Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro-

Endpoint summary

Description of key information

- Guinea Pig Maximization Test (GPMT): according to OECD TG 406, GLP, guinea pig, not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

When this study was performed, the LLNA did not yet exist.

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: Solid
- Lot/batch No.: Z-2281/1,2,4,5
- Expiration date of the lot/batch: April 01, 1997
- Storage condition of test material: at room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Prior to each intradermal treatment, the test substance was weighed into small glass containers, polyethylene glycol was added (w/w) and subsequently mixed using a mechanical stirrer and/or spatula.
- Prior to each epidermal treatment, the test substance was weighed into a mortar, vaseline was added (w/w) and subsequently mixed using a pestle.

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: Approx. 8 weeks
- Weight at study initiation: 318 - 450 grams
- Housing: 2 animals per cage
- Diet: standard guinea pig diet, including ascorbic acid (1600 mg/kg), ad libitum and hay, once weekly
- Water: tap-water, diluted with decalcified water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1992-09-15 To: 1992-10-16

Route:

intradermal

Vehicle:

polyethylene glycol

Concentration / amount:

5% (w/w) test substance, 0.1 mL/site

Day(s)/duration:

Day 1; once

Adequacy of induction:

not specified

Route:

intradermal

Vehicle:

polyethylene glycol

Concentration / amount:

vehicle control; 0.1mL/site

Day(s)/duration:

Day 1; once

Route:

intradermal

Vehicle:

other: Freunds' Complete Adjuvant (FCA)

Concentration / amount:

50:50 with distilled water, 0.1mL/site

Day(s)/duration:

Day 1; once

Route:

epicutaneous, semiocclusive

Vehicle:

other: vaseline

Concentration / amount:

50% (w/w) test substance; 0.5 mL/site

Day(s)/duration:

Day 8; 48h

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

epicutaneous, semiocclusive

Vehicle:

other: vaseline

Concentration / amount:

vehicle control

Day(s)/duration:

Day 8; 48h

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

other: vaseline

Concentration / amount:

10, 25, 50% (w/w) test substance

Day(s)/duration:

24h

Adequacy of challenge:

highest non-irritant concentration

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

other: vaseline

Concentration / amount:

vehicle control

Day(s)/duration:

24h

No. of animals per dose:

Preliminary study: 5
Main study: 20 (test item), 10 (control)

Details on study design:

RANGE FINDING TESTS:
- Intradermal injections: Four Intradermal injections (0.1 ml/site) of one guinea pig with a 5% (w/w) concentration of the test substance in polyethylene glycol. Erythema, necrosis and diameter of effects were recorded 24 and 48 hours later.
- Epidermal applications: The intradermally injected animal was also treated epidermally with approximately 0.5 ml of a 50% (w/w) concentration of the test substance in vaseline. The treated skin was assessed for erythema and oedema 24 and 48 hours after bandage removal.
Four other animals were exposed to 0.05 ml of a 50 %, 25 %, 10 % and 5 % (w/w) concentration of the test substance in vaseline, occlusively. The reaction sites were assessed for erythema and oedema 24 and 48 hours after bandage removal.

MAIN STUDY:
- Doses were chosen based on a preliminary study.

A. INDUCTION EXPOSURE
- Day of induction: Day 1 (intradermal injection); Day 8 (epidermal)
On day 7, approximately 24 hours prior to the epidermal induction application, the scapular area was pretreated with 10 % Sodium-Dodecyl-Sulfate (SDS) in vaseline. The SDS was massaged into the skin with a spatula without bandaging. This concentration of SDS enhances sensitisation by provoking a mild inflammatory reaction.
- Site: clipped area of the dorsal skin from the scapular region
- No. of exposures: 3 pairs (intradermal injection); 3 (epidermal)
- Exposure period: Once (intradermal injection); 48h (epidermal)
- Test groups: 5% (w/w) in polyethylene glycol (intradermal injection); 50% (w/w) in vaseline (epidermal)
- Control group: Freunds' Complete Adjuvant 50:50 with distilled water; polyethylene glycol (intradermal injection); vaseline (epidermal)
- Evaluation: Reaction sites were, assessed for erythema and oedema immediately after removal of the dressings.

B. CHALLENGE EXPOSURE
- Day of challenge: Two weeks after the epidermal induction application
- No. of exposures: Test and control guinea pigs
- Exposure period: 24h (the dressings and residual test substance were removed using a moistened tissue)
- Test groups: 10, 25, 50% (w/w) in vaseline
- Control group: Vaseline
- Site: 5 x 5 cm clipped area on the left flank of each guinea pig
- Evaluation (hr after challenge): 24 and 48 h (the sites were assessed for redness and swelling)
- Observations: Mortality/Viabillty/Toxicity (once daily); body weights (during acclimatisation and at termination of the study)
- Histopathology: All animals were sacrificed after the termination of observation by carbon dioxide asphyxiation. Immediately thereafter, the four challenge treated skin areas of the last challenge were cut using a surgical scissor. Each skin site was collected in a coded, perforated small cassette and per animal fixed in neutral phosphate buffered 4% formaldehyde solution. Sections were cut from the skin sites of experimental and control animals treated with the highest test substance concentration and with the vehicle. The sections were stained with haematoxylin and eosin. At least one section from each skin site was microscopically examined by a pathologist. No microscopically examination was performed on the skin sites treated with the intermediate concentrations of experimental and control animals.

