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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:
02 March, 2016 - 21 March, 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(May 2008, including most recent amendments)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 18.1 - 22.5 g
- 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

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25 and 60%
No. of animals per dose:
5
Details on study design:
WEIGHT OF EVIDENCE ANALYSIS:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected.

RANGE FINDING TESTS:
Two test item concentrations were tested; a 25% and 60% concentration. The highest concentration was the maximum concentration that could technically be applied.
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. 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 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 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 vehicle was chosen from the vehicles specified in the test guideline.

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 according to study plan.
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 (see attached background material).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
60%
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
795
Test group / Remarks:
10%
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
613
Test group / Remarks:
25%
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
862
Test group / Remarks:
60%

Any other information on results incl. tables

Results Pre-screen test:

Very slight erythema was noted for all pre-screen animals between Days 3 and 6. No signs of systemic toxicity were observed. White staining of the dorsal surface of the ears by test item remnants was noted for all animals. The staining did not hamper the scoring of the ears.

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 item concentration selected for the main study was a 60% concentration.

Other results - main study:

No irritation was observed in any of the animals.

White staining of the dorsal surface of the ears by test item remnants was noted all animals treated at 25% and 60%. The staining did not hamper the scoring of the ears.

 

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:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, isobutyl 4-hydroxybenzoate was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to and including 60% (v/v).
Executive summary:

A LLNA skin sensitisation study was performed with isobutyl 4-hydroxybenzoate according to OECD 429 test guideline and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 60% (v/v). Reliable positive and negative controls were included.

No irritation was observed in any of the animals. White staining of the dorsal surface of the ears by test item remnants was noted all animals treated at 25% and 60%. The staining did not hamper the scoring of the ears.

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 10, 25 and 60% were 795, 613 and 862 DPM, respectively. The mean DPM/animal value for the vehicle control group was 757 DPM and for the positive control (25%) was 4551 DPM.

The SI values calculated for the test item concentrations 10, 25 and 60% were 1.0, 0.8 and 1.1, respectively.

As the SI appeared not to be ≥ 3 when tested up to and including 60% (v/v), isobutyl 4-hydroxybenzoate was considered not to be a skin sensitiser and does not have to be classified according to Regulation (EC) No. 1272/2008 on the classification, labelling and packaging of substances and mixtures (including all amendments).