Reaction mass of carbon tetrachloride and chloroform and 1,2-dichloroethane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Carbon tetrachloride

EC Number:

200-262-8

EC Name:

Carbon tetrachloride

Cas Number:

56-23-5

Molecular formula:

CCl4

IUPAC Name:

tetrachloromethane

Constituent 2

Reference substance name:

TETRACHLORURE DE CARBONE

IUPAC Name:

TETRACHLORURE DE CARBONE

Details on test material:

- Name of test material (as cited in study report): CARBON TETRACHLORIDE
- Physical state: colorless liquid
- Analytical purity: 99.96
- Purity test date: 30 November 2009
- Lot/batch No.: R864 du 30/11/2009
- Expiration date of the lot/batch: 30 November 2010
- Storage conditions of test material: at room temperature (keep tightly closed in a dry and well-ventilated place), protected from humidity, heat and
light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 20.8 ± 0.7 g
- Housing: housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained (except for the 5 hours following the 3H-TdR injections) autoclaved sawdust (SICSA, Alfortville, France)
- Diet (e.g. ad libitum): conventional laboratory diet (SSNIFF R/M-H pelleted maintenance diet)
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: From 23 February 2010 to 08 March 2010

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50 and 100%.

No. of animals per dose:

4 animals.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum tested concentration of 50%.
- Irritation: non-irritant, whatever the concentration
- Lymph node proliferation response: incorporation of tritiated methyl thymidine

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: SI >= 3 with exclusion of excessive irritation

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.

Positive control substance(s):

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

Results and discussion

Positive control results:

HCA at the concentration of 25%: a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 17.10) were noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.51

Test group / Remarks:

Group 2: test item 25%

Results 2

Parameter:

SI

Value:

2.39

Test group / Remarks:

Group 3: test item 50%

Results 3

Parameter:

SI

Value:

6.1

Test group / Remarks:

Group 4: test item 100%

Results 4

Key result

Parameter:

EC3

Remarks:

(%)

Value:

58

Test group / Remarks:

test item

Results 5

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Group 1 - vehicle : 532.18 (DPM per group) and 66.52 (DPM per node) Group 2 - test item 25% : 801.13 (DPM per group) and 100.14 (DPM per node) Group 3 - test item 50% : 955.80 (DPM per group) and 159.30 (DPM per node) Group 4 - test item 100% : 3244.26 (DPM per group) and 405.53 (DPM per node)

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item, CARBON TETRACHLORIDE, demonstrated weak dermal sensitization potential in the murine Local Lymph Node Assay.

Executive summary:

The potential of the test item, CARBON TETRACHLORIDE, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA) was evaluated. Evaluation of local irritation was also carried out in parallel.

This study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, 20 female CBA/J mice were allocated to five groups:

. three treated groups of four animals each receiving the test item at the concentrations of 25%, 50% or 100% in a mixture of acetone/olive oil (4/1; v/v) (vehicle),

. one negative control group of four animals receiving the vehicle,

. one positive control group of four animals receiving the reference item, alpha-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% the vehicle.

 

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).

 

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

Results

The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum tested concentration of 50%.

 

Consequently, the concentrations selected for the preliminary test were 10%, 25%, 50% and 100%.

 

Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%).

 

No treatment-related mortality or clinical signs were observed during the main test.

Erythema was noted on day 6 in 1/4 females given 100%. No notable increase in ear thickness was observed in any treated animal.

 

A significant lymphoproliferation was noted in the positive control group given HCA at 25%. The study was therefore considered valid.

A dose-related increase in the SI was noted at all the concentrations and the threshold of 3 was exceeded at the concentration of 100%.

The results are presented in the following table:

 

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	25	non-irritant	1.51
Test item	50	non-irritant	2.39
Test item	100	non-irritant	6.10
HCA	25	-	17.10

 

In the absence of local irritation, the positive lymphoproliferative response observed was attributed to delayed contact hypersensitivity. The EC3 value for the test item, CARBON TETRACHLORIDE, is equal to 58%. Therefore, on the basis of relative skin sensitization potency, the test item was classified as weak sensitizer.

Conclusion

Under the experimental conditions of this study, the test item, CARBON TETRACHLORIDE, (batch No. R864), demonstrated weak dermal sensitization potential in the murine Local Lymph Node Assay and thus is classified skin sensitizer category 1B according to CLP and the GHS.