0.0
	10	2.9 + 1.0	0.0 + 0.0	0.0 + 0.0	0.0 + 0.0
	33	1.4 + 0.7	2.6 + 0.7	3.4 + 0.8	2.9 + 0.8

Extent of lesions was graded on a scale of 0 (absent) to 8 (mean values of 7 – 9 rats)

Conclusions:

The present study (Bruckner 1986) presents for CTC a LOAEL of 10 mg/kg bw in rats for subchronic repeated dose toxicity via oral exposure (12 weeks, treatment 5 d/week)

Executive summary:

The present study (Bruckner 1986) presents for CTC a LOAEL of 10 mg/kg bw in rats for subchronic repeated dose toxicity via oral exposure (12 weeks, treatment 5 d/week). Basis are biochemical parameters indicative for liver and kidney injury (moderate increase in SDH levels) and histological findings (mild centrilobular vacuolization). The corresponding NOAEL for subchronic oral toxicity is 1 mg/kg bw. The repeated dose toxicity of CTC (cited as carbon tertrachloride in the report) was evaluated in a 12 week oral toxicity study in rats following no official guideline. Male Sprague Dawley rats were treated with the test substance by oral gavage at 5 days a week. Approximately half of the animals of each dose group were killed at the end of the treatment period while the other half was kept for a recovery period of 13 days without any treatment before sacrifice. Blood samples were taken from 5 rats of each group at 2, 4, 6, 8, 10 and 12 weeks after study initiation, every rat was selected as blood donor 2 times throughout the study with an interval of 6 weeks. Three parameters of liver injury (OCT activity, SDH activity and GPT activity) and one parameter for kidney injury (blood urea nitrogen (BUN)) were determined. Liver and kidney lesions were analyzed histologically and graded with a scoring system. Body weights were recorded and relative organ weights of liver and kidney determined. No mortality was reported. Liver toxicity and nephrotoxicity parameters were not elevated for the two lower dose groups except for the SDH value in the mid dose group after 6 weeks and at the end of the treatment phase (fully reversible after the recovery period). In the high dose group lipid vacuolization, nuclear and cellular pleomorphism, bile duct hyperplasia and periportal fibrosis were apparent after the treatment period and only partly reversible within the recovery period). In the mid dose group (10 mg/kg bw) only lipid vacuolization was apparent and partly reversible within the recovery period. Based on these findings a LOAEL of 10 mg/kg bw (rat, subschronic (12 weeks), oral) can be derived. The corresponding NOAEL is 1 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

worst-case value used in the REACH dossier of CTC

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Remarks:

Combined Chronic Toxicity/ Carcinogenicity study

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

Limit test:

no

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:

not specified

Vehicle:

other: unchanged

Details on inhalation exposure:

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 (nominal)

Dose / conc.:

5 ppm (nominal)

Remarks:

(32 mg/m3)

Dose / conc.:

25 ppm (nominal)

Remarks:

(160 mg/m3)

Dose / conc.:

125 ppm (nominal)

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 453, 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 453, 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 453, 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 453, 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 453, 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

Description (incidence and severity):

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.

Mortality:

mortality observed, treatment-related

Description (incidence):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 effects observed

Description (incidence and severity):

No significant decrease of food consumption in any CTC-exposed group of either sex as compared with the clean-air-exposed group.

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

Description (incidence and severity):

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 findings:

effects observed, treatment-related

Description (incidence and severity):

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).

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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 pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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 sexes 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 in the male and female of dead rats exposed to 125 ppm, respectively. Incidences of hepatocellular adenomas and carcinomas and their combined incidences 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, mononuclear 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.

Key result

Dose descriptor:

LOAEC

Effect level:

5 ppm

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

Remarks on result:

other: The relevance of this LOAEC is questionnable.

