Reaction mass of 3,6-Bis(3-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, 3-(3-Chlorophenyl)-6-(4-chlorophenyl)-2,5-dihydro- pyrrolo[3,4-c]pyrrole-1,4-dione and 3,6-Bis(4-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charies River France, L'Arbresle Cedex, France
- Age at study initiation: approx. 11 weeks old
- Weight at study initiation: 18 - 28g
- Housing: Individual housing in labeled Macrolon cages (Ml type; height 12.5 cm) containing sterilized sawdust as bedding material (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren , The Netheriands).
- Diet: Free access to standard pelleted laboratory animal diet (code VRF 1, Altromin, Lage, Germany).
- Water: Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIll type; height 18 cm). Paper (Enviro-dri, TecniLab-BMl BV, Someren, The Netheriands) was supplied as cage-enrichment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7-22.1
- Humidity (%): 41 - 73
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

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

Remarks:

Acetone p.a.: Merck, Darmstadt, Germany; Olive oil: Sigma-Aldrich, Steinheim, Germany

Concentration:

5, 10, 25%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 25% test substance concentration was considered the highest concentration that could be prepared homogeneously to a visible acceptable level.
- Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration ( 10 or 25%) on three consecutive days. Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. Bodyweights were determined on day 3. The animals were sacrificed after the final observation and no necropsy was performed. Staining by the test substance prevented scoring for erythema. As no signs of necrosis were present, a 25% concentration was selected as highest concentration that could technically be used in the main study.

MAIN STUDY
- Test Item Prepatation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
- Three groups of five animals were treated with one test substance concentration ( 5, 10, 25%) per group. One group of five animals was treated with vehicle.
- Induction-Days 1, 2 and 3: The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
- Treatment - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of ³H-methyl thymidine (Amersham Biosciences, Buckinghamshire, UK). After approximately five hours, all animals were killed by intra peritoneal injection with pentobarbital (0.2 mL/animal Euthesate®; Sanofi Sante B.V., Maassluis, The Netheriands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
- Tissue processing for radioactivity - Day 6: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4°C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) at 4°C during the night.
- Radioactivity measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

OBSERVATIONS
- Mortality/Viability: Twice daily; Toxicity: At least once daily; Body weights: On days 1 (pre-treatment) and 6; Irritation: On day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Positive control substance(s):

other: alpha-hexylcinnamic aldehyde

Positive control results:

The SI values calculated for the substance concentrations 5, 10 and 25% were 2.1, 3.6 and 7.5, respectively. An EC3 value of 8.0% was calculated using linear Interpolation

Parameter:

SI

Remarks on result:

other: Concentration (% w/w); Stimulation Index ± SEM Control: 1 5%: 1.3 ± 0.5 10%: 1.6 ± 0.4 25%: 1.5 ± 0.6

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration (% w/w); Mean DPM ± SEM - Control: 130 ± 51 - 5%: 173 ± 71 - 10%: 206 ± 44 - 25% : 191 ± 88

Preliminary irritation study: Staining by the test substance prevented scoring for erythema. As no signs of necrosis were present, a 25% concentration was selected as highest concentration that could technically be used in the main study.

Main study:

- Induction phase: Skin effects seen after the third epidermal exposure: red staining was observed at all epidermally test subsstance treated skin sites. Red discolouration of the faeces was observed in group 3 and 4 animals, which was considered being related to uptake of the red test substance.

- Macroscopy of the nodes and surrounding area: The majority of nodes were considered normal in size, except for the nodes of animal 4 (in the vehicle control group), which were small (considered being related to low body weight), and the left node of animal 18 (25% concentration group), which was considered slightly enlarged.

- Body weights: The slight body weight loss, noted in some animals including animal 4 (lean appearance), was considered not toxicologically significant.

- Toxicity and Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation, LLNA:

In a GLP-compliant study, performed according to OECD guideline 429, the potential of the test substance to induce contact hypersensitivity in the mouse (Local Lymph Node Assay) was assessed (NOTOX 2006). Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, three groups of five experimental animals were epidermally exposed to test substance concentrations of 5%, 10% or 25% on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with ³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index is calculated for each group. The majority of nodes were considered normal in size, except for the nodes of animal 4 (vehicle control group), which were small (considered being related to low body weight), and the left node of animal 18 (25% concentration group), which was considered slightly enlarged. The slight body weight loss, noted in some animals including animal 4 (lean appearance), was considered not toxicologically significant. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5,10 and 25% were 173, 206 and 191, respectively. The mean DPM/animal value for the vehicle control group was 130. The SI values calculated for the substance concentrations 5,10 and 25% were 1.3,1.6 and 1.5, respectively. There was no indication that the test substance could elicit an SI ≥ 3. It was established that the EC3 value (if any) exceeds 25%. Based on these results the test substance would not be regarded as skin sensitizer.

Migrated from Short description of key information:
The test substance is not considered to be a skin sensitizer.

Justification for classification or non-classification

Based on the results of the skin sensitisation study, the test material does not need to be classified according to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 as amended for the thirteenth time in Regulation (EC) No. 2018/1480..