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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-compliant study,good qualitiy study, Klimisk 1 even no data on GLP status.

Data source

Reference
Reference Type:
publication
Title:
Thirteen-week inhalation toxicity of carbon tetrachloride in rats and mice
Author:
Nagano K, Umeda, Saito, Nishizawa, Ikawa, Arito H, Yamamoto S, Fukushima S;
Year:
2007
Bibliographic source:
J Occup Health 2007; 49: 249-259

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CCl4
- Analytical purity: > 99.8% (Wako Pure Chemical Industries, Ltd., Osaka, Japan)
- Impurities (identity and concentrations): no impurities detected with GC and IR-spectrometry

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Japan, Inc. (Kanagawa)
- Housing: individually in stainless steel wire hanging cages (150 mm x 216 mm x 176 mm)
- Diet (e.g. ad libitum): sterilized commercial pellet diet (CRF-1, Orienal Yeast Co., Tokyo) ad libidum
- Water (e.g. ad libitum): sterilized drinking water, ad libidum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24.5 - 25.1°C
- Humidity (%): 52.3 - 57.1%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not applicable, vapour of a volatile organic solvent
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chamber (4.3 cbm, glass and stainless steal) maximal load: 50 stainless steal wire mesh cages
- Method of holding animals in test chamber: stainless steal wire mesh cages, animals individualIy housed
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating vapor: vapor-air mixture was generated by bubbling clean air through liquid of CTC in a temperature-regulated glass flask, and by cooling it through a thermostatted condensor.The airflow containing the saturated vapor was diluted with clean air and then warmed in a thermostatted circulator which served to stabilize the vapor concentration by complete gasification of CTC and by buffering the pressure difference between the glass flask and the inhalation chamber. The vapor-air mixture flow rate was regulated with a flow meter, further diluted with humidity- and temperature-controlled clean air in a spiraling line mixer, and then supplied to the inhalation exposure chamber.
- Temperature, humidity, pressure in air chamber: 22 ± 2 °C, 55 ± 10 %, - 50 to - 150 Pa
- Air flow rate: 860 l/min
- Air change rate: 12 changes/h
- Method of particle size determination: not applicable (vapor of volatile solvent)
- Treatment of exhaust air: not reported


TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of CTC vapor were monitored by gas chromatography once every 15 min.
- Samples taken from breathing zone: yes


VEHICLE
- No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- gas chromatography, sampling every 15 min during exposure, no further details given
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hrs/day
5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
64, 192, 576, 1728 and 5184 mg/m³ (10, 30, 90, 270 and 810 ppm)
Basis:
analytical conc.
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: the lowest inhalation concentration of 64 mg/m³ (10 ppm) was selected in consideration of the validity of the present occupational exposure limit (OEL) value for CCld
- Rationale for animal assignment (if not random): not reported
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not reported
Positive control:
Not applied

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes (see Table 1)
- Time schedule for examinations: once a week


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes


FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the last week of the 13-wk exposure period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals: no data
- Parameters checked in table No. 2 were examined.
- further not-reported parameters might have been analyzed, as the authors reference OECD 413


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the last week of the 13-wk exposure period
- Animals fasted: Yes (overnight)
- How many animals: no data
- at least Parameters shown in table No. 2 were examined.
- further not-reported parameters might have been analyzed, as the authors reference OECD 413


URINALYSIS: Yes
- Time schedule for collection of urine: in the last week of the 13-wk exposure period
- Animals fasted: No data
- Parameters shown in table No. 2 were examined.
- further not-reported parameters might have been analyzed, as the authors reference OECD 413


NEUROBEHAVIOURAL EXAMINATION: No

RELATIVE ORGANS WEIGHT (see Table 1)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see table No. 3)
Other examinations:
The livers of 810 ppm-exposed and control groups of male rats were sectioned for examination of hepatic altered cell foci, a preneoplastic lesion, by immunohistochemical staining with anti-GST-P
Statistics:
Body weights, organ weights, and haematological and blood biochemical parameters: Dunnett's multiple comparison test with a p-value of 0.05
Pathological examinations and urinary parameters: Chi-square test with a p-value of 0.05