Critical effects observed:

not specified

See in endpoint 7.7 for the tables

Conclusions:

The study Nagano (2007) presents a LOAEC of 5 ppm (32 mg/m³) in rats for chronic (2 yrs) repeated dose toxicity via the inhalation exposure (treatment 6 h/d, 5 d/wk, 2 yrs)

Executive summary:

The study Nagano (2007) presents a LOAEC of 5 ppm (32 mg/m³) in rats for chronic (2 yrs) repeated dose toxicity via the inhalation exposure (treatment 6 h/d, 5 d/wk, 2 yrs). The LOAEC of 5 ppm is based on raised urinary protein levels in the low dose groups combined with rising incidence of nephrotoxicity in the higher dose groups.

Chronic toxicity of CTC was examined according to OECD guideline 453. F344 rats of both sexes were exposed by inhalation to 0, 5, 25, or 125 ppm (v/v) (= 0, 32, 160 or 800 mg/m³) CTC vapor for 2 yrs (6 h/d and 5 d/wk). Body weight development, food consumption and clinical signs were monitored during te course of the study. At the end of treatment, urinary parameters, haematology and clinical chemistry parameters were investigated. Furthermore, all animals underwent full macroscopic and microscopic pathological evaluation.

In the low dose group (5 ppm) neither reduced body weight gain nor reduced survival were obvious. As sole adverse effects raised urinary protein levels were detected for both sexes and a raised severity of eosinophilelic globules in the nasal cavity. In the mid dose group (25 ppm) had no significantly reduced survival rates and showed reduced body weight gain only in the last 20 to 30 weeks resulting in a decreased of about 10 % in the terminal body weights compared to controls. For the high exposure levels at 125 ppm the survival rates were significantly reduced along with a reduced body weight gain, significant after 13 wks (males) and 58 wks (females). The animals died either of various tumors including hepatocellular tumors or of chronic progressive nephropathy (CPN). Most of the animals (> 80 %) dying of CPN had hepatocellular tumors as well. Lung, liver and kidney weights were raised dose dependently in the both the mid dose and the high dose groups (significant for the mid dose groups, statistical significance not reached for the high dose group due to low survivor numbers). Biochemical parameters indicative of liver toxicity (AST, ALT, Urea nitrogen and for some groups also LDH and gamaGTP) were raised dose dependently in the mid dose and the high dose group, the effects being statistically significant were enough animals survived. In addition to these effects, in the mid and the high dose groups altered cell foci, fatty change, fibrosis and cirrhosis in the liver, raised severity of chronic progressive nephropathy uremic pneumonia (only high dose groups) and severity of eosinophilic globules in the nasal cavity were reported.

Carcinogenic effects are also discussed under chapter 7.7 carcinogenicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

32 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Carcinogenicity and chronic toxicity study ; weight-of-evidence approach value used in the REACH dossier of CTC

Organ:

kidney

liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Remarks:

Combined Chronic Toxicity/ Carcinogenicity study

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

Limit test:

no

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:

not specified

Vehicle:

other: unchanged

Details on inhalation exposure:

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 (nominal)

Dose / conc.:

5 ppm (nominal)

Remarks:

(32 mg/m3)

Dose / conc.:

25 ppm (nominal)

Remarks:

(160 mg/m3)

Dose / conc.:

125 ppm (nominal)

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 453, 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 453, 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 453, 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 453, 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 453, 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

Description (incidence and severity):

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.

Mortality:

mortality observed, treatment-related

Description (incidence):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 effects observed

Description (incidence and severity):

No significant decrease of food consumption in any CTC-exposed group of either sex as compared with the clean-air-exposed group.

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

Description (incidence and severity):

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 findings:

effects observed, treatment-related

Description (incidence and severity):

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).

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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 pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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 sexes 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 in the male and female of dead rats exposed to 125 ppm, respectively. Incidences of hepatocellular adenomas and carcinomas and their combined incidences 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, mononuclear 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.

Key result

Dose descriptor:

LOAEC

Effect level:

5 ppm

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

Remarks on result:

other: The relevance of this LOAEC is questionnable.

Critical effects observed:

not specified

See in endpoint 7.7 for the tables

Conclusions:

The study Nagano (2007) presents a LOAEC of 5 ppm (32 mg/m³) in rats for chronic (2 yrs) repeated dose toxicity via the inhalation exposure (treatment 6 h/d, 5 d/wk, 2 yrs)

Executive summary:

The study Nagano (2007) presents a LOAEC of 5 ppm (32 mg/m³) in rats for chronic (2 yrs) repeated dose toxicity via the inhalation exposure (treatment 6 h/d, 5 d/wk, 2 yrs). The LOAEC of 5 ppm is based on raised urinary protein levels in the low dose groups combined with rising incidence of nephrotoxicity in the higher dose groups.

