Pentanedioic acid, 2-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-, (2S)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May 2015 - 01 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Data source

Reference

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:

(July 2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(May 2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(March 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:

(2S)-2-(1,3-DIOXO-2,3-DIHYDRO-1H-ISOINDOL-2-YL)PENTANEDIOIC ACID

EC Number:

608-945-6

Cas Number:

340-90-9

Molecular formula:

C13H11NO6

IUPAC Name:

(2S)-2-(1,3-DIOXO-2,3-DIHYDRO-1H-ISOINDOL-2-YL)PENTANEDIOIC ACID

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Phthalyl-L-Glu
- Substance type: Organic
- Physical state: White powder
- Storage condition of test material: At room temperature
- pH (1% in water, indicative range): 2.46 – 2.41 (determined by WIL Research Europe)

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: 18-24g
- Housing: Animals were group housed in 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
- Temperature (°C): 21.9 – 22.1
- Humidity (%): 41 – 56
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 06 May 2015 to 01 June 2015

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0, 10, 25, 50%

No. of animals per dose:

5 (main study); 2 (preliminary test)

Details on study design:

RANGE FINDING TESTS:
In a pre-screen test two test substance concentrations were tested; a 50% and 25% concentration. The test system, procedures and techniques were identical as those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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 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 preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
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. There was no information available regarding the solubility or stability in vehicle.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

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 immediately after dosing) A description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy for gross macroscopic examination was performed according to protocol.

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-hexylcinnamicaldehyde 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

Parameter:

SI

Value:

1.2

Test group / Remarks:

10%

Results 2

Parameter:

SI

Value:

1.3

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

2

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 661, 730 and 1091 DPM, respectively. The mean DPM/animal value for the vehicle control group was 557 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 animals examined. Variations in ear thickness during the observation period were less than 25% from day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined. White staining of test substance remnants on the dorsal surface of the ears of all animals treated at 50% on days 1, 2 and 3, did not hamper scoring for erythema.

 

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 most experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since most changes were slight in nature and no concentration-related incidence was apparent.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines and GLP principles, the substance was considered not to be a skin sensitiser, as the SI was found to be < 3 when tested up to 50%.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and GLP principles. The test substance was tested in female mice at concentrations of 10, 25 or 50% w/w, by open application on the ears for three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (propylene glycol). No mortality occurred, and no clinical signs of systemic toxicity were observed in the treated animals. No irritation of the ears was observed in any of the animals examined, 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. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 661, 730 and 1091 DPM, respectively. The mean DPM/animal value for the vehicle control group was 557 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.2, 1.3 and 2.0, respectively. As the SI was found to be <3 when tested up to 50%, Phthalyl-L-Glu was considered not to be a skin sensitiser.