Chloroform

Endpoint summary

Description of key information

A wide range of acute oral LD50 values has been reported for chloroform in mice (36 to 1366 mg chloroform/kg body weight) and rats (450 to 2000 mg chloroform/kg body weight). Although, the mouse has been considered as the most sensitive species, the proposal for classification is based on a rat study (Chu et al. 1980) as requested by the EU and GHS regulation. In this study, the lowest LD50 of 908 mg/kg/body weight was found in male rats. A wide range of LC50 values has also been reported in mice and rats. An assessment can be made by a weight of evidence approach and the LC50 value determined in Bonnet et al. (1980) (9.17 mg/L/6 hours/ chloroform vapours) can be used to derive a 4-hour inhalation LC50 value of 10.5 mg/L. One dermal toxicity study, however non reliable, indicate that dermal application of a single dose of 3.98 g/kg for 24 hours did not cause mortality in rabbits (Torkelson et al. 1976).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Biobreeding Labs, Ottawa, Ontario, 150-200 g, acclimated for 1 week, temperature 21 +/- 2 °C, humidity 40-70 %, caged individually and allowed free access to food and water

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

administration at a constant volume of 5 mL/kg, animals were fasted overnight before dose administration

Doses:

546, 765, 1071, 1500, 2100 mg/kg body weight

No. of animals per sex per dose:

10 males, 10 females

Control animals:

no

Details on study design:

To establish the dose levels to be used in the LD50 studies, groups of two animals were administered test compounds at 250 and 3000 mg/kg and the mortality data were recorded. Then, the main LD50 studies were carried out with doses of 546-2100 mg/kg using 10 female and 10 male rats. Clinical observations were made for 14 days after dosing and a postmortem examination was performed on animals which died during this period. All surviving animals were killed at the end of 14 days and subjected to gross pathologic examination.

Statistics:

Statistical analysis of data was carried out using one-way analysis of variance and the Student-Newman-Keuls test. LD50 values were calculated using the probit analysis (Finney 1952).

Sex:

male

Dose descriptor:

LD50

Effect level:

908 mg/kg bw

95% CL:

750 - 1 082

Sex:

female

Dose descriptor:

LD50

Effect level:

1 117 mg/kg bw

95% CL:

843 - 1 514

Mortality:

See also Table 1: At the highest dose level, 4 males died within 24 hours after dosing and most animals were dead after 4 days post-administration. At lower doses animals died primarily on Day 3 to Day 4 post-administration.

Clinical signs:

other: Common clinical signs consisted of piloerection, sedation, flaccid muscle tone, ataxia, and prostration. Chloroform elicited dacryorrhea in some animals.

Gross pathology:

Increased relative weights of the liver and the kidneys. For lesions see Table 2

Other findings:

A clear trend towards increased cholesterol levels was seen in all treated animals. Inhibition of lactate dehydrogenase activities were seen in all treated groups. A significant decrease in the levels of liver proteins were observed in rats fed chloroform. Microsomal hydroxylase activity of female but not male rats was activated by administration of chloroform. A reduction in lymphocytes with significant decreased was noted in female rats. Decrease in haemoglobin and haematocrit values were observed in males and females fed chloroform.

Table 1: Mortality occurring in the acute oral toxicity study:

Dose (mg/kg)	Sex	Total animals dead*	Survival time (days after oral administration)							LD50 (mg/kg)
			1	2	3	4	5	6	7
546	Male	0	--	--	--	--	--	--	--	M:908(750-1082) F:1117(843-1514)
	Female	1	--	--	1	--	--	--	--
765	Male	5	1	--	1	1	2	--	--
	Female	3	--	--	1	2	--	--	--
1071	Male	6	2	--	2	--	2	--	--
	Female	6	--	2	2	--	--	2	--
1500	Male	9	--	--	5	1	2	1	--
	Female	5	--	1	3	1	--	--	--
2100	Male	10	4	--	3	1	1	1	--
	Female	9	--	--	4	5	--	--	--

*Animals dying before the end of the 14 day observation period

Table 2: Incidence of histological changes of rats which survived single oral doses of chloroform

Doses (mg/kg)	Liver		Kidneys
	Males	Females	Males	Females
546	0/2	1/2	02	0/2
765	2/2	0/2	1/2	1/2
1071	--	0/2	--	2/2
1500	0/1	0/2	0/1	1/2
2100	--	0/1	--	0/1

Numbers denote: number of animals showing lesions/animals examined

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Migrated information

Conclusions:

Based on the results of this study in the rat, chloroform is classified as harmful if swallowed.

