5-[4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid, L-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2015 - 20 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference 1

Reference Type:

other: study amendment

Title:

Unnamed

Year:

2021

Report date:

2021

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

5-[(S)-4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid

EC Number:

926-197-1

Cas Number:

2561176-48-3

Molecular formula:

C20 H15 N3 O9

IUPAC Name:

5-[(S)-4-Carboxy-4-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyrylamino]-2-nitro-benzoic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name (as cited in study report): P-Glupa-C
- Appearance: yellow powder
- Storage conditions: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 19-24 g
- Housing: Animals were group housed in labeled Makrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Environmental controls for the animal room were set to maintain 18 to 24°C, (actual daily mean range 22.1 – 22.5°C), a relative humidity of 40 to 70% (actual daily mean range 43 – 72%), at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
- During the last 32 hours of the in-life phase the actual daily ranges were: temperature 21.6-22.4°C; relative humidity 63.2 – 77%
- The deviations from the maximum level of relative humidity did not affect the integrity of the study as laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From:01 July 2015 to 20 July 2015

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25, 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
In a pre-screen test, two test substance concentrations were tested, a 25% and 50% concentration. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
INTERPRETATION
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI = 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions.
- Rationale for vehicle: The vehicle was selected on the basis of maximizing the solubility using the test substance data provided by the Sponsor and trial preparation results performed at WIL Research Europe.

Performed according to test guidelines:
- Days 1, 2 and 3: Induction (topical treatment of 25 µL/ear)
- Day 6: Injection of 20 µCi 3H-methyl thymidine. Five hours after the injection, all animals were killed and the ear lymph nodes were excised. A cell suspension of lymph nodes was prepared.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.
- Day 7: Radioactivity measurements

Observations:
- Mortality/Viability: Twice daily.
- Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Irritation: Once daily on Days 1-6 (on Days 1 - 3 within 1 hour after dosing)

Positive control substance(s):

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

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. The SI values calculated for the concentrations 5, 10 and 25% were 1.7, 3.0 and 9.1, respectively. An EC3 value of 10% was calculated using linear interpolation.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

4.9

Test group / Remarks:

25%

Results 2

Parameter:

SI

Value:

3

Test group / Remarks:

10%

Results 3

Parameter:

SI

Value:

7.1

Test group / Remarks:

50%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1923, 3108 and 4485 DPM, respectively. The mean DPM/animal value for the vehicle control group was 635 DPM.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the pre-screen animals. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Bald spots behind the ears were noted for both animals treated at 50% from Day 3 onwards. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration (maximum concentration as required in the test guidelines for solids).

Other results - main study:

- No irritation and no signs of systemic toxicity were observed in any of the animals. Bald spots behind the ears were noted for all animals treated at 50% from Day 3 onwards. Additionally, scaliness was noted for these animals on Day 6.

- All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

- No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study

- Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information (Category 1B) Criteria used for interpretation of results: EU

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, P-Glupa-C was considered to be a skin sensitiser, as the SI was shown to be >= 3 when tested up to and including 50%. The data showed a dose-response. An EC3 value of 10% was determined.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines with P-Glupa-C. Reliable positive and negative controls were included. Based on the results of a pre-screen test, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (N,N-Dimethyl formamide).

No irritation and no signs of systemic toxicity were observed in any of the animals. Bald spots behind the ears were noted for all animals treated at 50% from Day 3 onwards. Additionally, scaliness was noted for these animals on Day 6. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. No effect on body weight gain. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1923, 3108 and 4485 DPM, respectively. The mean DPM/animal value for the vehicle control group was 635 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 3.0, 4.9 and 7.1, respectively.

Based on these data, the substance is considered to be a skin sensitiser, as the SI was shown to be >= 3 when tested up to and including 50% and the data showed a dose-response. An EC3 value of 10% was determined. Based on these results, P-Glupa-C needs to be classified as skin sensitizer (Category 1B) according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.