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Description of key information

- Skin sensitisation: sensitising; LLNA/GPMT; RL2

- Respiratory sensitisation: no study available

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study concurrently conducted within the frame of an inter-laboratory validation study for the Local Lymph Node Assay.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Porcellus, Heathfield, Sussex, UK
- Weight at study initiation: 300-350 g
Route:
intradermal
Vehicle:
not specified
Concentration / amount:
0.1 %
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
not specified
Concentration / amount:
25.0%
Day(s)/duration:
between Day 6 and 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
not specified
Concentration / amount:
10.0%
Day(s)/duration:
between Day 12 and 14
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
control group: 4
test group: 10
Details on study design:
RANGE FINDING TESTS: Preliminary skin irritation studies were conducted to determine suitable concentrations of test chemical for induction and elicitation of sensitization.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous)
- Exposure period: Day 1 and between Day 6 and 8
- Test groups: test substance in FCA
- Control group: vehicle in FCA
- Site: shoulder region
- Frequency of applications: single intradermal and epicutaneous application, respectively.
- Duration: single injection and 48 h occluded patch
- Concentrations: 0.1% and 25%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: between Day 12 and 14
- Exposure period: 24 h
- Test groups: test substance
- Control group: test substance
- Site: one clipped and razored flank
- Concentrations: 10%
- Evaluation (hr after challenge): 24 and 48 h
Challenge controls:
No challenge controls
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole was not explicitly declared as positive control substance, but it was concurrently tested within the inter-laboratory validation study.
Positive control results:
2-Mercaptobenzothiazole (0.4% intradermal induction; 10.0% epicutaneous induction; 10.0% challenge) induced a positive response in 80% of the treated animals.
Key result
Reading:
other: Maximum challenge result 24-48 h after removing topical challenge patches.
Hours after challenge:
48
Group:
test group
Dose level:
10.0%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
other: Maximum challenge result 24-48 h after removing topical challenge patches.
Hours after challenge:
48
Group:
negative control
Dose level:
10.0%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
no indication of skin sensitisation

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

 

Guinea Pig Maximization Test (GPMT)

Test item

Animals

Intradermal induction conc. (%)

Epicutaneous induction conc. (%)

Challenge conc. (%)

GPMT result (% positive)*

2-Mercaptobenzothiazole

Test group

0.4

10

10

80

Control

0

0

10

0

Toluene diamine bismaleimide

Test group

0.1

25

10

70

Control

0

0

10

0

 *Maximum challenge result 24 -48 h after removal of challenge patches.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study, the test substance induced skin reactions (erythema) in 7/10 animals of the test group at 24-48 h after challenge patch removal. Therefore, the test substance is considered to be positive in the Guinea Pig Maximization Test and thus skin sensitising.
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
Qualifier:
equivalent or similar to
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)
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
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)
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.
Key result
Parameter:
SI
Value:
18.4
Test group / Remarks:
2.5% in DMF, Laboratory D
Key result
Parameter:
SI
Value:
11.9
Test group / Remarks:
5.0% in DMSO, Laboratory A
Key result
Parameter:
SI
Value:
>= 13.8 - <= 29.6
Test group / Remarks:
5.0% in DMF, Laboratory B-D
Key result
Parameter:
SI
Value:
12.2
Test group / Remarks:
10.0% in DMSO, Laboratory A
Key result
Parameter:
SI
Value:
>= 19.1 - <= 35.3
Test group / Remarks:
10.0% in DMF, Laboratory B-D
Key result
Parameter:
SI
Value:
11.8
Test group / Remarks:
25.0% in DMSO, Laboratory A
Key result
Parameter:
SI
Value:
>= 15.5 - <= 25.7
Test group / Remarks:
25.0% in DMF, Laboratory B-C

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

-

 

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a dermal sensitisation study comparable to OECD guideline 406 (May, 1981) with 2,4 -Bismaleimidotoluene 20 young adult Himalayan spotted guinea pigs were tested using the method of Magnusson and Kligman.

In a preliminary study intradermal injections with 1, 3 and 5% of the test material were made in order to identify irritant test article concentrations and the resulting dermal reaction were assessed 24 h later. Furthermore, epidermal applications have been conducted using patches of filter paper (2 x 2 cm) which were saturated with concentrations of 25, 15, 10 and 5% of the test article in ethanol and applied to the clipped and shaved flanks of each of four guinea-pigs in order to identify the highest non-irritating concentration for the challenge procedure. The dressings were removed after an exposure period of 24 h and the reaction sites were assessed for erythema and edema on a numerical basis according to the scale described above. Further examination of the sites was performed 24 h and 48 h after removal of the dressings. The highest non-irritating concentration used for challenge application was 25 % and the highest concentration producing mild to moderate reactions was determined to be 5 %.