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No death occured in any of the CCl4-exposed rats groups of either sexes
No CCl4-related clinical signs except for soiling of the perigenital and perinasal areas in male and female rats exposed to 270 ppm and above
BODY WEIGHT AND WEIGHT GAIN
Terminal body weight significantly decreased onlyin the 810-ppm exposed male rats


FOOD CONSUMPTION
No data

HAEMATOLOGY
White red blood cell counts significantly decreased in the 810 ppm-exposed rats of both sexes
Hemoglobin and hematocrit significantly decreased in male and female rats at 90 ppm and above

CLINICAL CHEMISTRY
Increase of AST, ALT and LDH in male rats exposed to 90 ppm and above and in female rats exposed to 30 ppm and above (although the increases in
AST and LDH in the 90 ppm-exposed male rats were not statistically significant)
ALP significantly increased in both sexes at 90 ppm and above
Total bilirubin significantly increased in male rats at 810 ppm and in female rats at 270 and 810 ppm.

URINALYSIS
Urinary protein significantly increased in male rats exposed to 270 and 810 ppm and in female rats exposed to 90 ppm and above.



ORGAN WEIGHTS
Significant increase in relative organ weight in livers of male rats exposed to 10 ppm and above and of female rats exposed to 30 ppm and above.
Relative kidney weights significantly increased in male rats exposed to 10 ppm and above, except 30 ppm, and of females exposed to 90 ppm and above.
Biologically meaningful increase in the relative kidney weight occured in both male and female rats exposed to 90 ppm and above (because a dose-dependent increase was observed in rats of both sexes exposed to 90 ppm and above and because the statistically increased relative kidney weight of
10 ppm-exposed male rats was attributed to a marginal decrease in the body weight.

GROSS PATHOLOGY
Granular liver surface in one female exposed to 90 ppm, in five males and 10 females exposed to 270 ppm and all male and female rats exposed to
810 ppm.

HISTOPATHOLOGY: NON-NEOPLASTIC
Non significant increase of fatty change (large droplets, greater in diameter than the hepatocellular nuclei) in 2 males and 2 females rats exposed to 10 ppm. Significant increase of fatty change in both male and female rats exposed to 30 ppm and above. Its averaged severity increased in a dose-dependent manner except for the female groups exposed to 270 and 810 ppm.
Significant increase in the incidence of fibrosis in male rats exposed to 270 and 810 ppm and in females at 90 ppm and above.
Cirrhosis in both male and female rats exposed to 270 and 810 ppm (incidence in the 270 ppm-exposed male rats not statistically significant).
Either fibrosis or cirrhosis was accompanied by increased mitosis and nuclear enlargement of hepatocytes, proliferation of bile duct and deposition of ceroid-like yellow pigment in the connective tissue area.
Altered cell foci, characterized by expansile nodules, in male rats exposed to 270 and 810 ppm, and in female rats at 90 ppm and above (incidences in the 270 ppm male rats and in the 90 ppm exposed females were not statistically significant). The altered cell foci were classified into acidophilic, basophilic or clear cell foci.
The cell foci seen in the 810 ppm-exposed male rats were also stained positively with the anti-GST-P anitobody. The altered cell foci were homogeneously composed of GST-P positive hepatocytes.
Localized glomerulosclerosis significantly increased in both male and female rats exposed to 810 ppm.

No histopathological changes observed in the respiratory tract, including the nasal cavity, larynx, trachea or lungs.

