Chlorobenzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD, EU and Japan Guidelines. Non-GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Chlorobenzene

EC Number:

203-628-5

EC Name:

Chlorobenzene

Cas Number:

108-90-7

Molecular formula:

C6H5Cl

IUPAC Name:

chlorobenzene

Details on test material:

- Name of test substance: Chlorobenzene
- Test-substance No.: 08/0386-2
- CAS No.: 108-90-7
- Batch Identification: S90576-359
- Form of application: solution in AOO (4+1, v/v)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

100, 50 and 25 %

No. of animals per dose:

5

Details on study design:

Three days after the last application the mice were injected into the tail vein with 20 μCi of 3Hthymidine in 250 μL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined.
Additionally, flow cytometric analysis using aliquots of the lymph node cell suspension was performed for determination of lymphocyte subpopulations (immunophenotyping). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

Statistics:

Statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.05, ## for p ≤ 0.01) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight and KRUSKAL-WALLIS test and WILCOXON test for flow cytometric endpoints.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

3H-thymidine incorporation - vehicle AOO: 1.00 - 25% in AOO: 1.91 - 50% in AOO: 3.23 - undiluted test substance: 5.30 Cell count - vehicle AOO: 1.00 - 25% in AOO: 0.97 - 50% in AOO: 1.34 - undiluted test substance: 1.61 Lymph node weight - vehicle AOO: 1.00 - 25% in AOO: 1.20 - 50% in AOO: 1.37 - undiluted test substance: 1.79 Ear weight - vehicle AOO: 1.00 - 25% in AOO: 1.08 - 50% in AOO: 1.18 - undiluted test substance: 1.25

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM / lymph node pair - vehicle AOO: 348-0 - 25% in AOO: 665.9 - 50% in AOO: 1,123.4 - undiluted test substance: 1,844.1

Any other information on results incl. tables

Body weight [g] on day 0 and day 5

- vehicle AOO: 20.1/20.3

- 25% in AOO: 20.2/21.1

- 50% in AOO: 20.2/20.3

- undiluted test substance: 19.9/20.4

Flow cytometry

The following tables show the mean values and the ratio of treated groups divided by concurrent control (SI) of the respective parameter with values > 1.25 in bold.

Mean values

	LY LLN GIGA/L	B220 LLN % LY	B/T LLN %	CD3 LLN % LY	CD4 LLN % CD3	CD8 LLN % CD3	CD 19 LLN % LY
AOO	1.24	8.75	9.91	91.01	55.53	30.5	7.44
25% in AOO	1.2	8.2	10.05	90.88	57.94	30.66	6.71
50% in AOO	1.66**	8.78	10.37	90.61	56.34	31.65	7.07
undiluted (1)	2.00**	9.02	15.37*	86.71*	54.69	28.67	7.33
Kruskal-Wallis + Wilcoxon-test, two sided, *p<= 0.05; **p<=0.01

Stimulation indices

	LY LLN GIGA/L	B220 LLN % LY	B/T LLN %	CD3 LLN % LY	CD4 LLN % CD3	CD8 LLN % CD3	CD 19 LLN % LY
AOO	1	1	1	1	1	1	1
25% in AOO	0.97	0.94	1.01	1	1.04	1.01	0.9
50% in AOO	1.34	1	1.05	1	1.01	1.04	0.95
undiluted (1)	1.61	1.03	1.55	0.95	0.98	0.94	0.99
(1) Calculation of B/T, CD3, CD4 and CD8 on basis of 4 animals due to outlier values.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the results of this study, the test substance chlorobenzene does not nedd to be classified for skin sinsitization according to Regulation (EU) No. 1272/2008 and Directive 67/548/EEC.