Chronic toxicity of CTC was examined according to OECD guideline 453. F344 rats of both sexes were exposed by inhalation to 0, 5, 25, or 125 ppm (v/v) (= 0, 32, 160 or 800 mg/m³) CTC vapor for 2 yrs (6 h/d and 5 d/wk). Body weight development, food consumption and clinical signs were monitored during te course of the study. At the end of treatment, urinary parameters, haematology and clinical chemistry parameters were investigated. Furthermore, all animals underwent full macroscopic and microscopic pathological evaluation.

In the low dose group (5 ppm) neither reduced body weight gain nor reduced survival were obvious. As sole adverse effects raised urinary protein levels were detected for both sexes and a raised severity of eosinophilelic globules in the nasal cavity. In the mid dose group (25 ppm) had no significantly reduced survival rates and showed reduced body weight gain only in the last 20 to 30 weeks resulting in a decreased of about 10 % in the terminal body weights compared to controls. For the high exposure levels at 125 ppm the survival rates were significantly reduced along with a reduced body weight gain, significant after 13 wks (males) and 58 wks (females). The animals died either of various tumors including hepatocellular tumors or of chronic progressive nephropathy (CPN). Most of the animals (> 80 %) dying of CPN had hepatocellular tumors as well. Lung, liver and kidney weights were raised dose dependently in the both the mid dose and the high dose groups (significant for the mid dose groups, statistical significance not reached for the high dose group due to low survivor numbers). Biochemical parameters indicative of liver toxicity (AST, ALT, Urea nitrogen and for some groups also LDH and gamaGTP) were raised dose dependently in the mid dose and the high dose group, the effects being statistically significant were enough animals survived. In addition to these effects, in the mid and the high dose groups altered cell foci, fatty change, fibrosis and cirrhosis in the liver, raised severity of chronic progressive nephropathy uremic pneumonia (only high dose groups) and severity of eosinophilic globules in the nasal cavity were reported.

Carcinogenic effects are also discussed under chapter 7.7 carcinogenicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

32 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Carcinogenicity and chronic toxicity study

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

As reported in the REACH dossier of Carbon tetrachloride, in all repeated-dose toxicity studies with carbon tetrachloride in experimental animals, liver toxicity was observed regardless of the species studied. In humans, damage to the liver was observed in acute poisoning cases. Suggestive evidence of hepatotoxicity was also seen in workers exposed to carbon tetrachloride for an extended period of time in the workplace. Thus, carbon tetrachloride is regarded to be a classic hepatotoxic substance and often used as a model compound to study mechanisms of liver toxicity and regeneration. Furthermore, induction of hepatic tumor formation was reported in rats and mice exposed orally or by inhalation to carbon tetrachloride. However, liver tumors were only seen in the presence of overt hepatotoxicity.

Oral Exposure

The only repeated dose toxicity study by oral route reported in this dossier is the one giving the NOAEL which was used in the REACH dossier of CTC.

The repeated dose toxicity of CTC was evaluated in a 12 week oral toxicity study in rats following no official guideline (Bruckner, 1986). Male Sprague Dawley rats were treated with the test substance by oral gavage at 5 days a week. Approximately half of the animals of each dose group were killed at the end of the treatment period while the other half was kept for a recovery period of 13 days without any treatment before sacrifice. Three parameters of liver injury (OCT activity, SDH activity and GPT activity) and one parameter for kidney injury (blood urea nitrogen (BUN)) were determined. Liver and kidney lesions were analyzed histologically and graded with a scoring system. Body weights were recorded and relative organ weights of liver and kidney determined. No mortality was reported. Liver toxicity and nephrotoxicity parameters were not elevated for the two lower dose groups except for the SDH value in the mid dose group after 6 weeks and at the end of the treatment phase (fully reversible after the recovery period). In the high dose group lipid vacuolization, nuclear and cellular pleomorphism, bile duct hyperplasia and periportal fibrosis were apparent after the treatment period and only partly reversible within the recovery period). In the mid dose group (10 mg/kg bw) only lipid vacuolization was apparent and partly reversible within the recovery period. Based on these findings a LOAEL of 10 mg/kg bw/d can be derived and the corresponding NOAEL is 1 mg/kg bw/d.

