1-cyclohexyl-1H-pyrrole-2,5-dione

Endpoint summary

Description of key information

The extrapolated EC3 value of IMILEX-C is 0.1%w/v and therefore is classified as; Skin Sens. cat 1A.

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

Study period:

14 July to 08 December 2017

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)

Deviations:

no

Remarks:

Deviation from study plan: Actual maximum temperature was 25.8°C instead of 22 ± 3°C. Actual maximum relative humidity was 79% instead of 30-70%. Mice of 1-3 weeks of age used instead of 8-12 weeks in Preliminary Experiment I. No adverse effect on study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: The Sponsor (Nippon Shokubai Co. Ltd.). Lot/batch Number: 5K21
- Expiration date of the lot/batch: 24 May 2018
- Purity test date: N-Cyclohexylmaleimide (98-100% wt), o-xylene (0-0.2% wt)

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (15-25ºC), protected from moisture and light
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: 50% w/v
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item weighed out to required doses and dissolved in 1% Pluronic solution
- Preliminary purification step (if any): Not stated
- Final dilution of a dissolved solid, stock liquid or gel: 0.25, 0.5, 1.0 and 2.5 % w/v
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid (solid dissolved in vehicle solution)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) n/a

OTHER SPECIFICS: n/a

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Specific Pathogen Free (SPF) at arrival
- Age at study initiation: 8 weeks old
- Weight at study initiation: 18.3 – 19.8 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
- Housing: Type II. polypropylene / polycarbonate group caging / mice were provided with glass tunnel-tubes. Bedding of certified wood chips was available to animals during the study
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 – 25.8°C
- Humidity (%): 30 - 79 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 8 weeks +/- 1 week) To: 9 weeks (+/- 1 week)

Vehicle:

other: 1% aqueous Pluronic

Remarks:

1% aqueous Pluronic® PE9200. Batch No. 45015356P0. Expiry: 27 August 2017.

Concentration:

0.25, 0.5, 1.0, 2.5% w/v test item concentration with 1% aqueous Pluronic

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS: Based on the observed ear swelling, an additional Preliminary Irritation/Toxicity Test was performed. Preliminary Experiment I test concentrations at 50% and 25% w/v. Preliminary Experiment II test concentrations at 10%, 5% and 2.5% w/v.

- Compound solubility: 50% w/v (maximum solubility)

- Irritation: In the Preliminary Experiment I (50% and 25% w/v) there was clearly indicated excessive local irritation (increase ≥ 25 %) for both animals of the 50% (w/v) and for both animals of the 25% (w/v) dose group on Day 6 and for both animals of the 50% (w/v) dose group on Day 3. In the Preliminary Experiment II there was clear indicatation of excessive local irritation (increase ≥ 25 %) for both animals of the 10% (w/v) and 5% (w/v) dose groups on Day 6.

- Systemic toxicity: In the Preliminary Experiment I (50% and 25% w/v), Test item precipitate or minimal amount of test item precipitate was observed on the ears of one animal of the 50% (w/v) dose group on Days 1-4 and the other animal of the 50% (w/v) dose group on Days 1-6, and for both animals of the 25% (w/v) dose group on Days 1-5. In the Preliminary Experiment II (10%, 5% and 2.5% w/v) no signs of systemic toxicity were observed. Minimal amount of test item precipitate was observed on the ears of one animal of the 10% (w/v) dose group on Day 3.

- Ear thickness measurements: In the Preliminary Experiment I (50% and 25% w/v), the revealing ear punch weights were out of the historical control range for both animals of the 50% (w/v) and for both animals of the 25% (w/v) dose groups, the increase was clearly above the limit of positive (≥ 25 %) for all animals of both dose groups. In the Preliminary Experiment II (10%, 5% and 2.5% w/v), the revealing ear punch weights were out of the historical control range for both animals of the 10% (w/v), for both animals of the 5% (w/v) and for both animals of the 2.5% (w/v) dose groups. The increase was clearly above the limit of positive (≥ 25 %) for all animals of the 10% (w/v) and 5% (w/v) and for one animal of the 2.5% (w/v) dose group; the mean for the 2.5% group was acceptable. The increased thickness and weight were not attributable to residual test item on the ears; they are considered to indicate an irritant response.

