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Toxicological information

Carcinogenicity

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Administrative data

Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Carcinogenicity and chronic toxicity in rats and mice exposed to chloroform by inhalation
Author:
Yamamoto S, Kasai T, Matsumoto M, Nishizawa T, Arito H, Nagano K, Matsushima T
Year:
2002
Bibliographic source:
Journal of Occupational Health 44, 283-293

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroform
EC Number:
200-663-8
EC Name:
Chloroform
Cas Number:
67-66-3
Molecular formula:
CHCl3
IUPAC Name:
chloroform
Test material form:
liquid

Test animals

Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals or test system and environmental conditions:
F-344/DuCrj rats of both sexes were obtained at the age of 4 weeks from Charles River Japan Inc., Kanagawa, Japan; animals were quarantined and acclimated for 2 weeks and were housed individually in stainless steel wire hanging cages in stainless steel chambers maintained at 23 +/- 2 °C, 55 +/- 10 % relative humidity, 12 air changes per hour; fluorescent lightning gave a 12 hours light/12 hours dark cycle; animals had free access to a pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo) and sterilised water

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Chloroform vapour-air mixture was produced by bubbling clean air through the liquid chloroform, further diluted with clean air, and supplied to the inhalation exposure chambers by the method and apparatus described by Kano et al. (2002, Journal of Occupational Health 44, 119-124).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
not specified, but reference is given to Kano et al. (2002, Journal of Occupational Health 44, 119-124).
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:10, 30, 90 ppmBasis:nominal conc.
No. of animals per sex per dose:
50 males and 50 females
Control animals:
yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:
Daily observations for clinical signs and mortality; animals were weighed and their food and water consumptions were measured weekly for the first 13 weeks and every 4 weeks thereafter
Sacrifice and pathology:
Animals found dead, in a moribund state or surviving to the end of the 2 year exposure received complete necropsy; urinary, haematological and blood biochemical parameters of all surviving animals were obtained from urine collected at the end of the 2-year exposure period and from blood samples taken at the end of the 2-year exposure period after overnight fasting; The urinary, haematological and blood biochemical parameters examined here were given in the OECD 453 guidelines. All organs were removed, weighed at necropsy and examined for macroscopically visible lesions. The histopathologically examined tissues were described in detail in Katagiri et al, 2001. The tissues for microscopic examination were fixed in 10% neutral buffered formalin, embedded in paraffin, and sections of all tissues and tumours were 5 micrometre thick, and stained with haematoxylin and eosin.
Statistics:
The incidence of non-neoplastic lesions and urinary data were analysed by chi-square test; the incidence of neoplastic lesions was analysed by Peto's test and Fisher's exact test; body weight, food consumption, and haematological and blood biochemical parameters were analysed by Dunnett test

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
no effects observed
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:
no effects observed
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:
There was no statistical difference in the 2-year survival rate between the exposed female groups and the control group, but the survival rate of the control male group significantly decreased as compared to the exposed male groups. The growth rate of the 90 ppm group of both sexes was significantly suppressed over the controls throughout the entire exposure period.Absolute and relative kidney weights of the 90 ppm female group were statistically higher than those of the controls.No statistically increase in the incidence of kidney tumours was seen in the exposed males and females, but a case of renal cell adenoma occurring in the 90 ppm female group was very rare as compared to the historical laboratory control data. Dose-dependent increases in the occurrence of nuclear enlargement of the proximal tubule and dilatation of the tubular lumen in the kidney were observed with exposure concentrations of 30 ppm and 90 ppm. The severity of chronic progressive nephropathy significantly decreased with increasing exposure concentrations in animals of both sexes.Hepatocellular adenomas were observed in the female rats, but the incidence was not exposure-related. There was a significantly increased incidence of vacuolated cell foci in the 90 ppm female group.Nasal lesions were observed at 5 ppm and above including thickening of the bone and atrophy and respiratory metaplasia of the olfactory epithelium. Lowered incidences of pituitary gland adenoma in the 90 ppm females and myocardial fibrosis in the 90 ppm males were observed.The exposed males exhibited significantly decreased serum levels of triglyceride, phospholipids and creatinine at 30 ppm and above and total cholesterol at 90 ppm. Serum levels of BUN significantly decreased in the three exposed male groups. GOT, GPT and gamma-GTP significantly increased in the exposed males. The exposed females exhibited a significant decrease in triglyceride and an increase in gamma-GTP at 90 ppm. Positive urinary glucose was observed in the 90 ppm male group and in the 10 ppm and above exposed females.

