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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21NOV2012 - 10DEC2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Oxooil LS13
- Substance type: organic
- Physical state: Clear colourless liquid
- Expiration date of the lot/batch: 11 October 2013 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen
- Hygroscopic: Yes, store in well-sealed container
- Volatile: Yes, vapour pressure: 500 Pa at 20°C
- Reactivity: Reactive to oxygen
- Specific Gravity / Density: 0.77 – 0.8

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. 9 weeks old)
- Weight at study initiation: 20-25g.
- 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
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Temporary deviations from the minimum level of daily mean relative humidity occurred, but laboratory historical data do not indicate an effect of the deviations.


IN-LIFE DATES: From 21NOV2012 - 10DEC2012

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25, 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two test substance 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 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 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. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. 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 formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
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 the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was performed according to protocol.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A Grubb’s outlier test (GraphPad Software, accessed online on 01 February 2013: http://www.graphpad.com/quickcalcs/Grubbs1.cfm) was conducted for the 50% group to assess whether any outliers (DPM/ animal values) were present.

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)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.6 and 2.8 respectively.
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 522, 591 and 1020 DPM respectively (EC3 value 14,5%). The mean DPM/animal value for the vehicle control group was 362 DPM.

Any other information on results incl. tables

Results Pre-screen test:

At a 100% concentration both animals showed up to grade 3 erythema of both ears (moderate to severe erythema) between days 2 and 6. This level of irritation exceeded the selection criterion (maximum erythema grade 2). On day 6, scales were noted on both ears of one animal. At a 50% concentration both animals showed very slight erythema of one or both ears between days 3 and 5. No signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period at 50 and 100% were less than 25% from Day 1 pre-dose values.

The auricular lymph nodes of the animals of two animals at a 100% concentration appeared larger in size as compared to the other group.

Other results - main study:

Very slight irritation of the ears was shown by all animals treated at a 50% concentration on days 3 and 4. This was considered not to have a toxicologically significant effect on the activity of the nodes.

All auricular lymph nodes of the animals of groups in the main experiment were considered normal in size, except for two animals dosed at 50%. No macroscopic abnormalities of the surrounding area were noted in 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. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered not to be a skin sensitiser, as the SI appeared not 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 up to 50%. No mortality occurred, no clinical signs of systemic toxicity were observed and body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Only minor skin irritation was seen on day 3 and 4 in animals treated with 50%.

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.6 and 2.8 respectively.

Based on these data, the test substance is not classified for sensitisation according to EC Regulation No. 1272/2008.