Chloroform

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Sensitization test of chloroform: Comparison of guinea pig maximization test (GPMT) and local lymph node assay (LLNA)

Author:

Matsuoka C, Kanazawa Y, Kojima K

Year:

2002

Bibliographic source:

Annual Report of Hatano Research Institute

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Principles of method if other than guideline:

The original study is available in Japanese only. The following evaluation is based on an English translation (Harlan Japan) of the report. The study was carried out according to the skin sensitisation test method applying the local lymph node assay, method B.42, suggested by the European Commission with acceptable restrictions.

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

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:

trichloromethane

Details on test material:

chloroform was purchased from Wako Pure Chemical Industries, Ltd.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

20 female mice of CBA/J strain (CBA/JNCrj, SPF) at 9 weeks of age; temperature between 21 and 25 °C, humidity between 40 and 75 %, air exchange rate approximately 15 times/hour, 12 hr light/12 hr darkness

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10%

No. of animals per dose:

5 animals

Details on study design:

4 groups: 1. 25 µL/ear chloroform; 2. 25 µL/ear acetone:olive oil (4:1); 3. 25 µL/ear 10% hexyl cinnamic aldehyde in chloroform; 4. 25 µL/ear; 10% hexyl cinnamic aldehyde in acetone:olive oil (4:1).
Application of test solutions to both auricles of the mice for three consecutive days. 3 days later, 3H-methyl thymidine (Amersham Pharmacia Biotech, Inc.) was administered intravenously (250 uL, 2.96 MBq/mL). Five hours later, animals were euthanised. The auricular lymph nodes were removed, in order to compare reactions to HCA with chloroform as vehicle and with acetone:olive oil as vehicle. Cells were isolated from the lymph nodes, cell suspensions prepared and radioactivity was measured with a beta scintillation counter.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean and standard deviation of the data were calculated and the groups were compared using student's t-test

Results and discussion

Positive control results:

The positive control substance hexyl cinnamic aldehyde was characterised to be sensitising in the local lymph node assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

Chloroform (10%)

Value:

2.48

Test group / Remarks:

chloroform in acetone:oilve oil (4:1) at 10%

Results 2

Key result

Parameter:

SI

Remarks:

Cinnamic aldehyde (positive control)

Value:

3.49

Test group / Remarks:

cinnamic aldehyde in acetone:olive oil (4:1)

Results 3

Key result

Parameter:

SI

Remarks:

negative control

Value:

1

Test group / Remarks:

negative control

Any other information on results incl. tables

In the local lymph node assay, chloroform tended to be higher abundant in the lymph nodes than the acetone/olive oil in the solvent control. The lympho-proliferative activity is used as an index of sensitisation in the LLNA, but since primary irritation also activates lymph cell proliferation through inflammatory cytokine effects, the reactions are said to be difficult to differentiate. Since Montelius et al. (1994) showed activation due to primary irritation in LLNA using methanol/chloroform as the vehicle, it is very likely that the reactions to chloroform seen in the Japanese LLNA study were due to primary irritation rather than sensitisation.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

In contrast to the positive control substance hexyl cinnamic aldehyde, chloroform was characterised to be not sensitising in the local lymph node assay.

Executive summary:

The skin sensitisation potential of chloroform was tested in a local lymph node assay with 20 female CBA/J mice according the test method B.42 suggested by the European Commission. Stimulation index determined for chloroform used in the vehicle acetone:olive oil (4:1) at a concentration of 10 % was 2.48, which was below the threshold of 3 indicating sensitising properties. In contrast, the positive control substance hexyl cinnamic aldehyde used in the vehicle acetone:olive oil (4:1) at a concentration of 10 % had a stimulation index of 3.49 and thus was identified as a skin sensitiser.

In the local lymph node assay the lymphoproliferative activity is used as an index of sensitisation, but since primary irritation also activates lymph cell proliferation through inflammatory cytokine effects, the reactions are said to be difficult to differentiate. Since Montelius et al. (1994) showed activation due to primary irritation in LLNA using methanol/chloroform as the vehicle, it is very likely that the reactions to chloroform seen in the Japanese LLNA study were due to primary irritation rather than sensitisation. In conclusion, chloroform should be considered as not sensitising to the skin.