2-[(3S)-2,6-dioxooxan-3-yl]isoindole-1,3-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2016 - 15 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

#440/2008, including most recent amendments

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:

2-[(3S)-2,6-dioxooxan-3-yl]isoindole-1,3-dione

EC Number:

607-813-5

Cas Number:

25830-77-7

Molecular formula:

C13 H9 N O5

IUPAC Name:

2-[(3S)-2,6-dioxooxan-3-yl]isoindole-1,3-dione

Test material form:

solid

Details on test material:

- Appearance: white solid
- Storage conditions: in refrigerator (2-8°C) protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: ca. 10 weeks
- Weight at study initiation: 18.6-24.5 g
- Housing: group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): set to maintain 18-24
- Humidity (%): set to maintain 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 24 May 2016 To: 15 June 2016

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0 (vehicle controls), 10, 25 and 50%

No. of animals per dose:

Main study: 5 females/dose
Pre-screen: 2 females/dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: soluble in N,N-dimethylformamide to at least 50% (maximal concentration required by the guideline)
- Irritation: No irritation was observed
- Systemic toxicity: No signs of systemic toxicity were noted
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White test item remnants were present on the dorsal surface of the ears of both animals at 25% and 50% (between Days 1 and 4), which did not hamper scoring of the skin reactions.
- Erythema scores: 0 at all time points in all animals

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- 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 using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch. The test item preparations (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. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
On 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 (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised.

Positive control substance(s):

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

Statistics:

Not performed

Results and discussion

Positive control results:

The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, are available and confirm the validity of the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

12

Variability:

± 2.0

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

5.9

Variability:

± 1.1

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

5.2

Variability:

± 1.4

Test group / Remarks:

50%

Results 4

Key result

Parameter:

SI

Value:

1

Variability:

± 0.2

Test group / Remarks:

0% (vehicle controls)

Results 5

Key result

Parameter:

SI

Value:

4.4

Variability:

± 0.9

Test group / Remarks:

25% alpha-hexylcinnamaldehyde (reliability check, positive control)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 9361, 4624 and 4054 DPM, respectively. The mean DPM/animal value for the vehicle control group was 780 DPM. The DMP/animal value in the reliability check with 25% alpha-hexylcinnamaldehyde was 4551 ± 643.

DETAILS ON STIMULATION INDEX CALCULATION
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
The response did not follow the expected dose-response relationship, more often seen in these kind of studies. The response of the mid and high dose groups might be less due to differences in skin penetration (less vehicle present).

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between >0 and 10%.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

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

Any other information on results incl. tables

The majority of auricular lymph nodes across all test item treated groups were considered slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In a GLP-compliant guideline study, the test substance induced the Stimulation Indices of 12.0, 5.9 and 5.2 as 10%, 25% and 50% solution in dimethylformamide, respectively. Based on the results of the study the substance is considered to be sensitizing to skin.

Executive summary:

In a GLP-compliant OECD Guideline 429 study (LLNA assay), the test substance at concentrations 10%, 25% and 50% solution in dimethylformamide induced the Stimulation Indices of 12.0, 5.9 and 5.2. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 9361, 4624 and 4054 DPM, respectively. The mean DPM/animal value for the vehicle control group was 780 DPM. 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. The majority of auricular lymph nodes across all test item treated groups were considered slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. The validity of the study was confirmed by reliability check with Alpha-hexylcinnamaldehyde performed not more than 6 months previously at the same test facility. The mean DPM/animal value for 25% alpha-hexylcinnamaldehyde was 4551 ± 643. The respective Stimulation Index was calculated to be 4.4. Based on the results of the study, the test substance is considered to be sensitizing to skin under the conditions of the assay and should be classified as Skin Sens. 1, H317 under Regulation 1272/2008/EC.