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Repeated dose toxicity: inhalation

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Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
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.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Carbon tetrachloride
EC Number:
200-262-8
EC Name:
Carbon tetrachloride
Cas Number:
56-23-5
Molecular formula:
CCl4
IUPAC Name:
tetrachloromethane
Details on test material:
- Name of test material (as cited in study report): Carbon Tetrachloride
- Analytical purity: analytical grade (purity > 99.8%
- Impurities (identity and concentrations): water < 0.01%, chlorine < 0.00005%, non volatile chemicals < 0.0003%

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results of examinations

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.

Effect levels

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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

See in endpoint 7.7 for the tables

Applicant's summary and conclusion

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.

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