Challenge controls:

vehicle

Positive control substance(s):

yes

Remarks:

Formaldehyde (A positive control experiment is carried out once a year as a sensitivity check of the test system. The most recent test was carried out in April 1991.)

Positive control results:

Clearly positive results were observed in the experimental animals after the challenge with 0.2% (w/w) FORMALDEHYDE in distilled water.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 (vehicle only)

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 (vehicle only)

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10, 25, 50%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10, 25, 50%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Histopathological examination revealed no treatment related differences between the 50% test substance concentration treated- and control-skin areas in both experimental and control animals.

Remarks on result:

no indication of skin sensitisation

TOXICITY SYMPTOMS / MORTALITY:

No symptoms of systemic toxicity were observed in the animals of the main study and no mortality occurred during the preliminary- and main-study.

 

BODY WEIGHTS:

The average body weight gain of experimental and control animals was considered to be similar.

 

CHALLENGE:

No skin reactions were evident in response to any of the test substance concentrations and the vehicle control. However, many challenge-sites were difficult to score due to red  staining of the skin by the test substance.

 

HISTOPATHOLOGY

CONTROL GROUP:
- 50% concentration: infiltrates of mononuclear cells in all animals and acanthosis and parakeratosis in some animals.
- Vaseline (vehicle): infiltrates of mononuclear cells in the majority of animals and very slight acanthosis in some animals.
EXPERIMENTAL GROUP:
- 50% concentration: infiltrates of mononuclear cells in the majority of animals and acanthosis and parakeratosis in some animals.
- Vaseline (vehicle): infiltrates of mononuclear cells in the majority of animals and acanthosis and parakeratosis in some animals.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test substance is considered not skin sensitizing.

Executive summary:

In order to assess the cutaneous allergenic potential of the test substance (purity 97.5%), a Maximisation Test was performed in 30 (20 test and 10 control) female albino guinea pigs, in accordance with OECD Guideline 406 and GLP. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, animals were intradermally injected with 5% test substance concentration in polyethylene glycol (PEG) on day 1. On day 7 all animals were treated with 10% SDS, followed by epidermal exposure to a 50% concentration of the test substance in vaseline 24 h later. Control animals were similarly treated with each vehicle alone. Two weeks after the epidermal application (induction), all animals were challenged by epidermal exposure to a semiocclusive dressing with the test substance in vaseline (vehicle) at concentrations of 10, 25 and 50% (w/w) and the vehicle control, respectively. The challenge reactions were assessed 24 and 48 hours after bandage removal. The epidermal exposure to the test substance in the challenge phase did not result in positive sensitisation reactions in response to any of the test substance concentrations and the vehicle, respectively. Red staining was observed at the test substance treated skin sites, which made scoring of erythema difficult. Therefore, treated skin was evaluated by histopathology for infiltration of immune cells. Histopathological examination revealed no treatment related differences between the 50% test substance concentration treated- and control-skin areas in both experimental and control animals. Moreover, no differences were found in the microscopical findings between experimental and control animals. From these results it was concluded that no sensitisation was induced by the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In order to assess the cutaneous allergenic potential of the test substance (purity 97.5%), a Maximisation Test was performed in 30 (20 test and 10 control) female albino guinea pigs, in accordance with OECD Guideline 406 and GLP. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, animals were intradermally injected with 5% test substance concentration in polyethylene glycol (PEG) on day 1. On day 7 all animals were treated with 10% SDS, followed by epidermal exposure to a 50% concentration of the test substance in vaseline 24 h later. Control animals were similarly treated with each vehicle alone. Two weeks after the epidermal application (induction), all animals were challenged by epidermal exposure to a semiocclusive dressing with the test substance in vaseline (vehicle) at concentrations of 10, 25 and 50% (w/w) and the vehicle control, respectively. The challenge reactions were assessed 24 and 48 hours after bandage removal. The epidermal exposure to the test substance in the challenge phase did not result in positive sensitisation reactions in response to any of the test substance concentrations and the vehicle, respectively. Red staining was observed at the test substance treated skin sites, which made scoring of erythema difficult. Therefore, treated skin was evaluated by histopathology for infiltration of immune cells. Histopathological examination revealed no treatment related differences between the 50% test substance concentration treated- and control-skin areas in both experimental and control animals. Moreover, no differences were found in the microscopical findings between experimental and control animals. From these results it was concluded that no sensitisation was induced by the test substance.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No. 1272/2008as amended for the thirteenth time in Regulation (EC) No. 2018/1480.