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Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Safety evaluation of toothpaste containing chloroform III. Long-term study in Beagle dogs
Author:
Heywood R, Sortwell RJ, Noel PRB, Street AE, Prentice DE, Roe FJC, Wadsworth PF, Worden AN, Van Abbé NJ
Year:
1979
Bibliographic source:
Journal of Environmental Pathology and Toxicology 2: 835-851

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
GLP compliance:
no
Limit test:
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:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
Pure bread dogs, initially 18 to 24 weeks old, were housed singly in kennels. All the dogs were clinically examined and inoculated against distemper, canine hepatitis and leptospirosis as well as being dose with piperazine adipate as an anthelmintic. Inoculatin and anthelmintic treatment were repeated annually. The dogs were fed weighed amounts (200 g) of a dry diet at fixed times twice daily. When new food was offered, any residue from the previous meal was removed. From week 300, the daily food ration of dogs considered to be obese was reduced from 400 to 300 g. Water was freely available at all times and fresh milk (200 mL) offered to each dog on weekday mornings for the first 6 months.

Administration / exposure

Route of administration:
oral: capsule
Vehicle:
other: toothpaste base (including peppermint oil and eucalyptol) given orally by gelatin capsule
Details on oral exposure:
Dogs received a dose of chloroform contained in tooth paste for 7 days per week during the main study.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
7.5 years
Frequency of treatment:
6 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:0, 15 and 30 mg/kg body weigth/dayBasis:other: nominal in capsule
No. of animals per sex per dose:
8 males, 8 females in the 30 and 15 mg/kg body weight dose groups and in untreated and alternative non-chloroform toothpaste groups; 16 males, 16 females in the 0 mg/kg body weight dose group
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
From observations made in preliminary studies and to ensure that the upper dose level would reach the threshold for early toxic effects but with many dogs as possible remaining alive and well for at least 7 years

Examinations

Observations and examinations performed and frequency:
Clinical signs: daily; food consumption: daily; water consumption: intermittently; body weight: once per week; opthalmoscopy: 3-month intervals; thorough clinical examination: at least twice yearly; comprehensive range of laboratory investigations: during pre-treatment period, after 6, 13 weeks, 6.9 and 12 months, then every 6 months; serum enzyme studies: in the later stages of the treatment;
Sacrifice and pathology:
Between weeks 395 and 399, each dog still alive received an intravenous injection of sodium pentobarbitone; immediately after killing or as soon as possible in the case of animals found dead, full macroscopic examination was made of all tissues and any abnormalities were noted; principal organs were removed and weighed; small portions of these and a wide range of other tissues along with portions of any lump or tumour were placed in fixative; sections were stained with haemotoxylin and eosin; examination, by electron microscopy, was conducted on liver and kidney sections

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
LOAEL
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: increased serum levels of alanine aminotransferase and increased incidence of aggregations of vacuolated histiocytes forming "fatty cysts" in the liver of treated dogs

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Group mean values in biochemical analyses of different groups treated with chloroform, untreated control groups and groups receiving alternative toothpaste (male and female dogs combined)

Treatment (mg/kg/d)

30

15

Vehicle control

Untreated

Alternative toothpaste

No. of dogs

15

15

27

12

15

Week

374 a)

395 b)

374

395

374

395

374

395

374

395

Urea (mg %)

30

28

32

26

31

31

33

27

34

29

Glucose (mg %)

99

99

100

100

101

98

99

95

102

98

Total serum proteins (g %)

5.9

6.1

6.1

6.2

6.3

6.0

6.3

6.0

6.4

6.1

Albumin

2.9

2.8

3.0

2.8

3.1

2.8

3.2

2.9

3.2

2.9

alpha1 globulin

0.3

0.3

0.3

0.3

0.3

0.4

0.3

0.3

0.3

0.3

alpha2 globulin

0.7

0.8

0.7

0.8

0.7

0.8

0.7

0.7

0.7

0.8

beta globulin

1.6

1.7

1.7

1.7

1.7

1.6

1.6

1.6

1.7

1.6

gamma globulin

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.4

0.6

0.4

A/G ratio

0.98

0.86

0.98

0.87

0.99

0.91

1.04

0.97

1.00

0.94

SAP (KA units)

