Dibutyl itaconate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28OCT2015 - 23NOV2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

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:

(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, including most recent amendments)

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:

Dibutyl itaconate

EC Number:

218-451-9

EC Name:

Dibutyl itaconate

Cas Number:

2155-60-4

Molecular formula:

C13H22O4

IUPAC Name:

1,4-dibutyl 2-methylidenebutanedioate

Test material form:

liquid

Details on test material:

- Appearance: colourless liquid
- 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: 20.3-24.2
- 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 (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28OCT2015 to 23NOV2015

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 25, 50, 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TEST:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. Two test item concentrations were tested; a 50% and 100% concentration. The test system, procedures and techniques were identical to 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. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). 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. Animals were sacrificed after the final observation.

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 item 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 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.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

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) according to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy for gross macroscopic examination was performed.

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

Key result

Parameter:

SI

Value:

1.2

Variability:

± 0.2

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

1.6

Variability:

± 0.4

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

1.9

Variability:

± 0.4

Test group / Remarks:

100%

Cellular proliferation data / Observations:

Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 1147 (± 140), 1542 (± 284) and 1851 (± 254) DPM, respectively. The mean DPM/animal value for the vehicle control group was 993 DPM.

Any other information on results incl. tables

Results Pre-screen test:

Very slight erythema was noted for the animals treated at 100% on day 3. Variations in ear thickness during the observation period were less than 25% from day 1 pre-dose values. No signs of systemic toxicity were observed in any of the pre-screen animals.

Based on these results, the highest test item concentration selected for the main study was a 100% concentration.

Other results - main study:

Very slight irritation of the ears was shown by some animals treated at 50% and all animals treated at 100% at day 2 and day 3. This was considered not to have a toxicologically significant effect on the activity of the nodes.

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:

GHS criteria not met

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines and GLP principles, DBI was found not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 100% v/v.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 25%, 50% and 100% v/v. Very slight irritation of the ears was shown by some animals treated at 50% and all animals treated at 100% at day 2 and day 3. This was considered not to have a toxicologically significant effect on the activity of the nodes. 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. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 1147, 1542 and 1851 DPM, respectively. The mean DPM/animal value for the vehicle control group was 993 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 1.2, 1.6 and 1.9, respectively. As the SI appeared not to be ≥ 3 when tested up to 100% v/v, DBI was considered not to be a skin sensitiser.