 Inhalation Exposure

Case reports of acute high-level exposures to carbon tetrachloride vapor or long-term occupational exposure provide evidence of mainly hepatotoxic and, to a lesser extent, nephrotoxic effects of carbon tetrachloride in humans. Observations indicative of an effect on the liver include jaundice, increased serum enzyme levels, and, in fatal cases, necrosis of the liver. A cross-sectional epidemiology study of hepatic function in workers exposed to carbon tetrachloride found suggestive evidence of an effect of occupational carbon tetrachloride exposure on serum enzymes indicative of hepatic effects at workplace concentrations in the range of 6.3-24.5 mg/m³. Serum enzyme activity levels are routinely employed as biochemical indices of this toxicity and serve as reliable indicators of hepatic damage.

 

In the Nagano studies reported in this dossier, rats and mice were exposed by inhalation to 0, 5, 25 or 125 ppm (v/v) (= 0, 32, 160 or 800 mg/m3) CTC vapor for 90 days or 2 years. Body weight development, food consumption and clinical signs were monitored during te course of the study. At the end of treatment, urinary parameters, haematology and clinical chemistry parameters were investigated. Furthermore, all animals underwent full macroscopic and microscopic pathological evaluation.

The NOAEC of 5 ppm (32 mg/m3) from the 2 -year study in rats was used in the CTC dossier for repeated dose toxicity by inhalation. In the low dose group (5 ppm) neither reduced body weight gain nor reduced survival were obvious. As sole adverse effects raised urinary protein levels were detected for both sexes and a raised severity of eosinophilelic globules in the nasal cavity. In the mid dose group (25 ppm) had no significantly reduced survival rates and showed reduced body weight gain only in the last 20 to 30 weeks resulting in a decreased of about 10 % in the terminal body weights compared to controls. For the high exposure levels at 125 ppm the survival rates were significantly reduced along with a reduced body weight gain, significant after 13 wks (males) and 58 wks (females). The animals died either of various tumors including hepatocellular tumors or of chronic progressive nephropathy (CPN). Most of the animals (> 80 %) dying of CPN had hepatocellular tumors as well. Lung, liver and kidney weights were raised dose dependently in the both the mid dose and the high dose groups (significant for the mid dose groups, statistical significance not reached for the high dose group due to low survivor numbers). Biochemical parameters indicative of liver toxicity (AST, ALT, Urea nitrogen and for some groups also LDH and gamaGTP) were raised dose dependently in the mid dose and the high dose group, the effects being statistically significant were enough animals survived. In addition to these effects, in the mid and the high dose groups altered cell foci, fatty change, fibrosis and cirrhosis in the liver, raised severity of chronic progressive nephropathy uremic pneumonia (only high dose groups) and severity of eosinophilic globules in the nasal cavity were reported.

Dermal Exposure

No study available

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

Justification for classification or non-classification

Harmonized classification

The registered substance has no harmonized classification according to the Regulation (EC) No 1272/2008 (CLP).

However, the constituent Carbon tetrachloride has an harmonized classification for repeated dose toxicity STOT-RE 1 (H372**) according to the Regulation (EC) No. 1272/2008, and specific concentrations:

- STOT-RE 1 (H372): C >= 1%

- STOT-RE 2 (H373): 0.2 =< C < 1%

Self-classification:

Based on the available data, no additional classification is proposed for the constituent carbon tetrachloride and based on the rules for classification of mixtures, the registered substance is classified as STOT-RE 1 (H372) and STOT-RE 2 (H373) regarding the specific target organ toxicity after a dose-repeated exposure by inhalation and oral route, respectively, according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

There were no data regarding dermal route for this constituent.