- Erythema scores: Preliminary Experiment I - Slight erythema was observed for both animals of the 50% (w/v) dose group on Days 3-4 and for both animals of the 25% (w/v) dose group on Days 3-5. Well defined erythema was observed for both animals of the 50% (w/v) dose group on Day 5. Preliminary Experiment II - no erythema observed.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferation Assay
- Criteria used to consider a positive response: A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION: The test item was weighed and formulations prepared daily on a weight:volume basis
(as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd. During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. A cut pipette tip was used for the treatment. There were 4 animals per dose treatment. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, each mouse was intravenously injected via the tail vein with 250 μL of sterile Phosphate Buffered Saline (PBS) containing approximately 20 μCi of 3^HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not specified

Positive control results:

No mortality cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 10.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Key result

Parameter:

EC3

Value:

ca. 0.1

Remarks on result:

other: Category 1 skin sensitizer (sub-category 1A).

Remarks:

Known to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay

Parameter:

SI

Value:

21.8

Test group / Remarks:

IMILEX-C 0.25% w/v in 1% Pluronic

Parameter:

SI

Value:

37.3

Test group / Remarks:

IMILEX-C 0.5%w/v in 1% Pluronic

Parameter:

SI

Value:

51.8

Test group / Remarks:

IMILEX-C 1.0%w/v in 1% Pluronic

Parameter:

SI

Value:

47.1

Test group / Remarks:

IMILEX-C 2.5%w/v in 1% Pluronic

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : The appearance of the lymph nodes was normal in the negative control group. Larger than normal lymph nodes were observed in the 2.5% (w/v), 1% (w/v), 0.5% (w/v) and 0.25% (w/v) test item treated dose groups and in the positive control group.

DETAILS ON STIMULATION INDEX CALCULATION : Stimulation index (SI) values were 47.1, 51.8, 37.3 and 21.8 at concentrations of 2.5% (w/v), 1% (w/v), 0.5% (w/v) and 0.25 % (w/v), respectively. Stimulation index for each treatment group was calculated by: (SI = disintegrations per minute (DPN) value of a treated group divided by the DPN value of the negative control group).

EC3 CALCULATION : Since the test item gave a positive response and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3). The calculation of the EC3 value was conducted by log-linear interpolation.

CLINICAL OBSERVATIONS: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained. No mortality or systemic clinical signs were observed during the main study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application.

BODY WEIGHTS: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3^HTdR injection) with a precision of ± 0.1 g. No marked body weight losses (≥5%) was observed on the mean body weight changes in any groups during the main study.

EAR THICKNESS: The detected ear thickness values were within the acceptable range (increase was below the threshold limit of 25%) at 2.5% (w/v) (except one animal, only left ear), 1% (w/v), 0.5% (w/v) and 0.25% (w/v) dose groups on Day 6. The revealing ear punch weights were out of the historical control range for all animals of the 2.5% (w/v) and 1% (w/v) dose groups, however the biopsy weights were within the acceptable range for all four treated groups (a positive response being over 28.16 mg (≥25%). No results were over this).

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The skin sensitisation potential of IMILEX-C (1-cyclohexyl-1H-pyrrole-2,5-dione; CAS 1631-25-0) following dermal exposure in mice was performed as a Local Lymph Node Assay. The study was performed in vivo with vertebrate animals as no full regulatory in vitro alternative was available.

Under the conditions of the present assay, IMILEX-C, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The extrapolated EC3 value of IMILEX-C is 0.1% (w/v). The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1A).

Executive summary:

The skin sensitisation potential of IMILEX-C (1-cyclohexyl-1H-pyrrole-2,5-dione; CAS 1631-25-0) following dermal exposure in mice was performed as a Local Lymph Node Assay. The study was performed in vivo with vertebrate animals as no full regulatory in vitro alternative was available.

In the main assay, twenty-four female CBA/CaOlaHsd mice were allocated to six groups of four animals each for the test item concentration doses (formulated in 1% Pluronic) of 2.5% (w/v), 1% (w/v), 0.5% (w/v) and 0.25 % (w/v). The negative control group received the vehicle (1% Pluronic) only, and the positive control group received 25 % (w/v) HCA (dissolved in 1% Pluronic). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

No mortality or systemic clinical signs were observed during the main study. No test item precipitate was observed on the ears of the experimental animals. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight changes in the main study. The stimulation index values were 47.1, 51.8, 37.3 and 21.8 at concentrations of 2.5% (w/v), 1% (w/v), 0.5% (w/v) and 0.25% (w/v), respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of the present assay, IMILEX-C, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The extrapolated EC3 value of IMILEX-C is 0.1% (w/v). The following classification/labelling is triggered: Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1A).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The extrapolated EC3 value of IMILEX-C is 0.1%w/v and therefore is classified as; Skin Sens. cat 1A.