30

26

20

17

14

16

12

14

10

13

SGPT mU/mL

102

111

66

48

51

128

50

56

57

87

SGOT mU/mL

33

33

27

29

25

32

22

32

25

31

LAP (GR units)

90

72

82

62

73

54

81

56

82

60

Bilirubin (mg %)

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

Erythrocyte cholinesterase (delta pH/h)

0.73

0.73

0.78

0.81

0.77

0.81

0.77

0.82

0.65

0.71

Plasma (delat pH/h)

1.08

0.91

1.00

0.88

1.00

0.87

1.02

0.86

1.05

0.91

gamma GT (mU/mL)

3.6

3.3

2.9

2.5

2.9

2.1

2.5

1.7

2.8

1.8

GDH (mU/mL)

7.2

5.3

6.5

3.9

4.9

4.0

3.9

3.0

4.9

3.0

ICD (B&B units)

11.8

11.4

11.2

10.7

9.9

12.9

9.2

11.7

10.6

12.9

a) end of treatment; b) end of recovery period; A/G: Albumin/Globulin; SAP: Serum Amyloid P component; SGPT: Serum Glutamic Pyruvic Transaminase (also Alanine Aminotransferase); SGOT: Serum Glutamic Oxaloacetic Transaminase (also Aspartate Transaminase); gamma GT (Gamma Glutamyl Transferase); GDH: Glutamate Dehydrogenase; ICD: Isocitrate Dehydrogenase

Table 2: Changes of alanine aminotransferase (ALAT) levels throughout the main study

Treatment (mg/kg/d)

Group mean ALAT (mU/mL)

Pre-reatment

Posttreatment (weeks)

6

13

26

39

52

78

104

130

156

182

208

234

260

286

312

338

364

372

14

19

30 mg/kg/d

24

34 a)

37 b)

58 c)

63 c)

52 c)

73 c)

64 c)

51 c)

76 c)

108 c)

91 c)

80 c)

147 c)

138 c)

128 c)

134 c)

104 c)

102 c)

105 c)

111

15 mg/kg/d

22

29

30

33

39

32

43

45

34 b)

46 b)

61 b)

55 b)

55 a)

95 c)

93 b)

89 c)

73 a)

79 a)

66

53

48

Vehicle control

22

29

29

30

39

29

35

40

22

30

33

40

34

33

65

47

49

50

51

56

128

Alternative toothpaste

28

31

31

33

40

28

37

36

21

30

34

36

35

50

47

52

47

59

57

48

87

Untreated

24

30

30

30

38

27

37

37

21

29

33

30

33

32

48

50

44

50

50

53

56

a) comparison with untreated group; p 0.05; b) comparison with untreated group; p 0.01; c) comparison with untreated group; p 0.001

Table 3: Liver changes (nodules of altered hepatocytes and “fatty cysts”)

Treatment (mg/kg/d)

Sex

No. of dogs examined histologically at end of experiment

No. with nodules

No. with “fatty cysts”

Occasional or minimal

Moderate or marked

30

Male

7

0

1

6

Female

8

4

0

7

15

Male

7

1

0

6

Female

8

1

2

3

Vehicle control

Male

15

0

7

1

Female

12

3

3

0

Untreated

Male

7

1

2

0

Female

5

1

1

0

Alternative toothpaste

Male

8

0

2

0

Female

7

1

0

0

Applicant's summary and conclusion

Conclusions:
This long-term study investigating chronic effects of chloroform applied orally to dogs provided a LOAEL value of 15 mg/kg body weight/day.
Executive summary:

A combined repeated dose and carcinogenicity study was carried out with chloroform orally administered to male and female Beagle dogs over a period of more than 7 years. The study was comparable to the principles set out in the testing guideline for method B.33 suggested by the European Commission with minor restrictions. The chloroform was contained in toothpaste and administered in form of a gelatine capsule.

Chronic effects related to chloroform exposure included a significantly increased level of alanine aminotransferase level in the high dose group throughout the study and in the lower-dose group during the later phase of the study and increased incidence of aggregations of vacuolated histiocytes forming "fatty cysts" in the liver of treated dogs.

A NOAEL value could not be established and the LOAEL value found in this study was 15 mg/kg body weight/day established for female and male dogs together.