(2H)chloroform

Repeated dose toxicity: oral

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

Test material information

Constituent 1

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.