In the main study 10 animals were exposed to intradermal 5% and epicutaneously 25 % 2,4 -Bismaleimidotoluene for 48 h. After 14 days of recovery the epicutaneous application was repeated and the sensitisation potential was determined.

One female animal died spontaneously on day 3 of the study. Necropsy was performed for that animal: the lungs were found not collapsed , discoloured and reddish. In the control group erythema and edema were found from day 2 to 5; necroses were found from day 6 to 8; desiccation was found from day 10 to 16 and exfoliation from day 15 to 25. In the test group the same symptoms were found and additionally in the epidermal induction area staining was obvious from day 11 to 12 and on day 23. No other symptoms were found and body weights were not adversely affected.

After challenge no visible changes of the treated skin sites were observed in the test group compared to the control group animals 24 and 48 h after patch removal (= grade 0).

The test material produced a response in 0% of animals. According to CLP, EU GHS (Regulation (EC) No 1272/2008), a response of at least 15% of the test animals of an adjuvant type guinea pig test method for skin sensitisation is considered as positive.

2,4 -Bismaleimidotoluene was not a dermal sensitiser in this study.

In an inter-laboratory validation study for the Local Lymph Node Assay (LLNA), 2,4-Bismaleimidotoluene was tested for its skin sensitising potential following a protocol similar to OECD guideline 429 at four different testing facilities (Laboratory A to D). In addition, a Guinea Pig Maximization Test (GPMT) following a protocol similar to OECD guideline 406 was conducted at Laboratory A.

For the LLNA, the test substance was diluted in DMSO (Laboratory A) or DMF (Laboratory B-D) as vehicle. Test concentrations were 2.5, 5 and 10 % at Laboratory D, and 5, 10 and 25% at Laboratory A-C. Groups of four female CBS/Ca mice received 25 µL of one of the three concentrations on the dorsum of both ears daily for three consecutive days. Control animals received an equal volume of vehicle. Five days after exposure initiation, all animals were injected i.v. 250 µL of phosphate-buffered saline (PBS) containing 20 µCi of ³H-methyl thymidine (³HTdR at specific activity of 2 Ci/mmol). After 5 h, animals were sacrificed and the draining auricular lymph nodes were excised and pooled for each test group. Single cell suspensions of lymph node cells (LNC) were prepared and, following centrifugation and washing with PBS, resuspended in 3 mL 5% trichloroacetic acid (TCA). After overnight incubation at 4°C, precipitates were centrifuged, resuspended in 1 mL TCA and transferred to 10 mL of scintillation fluid. Incorporation of ³HTdR was measured by means of β-scintillation counting. LNC proliferation was expressed as number of radioactive disintergatons per minute (dpm) per lymph node for each test group. The ratio of ³HTdR incorporation of test lymph nodes relative to control lymph nodes (T/C ratio) was calculated for each experimental group.

The test substance induced a greater than threefold ³HTdR incorporation (T/C ration >3) at all test concentrations and testing facilities. Therefore, the test substance was considered to be positive in the Local Lymph Node Assay and thus skin sensitising.

For the GPMT, preliminary skin irritation studies were performed in order to determine the suitable test concentrations for induction and challenge. In the main, 10 Dunkin Hartley guinea pigs were intradermally induced with the test substance at 0.1%. Six to eight days later, animals were epicutaneously induced with the test substance at 25% by means of a 48 h occluded patch. A group of 4 control animals were similarly treated with the vehicle. Twelve to fourteen days later, test and control animals were topically challenged with the test substance at 10% for 24 h using an occlusive patch. The skin sensitisation potential was determined by visual assessment of erythema at the challenge treated sites 24 and 48 h after patch removal.

The test substance induced skin reactions (erythema) in 7/10 test animals and in 0/4 control animals. Therefore, the test substance is considered to be positive in the Guinea Pig Maximization Test and thus skin sensitising.

Taken together, the results of the inter-laboratory validation study indicate that 2,4-Bismaleimidotoluene is a skin sensitizer. The negative results obtained in the GLP-compliant OECD 406 study are likely to be due to the use of a vehicle (ethanol), in which the test substance may be less soluble than in other vehicles such as DMSO and DMF.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfiled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.

Justification for classification or non-classification

Based on relevant, reliable and adequate data, 2,4 -Bismaleimidotoluene fulfills the criteria for classification and labelling according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) with respect to skin sensitisation.