Effect levels

Dose descriptor:
LOAEC
Effect level:
64 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: LOAEC refered to the endpoints: -liver fatty change with large droplets in rats (male and female), with increase of relative liver weight (in male rats)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Repeated inhalation exposure of rats of both sexes to CTC vapor at 10 to 810 ppm for 13 wk induced mild to severe hepatoxicity including a preneoplastic lesion, as well as renal and hematological toxicities to a less severe extent. However, inhalation exposure of both sexes to CTC vapor did not produce any histopathological change in the respiratory tracts, including the nasal cavity, larynx, trachea and lungs. The present study confirmed the pathological findings of earlier inhalation studies, by demonstrating the increased liver weight, enhanced release of transaminases into plasma and fatty degeneration, fibrosis and cirrhosis. Inhalation exposure to high levels of CTC for 13 wk induced the hepatic altered cell foci mirrored by GST-P-positively stained hepatocytes as a preneoplastic lesion, while the low levels of CCl4 designated 10ppm for the most sensitive sign as fatty change, cytoplasmic globules and increased liver weight.

Table 1: Terminal body weight, and relative liver and kidney weights of rats exposed to CCl4 vapor by inhalation for 13 wk

Male

Group (ppm)

Control

10

30

90

270

810

No. of animals examined

10

10

10

10

10

10

Body weight (g)

301

296

304

302

289

244

Organ weight

Liver (%)

2.44

2.59**

2.89**

3.27**

3.91**

4.23**

Kidney (%)

0.57

0.61**

0.59

0.63**

0.70**

0.83**

Contd.

Female

Group (ppm)

Control

10

30

90

270

810

No. of animals examined

10

10

10

10

10

10

Body weight (g)

176

178

185

184

175

169

Organ weight

Liver (%)

2.33

2.47

3.19**

4.41**

4.59**

4.76**

Kidney (%)

0.65

0.65

0.65

0.72**

0.83**

0.91**

Values indicate means. Significant difference; +: p 0.05 **: p0.01 by Dunnett's test.

Table 2. Hematological, blood biochemical and urinary parameters of the rats exposed to CCl4 vapor by inhalation for 13 wk

Male

Group (ppm)

Control

10

30

90

270

810

Hematology

No. of animals examined

10

10

10

10

10

10

Red blood cell (106/μl)

10.16

10.27

10.36

10.27

10.01

7.92**

Hemoglobin (g/dl)

17.0

17.1

16.8

16.0**

14.8**

12.0

Hematocrit (%)

48.1

48.3

47.9

45.9**

43.1**

34.7**

Blood chemistry

No. of animals examined

10

10

10

10

10

10

AST (IU/l)

76

81

79

118

459**

1'465**

ALT (IU/l)

25

27

30

67**

303**

465**

LDH (IU/l)

350

353

335

565

739*

642

ALP (IU/l)

266

271

284

294**

497**

1'098**

Total bilirubin (mg/dl)

0.14

0.14

0.11

0.14

0.18

0.48

Urinalysis

No. of animals examined

10

10

10

10

10

10

Protein a

0

0

0

0

7**

10**

Contd.

Female

Group (ppm)

Control

10

30

90

270

810

Hematology

No. of animals examined

10

10

9b

10

10

10

Red blood cell (106/μl)

9.08

9.32

9.67**

9.26

9.06

8.29**

Hemoglobin (g/dl)

16.4

16.8

16.3

15.1**

14.4**

12.8**

Hematocrit (%)

46.0

47.0

46.8

42.8**

41.0**

36.7**

Blood chemistry

No. of animals examined

10

10

10

10

10

10

AST (IU/l)

65

72

100**

233**

364**

897**

ALT (IU/l)

19

25

38**

111**

146**

322**

LDH (IU/l)

294

265

438*

471**

501**

404

ALP (IU/l)

183

204

196

284**

427**

725**

Total bilirubin (mg/dl)

0.17

0.16

0.14

0.17

0.25*

0.36**

Urinalysis

No. of animals examined

10

10

10

10

10

10

Protein a

0

0

0

7**

10**

10**

Values indicate means. Significant difference; *: p 0.05 **: p0.01 by Dunnett's test.AST: aspartate aminotransferase, : alanine aminotransferase, LDH: lactate dehydrogenase, ALP: alkaline phosphatase. a: Number of animals whose urinary protein level exceeded 100 mg/dl was counted. b: blood collection failed for one rat