Executive summary:

The acute oral toxicity of chloroform, solubilised in corn oil, was tested in female and male Sprague-Dawley rats (10 animals per dose group) in accordance with the OECD Guideline No. 401 with minor restrictions. Chloroform was administered at doses of 546, 765, 1071, 1500 and 2100 mg/kg body weight by oral gavage. Animals were then observed for 14 hours, clinical signs were noted and animals which were dying during the observation period or were killed after 14 days were submitted to necropsy and gross pathology. The mean LD50 values determined for male rats was 908 mg/kg body weight, that of female rats was 1117 mg/kg. Following the GHS classification system, chloroform on the basis of the present study is classified into Toxicity Category IV, receiving the hazard statement "harmful if swallowed".

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

908 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male rats received whole-body exposure to chloroform vapours for 6 hours and were observed for 14 days subsequently.

GLP compliance:

no

Test type:

traditional method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Rats had a body weight between 130 and 200 g.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gas chromatograph

Duration of exposure:

6 h

No. of animals per sex per dose:

12 male rats

Control animals:

no

Details on study design:

After exposure animals were observed for 14 days. All animals were subject to necropsy and gross pathology.

Statistics:

The LD50 value and the 95% confidence interval were determined based on the method of Bliss (1938).

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

9.17 mg/L air

Based on:

test mat.

95% CL:

> 8.69 - < 9.7

Exp. duration:

6 h

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Based on the results of this study, the 6 -h LC50 value determined was 9.17 mg/L. Upon recalculation, the 4 -h LC50 would be 10.5 mg/L.

Executive summary:

The acute inhalation toxicity of chloroform was tested in male Sprague-Dawley rats exposed to chloroform vapours during 6 hours followed by an observation period of 14 days; the 6 -h LC50 value determined was 9.17 mg/L. Upon recalculation, the 4 -h LC50 would be 10.5 mg/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

10 500 mg/m³ air

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute oral toxicity

The acute lethal toxicity varies depending on species, strain, sex and vehicle. In old, not well-documented studies, the oral LD50 values in mice range from 36 to 1366 mg chloroform per kg body weight (Pericin and Thomann 1979), whereas in rats the values range from 450 to 2000 mg chloroform per kg body weight. The study in Sprague Dawley rats by Chu et al. (1980) is considered to be the key study, which provided an oral LD50 value of 908 mg/kg body weight in male rats. According to the CLP Regulation (EC) No 1272/2008, chloroform has been classified for acute oral toxicity in category 4 with the hazard statement harmful if swallowed.

Acute inhalation toxicity

Depression of the central nervous system is the dominant symptom of acute inhalation toxicity of chloroform. The available rodent studies on the acute inhalation toxicity of chloroform give evidence that chloroform exhibits low acute inhalation toxicity. The study performed in male Sprague Dawley rats (Bonnet et al. 1980) resulted in an acute inhalation LC50 value of 9.2 mg/L after 6 hours of exposure. For classification, the relevant duration of exposure is 4 hours. Applying Haber’s law a corrected LC50 value for acute inhalation toxicity of 10.5 mg/L is calculated for chloroform. This value leads to the classification as acute category 4 with the hazard statement harmful if inhaled according to Regulation (EC) No 1272/2008.

Acute dermal toxicity

Only one study on the dermal toxicity of chloroform is available, which is considered as not reliable. A single dermal application of 3.98 g/kg for 24 hours under impermeable plastic cuff held tightly around the clipped bellies of two rabbits did not result in any deaths (Torkelson et al. 1976). However, no additional study on the acute dermal toxicity of chloroform is necessary.

Justification for selection of acute toxicity – oral endpoint

For oral toxicity results from a reliable study in Sprague Dawley male rats (Chu et al. 1980) provided an oral LD50 value of 908 mg/kg body weight.

Justification for selection of acute toxicity – inhalation endpoint

A study performed in Sprague Dawley rats (Bonnet et al. 1980) resulted in an acute inhalation LC50 value of 9.2 mg/L after 6 hours of exposure corresponding to 10.5 mg/L for 4 hours of exposure by applying Haber's law correction.

Justification for selection of acute toxicity – dermal endpoint

There were no reliable data for the dermal route.

Justification for classification or non-classification

Chloroform exhibits rather low acute toxicity. According to Commission Regulation (EU) No 944/2013 of 2 October 2013, amending Annex VI to the Regulation (EC) No 1272/2008 (CLP), chloroform is classified Category 4 for the oral route: "Harmful", with the hazard statement: harmful if swallowed (H302). For the inhalation route, chloroform is classified as Category 3 "Toxic" with the hazard statement: toxic if inhaled (H331).

Based on acute toxicity data, the registrant's self-classification for chloroform is Category 4, "Harmful", with the hazard statements: harmful if swallowed (H302) and harmful if inhaled (H332).