Indoline-2,3-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

Study period:

From March 10, 2015 to May 29, 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 429 and EU Method B.42. GLP study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Harlan Laboratories S.r.l San Pietro al Natisone (UD) Zona Industriale Azzida, 57, 33049 Italy.
- Strain: CBA/CaOlaHsd (On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used).
- Age at study initiation: 10 weeks old
- Weight at study initiation: 21.2 – 23.0 g (the weight variation in animals in the study did not exceed ± 20% of the mean weight)
- Housing: Group caging / mice were provided with glass tunnel-tubes; Cage Type II (polypropylene / polycarbonate); Bedding was available to animals during the study.
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 680 2237 / 814 3108, Expiry date: March 2015 / August 2015) produced by Sniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): tap water from the municipal supply from 500 mL bottle, ad libitum.
- Acclimation period: 3 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4-24.9°C
- Humidity (%): 27-60 %
- Air changes (per hr): Ventilation: 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: 11 March 2015; To: 14 April 2015.

Vehicle:

dimethylformamide

Remarks:

(DMF)

Concentration:

50, 25 and 10% (w/v).

No. of animals per dose:

20 animals: 4 animals/group distributed in 3 treated groups, 1 positive control group and 1 solvent control group.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest achievable concentration for the test based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 50% (w/v).
- Irritation: Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6. Slightly increased ear thickness values were detected in some cases, but below the limit of positivity in all cases.
- Lymph node proliferation response: The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in both dose groups.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay - Proliferation assay
- Criteria used to consider a positive response: Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION:
The solubility of the test item was examined in a short Preliminary Compatibility Test. The standard OECD vehicles were assessed: Acetone : Olive oil 4:1 (v:v) mixture (AOO), N,N-Dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200 (1% Pluronic). The 100% (w/v) formulation was not achievable using any of these vehicles. The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be DMF and the highest achievable concentration was 50% (w/v).
Animals were topically dosed with 25 μL of the appropriate formulation on the dorsal surface of each ear. 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.
Each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Individual body weights were recorded on Day 1 and on Day 6. Ear thickness was measured on Day 1, 3 and 6 and after the euthanasia.

Positive control substance(s):

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

Positive control results:

The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (DMF) using CBA/CaOlaHsd mice. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 9.5) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Parameter:

SI

Remarks on result:

other: The stimulation index values were 1.9, 3.5 and 3.1 at concentrations of 50, 25 and 10% (w/v), respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The disintegrations per minute (DPM) were 7807, 14068 and 12359 at concentrations of 50, 25 and 10% (w/v), respectively.

The test item is regarded as a sensitizer if both of the following criteria are fulfilled:

- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.

- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

No mortality or signs of systemic toxicity were observed during the study, either the preliminary or the main test. There were no indications of any irritancy at the site of application. No marked body weight loss (>5%) was detected for any animals except of one animal in the 25% (w/v) group and one animal in the positive control group, but based on the body weights of the other animals in these groups, these facts were considered as animal variability.

	Mean initial body weight (g)	Mean terminal body weight (g)	Change (%)
Negative (vehicle) control (DMF)	21.8	21.9	0.6
Indoline-2,3-dione 50% (w/v)	21.8	22.2	1.7
Indoline-2,3-dione 25% (w/v)	21.7	22.0	1.4
Indoline-2,3-dione 10% (w/v)	21.8	22.1	1.3
Positive control (25% (w/v) HCA in DMF)	21.9	21.4	-2.1

Test group name	Measured DPM/group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	33 35	-	-	-	-
Negative (vehicle) control (DMF)	4080	4046.0	8	505.8	1.0
Indoline-2,3-dione (50% (w/v) in DMF)	7841	7807.0	8	975.9	1.9
Indoline-2,3-dione (25% (w/v) in DMF)	14102	14068.0	8	1758.5	3.5
Indoline-2,3-dione (10% (w/v) in DMF)	12393	12359.0	8	1544.9	3.1
Positive control (25% (w/v) HCA in DMF)	38471	38437.0	8	4804.6	9.5

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under the conditions of the present assay, Indoline-2,3-dione, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of Indoline-2,3-dione following dermal exposure, according to OECD Guideline 429, EU Method B.42 and following GLP. The Preliminary Irritation / Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (50 and 25% (w/v) in DMF). Based on the observations recorded in the preliminary test, 50% (w/v) was selected as top dose for the main test. In the main assay, 20 female mice were allocated to 5 groups of four animals each: 3 groups received the test item at 50, 25 and 10% (w/v) concentrations; 1 group received DMF (vehicle control); and 1 group received 25% (w/v) α-Hexylcinnamaldehyde (HCA) (positive control). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate Stimulation Indices (SI). No mortality or signs of systemic toxicity were observed during the study. No treatment related body weight loss was observed. Test item precipitate / minimal amount of test item precipitate was observed on the ears of the animals in the 50% (w/v) dose group. There were no indications of any irritancy at the site of application. The stimulation index values were 1.9, 3.5 and 3.1 at concentrations of 50, 25 and 10% (w/v), respectively (surpassing the threshold of 3). The extrapolated EC3 value was 8.0% (w/v). A significant lymphoproliferative response (stimulation index value of 9.5) was noted for the positive control chemical, this result confirmed the validity of the assay. In conclusion, under the conditions of the present assay, Indoline-2,3-dione, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study: A Local Lymph Node Assay was performed using female CBA mice, in accordance to OECD Guideline 429, EU Method B.42 and following GLP. A Preliminary Irritation/Toxicity test was performed and the top dose of 50% (w/v) on N,N-dimethylformamide was selected for the main test. Twenty animals were distributed in 5 groups: Solvent control, Positive control (25% (w/v) HCA) and 3 dose level groups (10, 25 and 50% (w/v). The test item solutions were applied on the dorsal surface of ears of experimental animals for 3 consecutive days. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No signs of systemic or local toxicity, mortality, or body weight loss were observed during the study. The stimulation index values were 1.9, 3.5 and 3.1 at concentrations of 50, 25 and 10% (w/v), respectively. The extrapolated EC3 value was 8.0% (w/v).

In conclusion, under the conditions of the present assay, Indoline-2,3-dione, tested in a suitable vehicle, showed to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Migrated from Short description of key information:
Key study: OECD guideline 429. GLP study.
In this study Stimulation Indices (S.I) of 3.1, 3.5 and 1.9 (surpassing the threshold of 3) were determined with the test item at concentrations of 10, 25 and 50% (w/v) in N,N-dimethylformamide, respectively. The test item is considered to have skin sensitization properties in this assay.

Justification for selection of skin sensitisation endpoint:
Only one study available (OECD 429, GLP study).

Justification for classification or non-classification

Based on the available information the test substance is classified as skin sensitizer Category 1B according to CLP Regulation (EC) no 1272/2008.