Effect levels

Dose descriptor:
NOAEL
Effect level:
10 ppm
Sex:
male/female
Basis for effect level:
other: Histopathological changes in the kidneys
Remarks on result:
other: Effect type: toxicity (migrated information)

Any other information on results incl. tables

Table 1: Incidence of neoplastic lesions in the rats exposed to chloroform vapour for 104 weeks

Male

Female

Group

Control

10 ppm

30 ppm

90 ppm

Control

10 ppm

30 ppm

90 ppm

Number of animals examined

50

50

50

50

50

50

50

49

Liver

Hepatocellular adenoma

0

0

0

0

1

0

2

1

Kidneys

Renal cell adenoma

0

0

0

0

0

0

0

1

Pituitary gland

Adenoma

22

23

21

17

24

20

18

11*

*: significant difference at p 0.05 by Fisher exact test

Table 2: Incidence of selected non-neoplastic lesions in the liver and kidneys of rats exposed to chloroform vapour for 104 weeks

Male

Female

Group

Control

10 ppm

30 ppm

90 ppm

Control

10 ppm

30 ppm

90 ppm

Number of animals examined

50

50

50

50

50

50

50

49

Liver

Total altered cell foci

11

16

16

18

15

9

20

26

Clear cell foci

4

4

5

6

4

1

2

7

Acidophilic cell foci

2

5

2

3

0

1

0

1

Basophilic cell foci

4

6

8

8

7

5

10

4

Mixed cell foci

1

1

1

1

4

2

6

9

Vacuolated cell foci

0

0

0

0

0

0

2

5*

Kidneys

Nuclear enlargement: proximal tubules

0

0

5*

32**

0

0

6*

34**

Dilatation: tubular lumen

0

0

9*

27**

0

0

5*

38**

Chronic progressive nephropathy b) +

3

11*

10*

17**

8

19**

27**

15**

2 +

6

10

24

14

15

7

5

3

3 +

19

15

8

2

14

3

3

1

4 +

19

8

2

1

4

2

0

2

Significant different at p 0.05 (*) and p 0.01 (**) by Chi square test; b) the severity of chronic progressive nephropathy was classfied into four different grades according to the criteria described by Kawai (1980)

Table 3: Serum levels of blood biochemical parameters and urinalysis in rats exposed to chloroform vapour for 104 weeks

Male

Female

Group

Control

10 ppm

30 ppm

90 ppm

Control

10 ppm

30 ppm

90 ppm

Number of animals examined

27

39

36

38

37

35

40

34

Biochemistry

Total protein (g/dL)

6.7

7.1

7.0

6.9

7.0

7.4*

7.3

7.2

Glucose (mg/dL)

162

169

165

154

170

168

164

160

Total cholesterol (mg/dL)

173

164

153

125**

142

131

135

149

Triglyceride (mg/dL)

222

167

146*

87**

191

126

109

94**

Phospholipids (mg/dL)

289

268

241*

196**

280

252

255

271

Creatinine (mg/dL)

0.9

0.6

0.6**

0.7**

0.5

0.5

0.5

0.5

BUN (mg/dL)

28.6

20.6**

18.3**

23.2**

17.9

18.5

18.5

18.6

GOT (IU/L)

67

79

81

98*

128

113

124

144

GPT (IU/L)

21

25*

24

26*

37

40

39

48

gamma-GTP (IU/L)

4

8*

10**

7*

4

4

5

6**

LDH (IU/L)

164

239

179

311

297

245

239*

344

ALP (IU/L)

215

265

283

243

152

133

157

174

Urinalysis

Glucose

0/27

0/39

2/37

20/39**

2/41

8/37*

29/41**

24/34**

Occult blood

3/27

6/39

4/37

10/39

2/41

1/37

4/41

2/34

Values shown are the means for each group and the urinalysis data indicate the number of animals having positive glucose or occult blood/total number of animals examined. Significant differences at p 0.05 (*) and p 0.01 (**) by Dunnett's test for biochemistry and by Chi-square test for urinalysis. BUN: blood urea nitrogen; GOT: glutamate oxaloacetate transminase; GPT: glutamate pyruvate transaminase: ALP: alkaline phosphatase; gamma-GTP: gamma-glutamyl transpeptidase; LDH: lactate dehydrogenase

Applicant's summary and conclusion

Conclusions:
Inhalation exposure of male and female F-344 rats to 10, 30 and 90 ppm chloroform vapour did not result in any statistically significant, exposure-related increase in the incidence of liver and kidney tumours. Exposure to chloroform at 30 and 90 ppm induced nuclear enlargement of the proximal tubules and dilatation of the tubular lumen without development of renal tumours. No significantly increased incidence of histopathological lesions in the liver was found. Slight but significant increases in the serum levels of GPT and gamma-GTP occurred in exposed male rats from 10 ppm exposure concentration. Although these two biomarkers are biologically significant, (indicative of liver cell necrosis and regeneration), they were not considered to determine a LOAEL since no dose-relationship was observed.
Executive summary:

A combined chronic toxicity/carcinogenicity study was carried out with chloroform using female and male F-344 rats receiving inhalation exposure to chloroform vapours of 10, 30 and 90 ppm during a period of 2 years. The study was in accordance with the method B.33 suggested by the European Commission with minor restrictions.

The growth rate of female and male rats exposed to 90 ppm chloroform vapour was suppressed over the controls. Nasal lesions were observed at exposure concentrations of 10 ppm and above. The 2 -year inhalation exposure to 10, 30 or 90 ppm did not result in any significant increase in the incidence of liver and kidney tumours in both sexes. Exposure to chloroform at 30 or 90 ppm induced nuclear enlargement of the proximal tubules and dilatation of the tubular lumen without development of renal tumours. No significantly increased incidence of histopathological lesions in the liver was found. The NOAEL value for the histopathological changes in the kidneys was found to be 10 ppm.