Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March 2016 - 20 April 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:

No. 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:

Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

EC Number:

245-048-5

EC Name:

Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

Cas Number:

22509-74-6

Molecular formula:

C11H9NO4

IUPAC Name:

ethyl 1,3-dioxo-2,3-dihydro-1H-isoindole-2-carboxylate

Test material form:

solid

Details on test material:

- Appearance: white solid
- Storage conditions of test material: in refrigerator (2-8°C)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in propylene glycol, no data available on stablity in the solvent

OTHER SPECIFICS:
pH (1% in water) 4.36 – 3.38 (determined by WIL Research Europe)

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 old
- Weight at study initiation: 19.8-23.1 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): 21.78-21.9
- Humidity (%): 41-45
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 17 March 2016 To: 20 April 2016

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Main study: 0 (vehicle controls), 10, 25 and 50%
Pre-screen: 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 propylene glycol up to the maximal concentration required by the guideline (50%)
- Irritation: Very slight erythema was noted for the animals treated at 50% on Day 3 only. White staining of the dorsal surface of the ears by test item remnants was noted for all pre-screen animals between Days 1 and 4 but did not hamper the scoring of the ears.
- 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.
- Erythema scores: 1 in both animals of 50% group on day 3. At all other time points the erythema scores were 0 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 WIL Research Europe.
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 (1.036). Homogeneity was assessed by visual inspection of the solutions.
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 six-month reliability check with Alpha-hexylcinnamaldehyde performed at WIL Research is available and indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

17.3

Variability:

± 3.8

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

13.6

Variability:

± 3.1

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

11.1

Variability:

± 3.4

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 (positive control, reliability check)

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 10797, 8510 and 6911 DPM, respectively. The mean DPM/animal value for the vehicle control group was 625 DPM. In the reliability check, the DPM for 25% solution alpha-hexylcinnamaldehyde was 4551 ± 643.

DETAILS ON STIMULATION INDEX CALCULATION
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 which is more often seen in this kind of studies. The response in the higher 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

Main study:

The very slight irritation and/or scaliness of the ears as shown by all animals treated at 50% and one animal treated at 10% was considered not to have a toxicologically significant effect on the activity of the nodes. White staining of test item remnants on the dorsal surface of the ears of all experimental animals did not hamper scoring for erythema.

All except one auricular lymph nodes of the animals of the experimental groups were considered enlarged in size. 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 item induced the Stimulation Indices of 17.3, 13.6 and 11.1 when tested as 10%, 25% and 50% in propylyne glycol, respectively. Based on the results of the study, the test item is considered to be sentising to skin.

Executive summary:

In a GLP-compliant OECD guideline 429 study (LLNA assay), the test item was tested as 10%, 25% and 50% solution in propylene glycol in female CBA/J mice (5/group). The mean DPM/animal values were 10797, 8510 and 6911, respectively, vs. 625 in vehicle controls. The calculated Stimulation Indices were 17.3, 13.6 and 11.1 for 10%, 25% and 50% solution of the test item, respectively. There were no mortalities or clinical signs of systemic toxicity. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The very slight irritation and/or scaliness of the ears was seen in all animals treated at 50% and in one animal treated at 10%. The validity of the study was confirmed by the reliability check with alpha-hexylcinnamaldehyde, performed not more than 6 months previously at the test facility. The mean DMP/animal value obtained for 25% alpha-hexylcinnamaldehyde solution was 4551 ± 643; the respective Stimulation Index was 4.4. Based on the results, the test substance is considered to be sensitizing to skin under the conditions of the study and should be classified as Skin. Sens.1, H317 according to Regulation 1272/2008/EC.