Table 3. Incidences and severities of selected histopathological lesions in the rats exposed to CCl4 vapor by inhalation for 13 wk

Male

Group (ppm)

Control

10

30

90

270

810

No. of animals examined

10

10

10

10

10

10

Liver
Fatty change: small droplet

0

0

0

0

0

0

Fatty change: large droplet

0

2

(1.0)

10**

(1.1)

10**

(1.5)

10**

(2.0)

10**

(2.1)

Fibrosis

0

0

0

0

10**

10**

Cirrhosis

0

0

0

0

2

10**

Altered cell foci

0

0

0

0

3

10**

Acidophilic cell foci

0

0

0

0

2

7**

Basophilic cell foci

0

0

0

0

1

6**

Clear cell foci

0

0

0

0

0

0

Kidney

Glomerulo­sclerosis:

localized

0

0

0

0

0

10**

Contd.

Female

Group (ppm)

Control

10

30

90

270

810

No. of animals examined

10

10

10

10

10

10

Liver
Fatty change: small droplet

8

(1.0)

8

(1.0)

0

0

0

0

Fatty change: large droplet

0

2

(1.0)

10**

(2.0)

10**

(2.5)

10**

(2.0)

10**

(1.5)

Fibrosis

0

0

0

5**

5**

9**

Cirrhosis

0

0

0

0

10**

9**

Altered cell foci

0

0

0

3

10**

10**

Acidophilic cell foci

0

0

0

1

6**

6**

Basophilic cell foci

0

0

0

1

0

0

Clear cell foci

0

0

0

1

9**

10**

Kidney

Glomerulo­sclerosis:

localized

0

0

0

0

0

10**

Values indicate number of animals bearing lesion. The values in parentheses indicate the average of severity grade index of the lesion. The average of severity grade was calculated with the following equation. Σ (grade x number of animals with grade) / number of affected animals. Grade 1=slight, 2=moderate, 3=severe. Significant difference; *: p 0.05 **: p0.01 by chi-square test

Applicant's summary and conclusion

Conclusions:
The present study (Nagano 2007) presents for CTC a LOAEC of 64 mg/m³ (10 ppm) in rats for subchronic repeated dose toxicity via inhalation exposure (Treatment: 6 h/d, 5 d/week, 13 weeks)
Executive summary:

The present study (Nagano 2007) presents for CTC a LOAEC of 64 mg/m³ (10 ppm) in rats for subchronic repeated dose toxicity via inhalation exposure (Treatment: 6 h/d, 5 d/week, 13 weeks). Basis are organ weight effects and fatty change in liver .

Subchronic toxicity of CTC was examined according to OECD guideline 413. F344 rats of both sexes were exposed by inhalation to 0, 10, 30, 90, 270 or 810 ppm (v/v) CTC vapor for 13 wk (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 anc microscopic pathological evaluation.

In the high exposure levels at 270 and 810 ppm, altered cell foci in the livers, and fibrosis and cirrhosis in the rat liver were observed. Hematoxylin and eosin-stained altered cell foci of rats were recognized as glutathione-S-transferase placental form (GST-P) positive foci, which are preneoplastic lesions of hepatocarcinogenesis. The most sensitive endpoint of CTC-induce toxicity was fatty change with large droplets in rats of both sexes,  as well as increased relative liver weight in male rats. Those endpoints were manifested at 10 ppm and the LOAEL was determined as 10 ppm for the hepatic endpoints in rats. Enhanced cytolytic release of liver transaminase into plasma in rats was observed at medium and high levels of inhalation exposure. Both CTC-induced hepatoxicity and nephrotoxicity were observed, but nephrotoxicity was manifested at higher exposure concentrations than hepatotoxicity.