1,1'-(4-methyl-1,3-phenylene)bis-1H-pyrrole-2,5-dione

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:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

The local lymph node assay: Results of a final inter-laboratory validation under field conditions

Author:

Scholes, EW et al.

Year:

1992

Bibliographic source:

Journal of Applied Toxicology, Vol. 12(3), 212-222

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Inter-laboratory validation study comprising four testing facilities (Laboratory A to D).

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(4-methyl-1,3-phenylene)bis-1H-pyrrole-2,5-dione

EC Number:

229-175-3

EC Name:

1,1'-(4-methyl-1,3-phenylene)bis-1H-pyrrole-2,5-dione

Cas Number:

6422-83-9

Molecular formula:

C15H10N2O4

IUPAC Name:

1,1'-(4-methyl-1,3-phenylene)bis-1H-pyrrole-2,5-dione

Test material form:

not specified

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Laboratory B obtained animals from the Barriered Animal Breeding Unit, Alderley Park; Laboratories A, C and D obtained animals from Harlan Olac Ltd., Bicester, Oxon.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: ca. 8-12-weeks

Study design: in vivo (LLNA)

Vehicle:

other: Laboratory A: DMSO; Laboratory B-D: DMF

Concentration:

Laboratory A-D: 2.5, 5.0, 10.0 and 25.0%

No. of animals per dose:

Laboratory A-D: 4

Details on study design:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (radioactive)
- Criteria used to consider a positive response: The proliferative activity was expressed as the number of radioactive disintegrations per minute (dpm) per lymph node for each test group. The ratio of ³HTdR incorporation by lymph node cells of test lymph nodes relative to that for control lymph nodes test/control (T/C) ratio was calculated for each experimental group. The test item was considered positive in the LLNA if the following criteria were fulfilled:
- exposure to at least one test item concentration resulted in an at least threefold ³HTdR incorporation compared to the control group.
- the data were not incompatible with a conventional biological dose response.
A test item fulfilling these criteria was considered ‘a sensitizer’. If the test item failed cause a threefold or greater increase in ³HTdR incorporation was considered ‘not a strong sensitizer’.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of 4 mice received 25 µL of one of three concentrations of the test item on the dorsum of both ears daily for three consecutive days. Control mice were treated with equal volume of vehicle.
Five days after start of exposure, all animals were injected i.v. via the tail vein with 250 µL of phosphate-buffered saline containing 20 µCi of [³H]methyl thymidine (³HTdR: specific activity 2 Ci per mmol). Five hours later, animals were sacrificed and the draining (auricular) lymph nodes were excised and pooled for each experimental group. Single-cell suspensions of lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 mesh size). The pooled lymph node cells were pelleted by centrifugation at 190 g for 10 min, washed twice with 10 mL of phosphate-buffered saline and resuspended in 3 mL of 5% trichloroacetic acid. After overnight incubation at 4 °C, precipitates were recovered by centrifugation, resuspended in 1 mL of trichloroacetic acid and transferred to 10 mL of scintillation fluid. ³HTdR incorporation was measured by beta-scintillation counting.

Positive control substance(s):

mercaptobenzothiazole (CAS No 149-30-4)

Results and discussion

Positive control results:

2-Mercaptobenzothiazole (10.0, 25.0 and 50% in DMF) induced a greater than threefold increase in ³HTdR incorporation at all test concentrations in all four testing facilities.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

18.4

Test group / Remarks:

2.5% in DMF, Laboratory D

Results 2

Key result

Parameter:

SI

Value:

11.9

Test group / Remarks:

5.0% in DMSO, Laboratory A

Results 3

Key result

Parameter:

SI

Value:

>= 13.8 - <= 29.6

Test group / Remarks:

5.0% in DMF, Laboratory B-D

Results 4

Key result

Parameter:

SI

Value:

12.2

Test group / Remarks:

10.0% in DMSO, Laboratory A

Results 5

Key result

Parameter:

SI

Value:

>= 19.1 - <= 35.3

Test group / Remarks:

10.0% in DMF, Laboratory B-D

Results 6

Key result

Parameter:

SI

Value:

11.8

Test group / Remarks:

25.0% in DMSO, Laboratory A

Results 7

Key result

Parameter:

SI

Value:

>= 15.5 - <= 25.7

Test group / Remarks:

25.0% in DMF, Laboratory B-C

Any other information on results incl. tables

Table 1. LLNA results obtained at the collaborating laboratories for the inter-laboratory trial

	LLNA result (T/C ratio)
Test item	Concentration (%)	Laboratory A (vehicle)	Laboratory B (vehicle)	Laboratory C (vehicle)	Laboratory D (vehicle)
2-Mercaptobenzothiazole	10.0	4.5 (DMF)	9.8 (DMF)	5.2 (DMF)	10.0 (DMF)
	25.0	4.6	9.5	9.1	10.8
	50.0	5.5	8.9	4.8	8.1
Toluene diamine bismaleimide	2.5	- (DMSO)	- (DMF)	- (DMF)	18.4 (DMF)
	5.0	11.9	13.8	16.3	29.6
	10.0	12.2	19.1	25.3	35.3
	25.0	11.8	15.5	25.7	-

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this inter-laboratory validation study, the test substance induced a greater than threefold increase in the in the ³HTdR incorporation at all test concentrations in all four testing facilities. Therefore, the test substance is considered to be positive in the Local Lymph Node Assay and thus skin sensitising.