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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:
July 16, 2013 - August 5, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Off-white powder
- Storage condition of test material: At room temperature in the dark
Specific details on test material used for the study:
pH (1% in water, indicative range): 6.0 - 5.8

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
(main study: approx. 9 weeks old; range finding tests: approx. 9-10 weeks old)
- Weight at study initiation:
Main study: 19-23 g; range finding tests: 20-27 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): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Variations to these conditions occurred (i.e. maximum relative humidity between 71 and 91% on 14 days during acclimatization, prescreen and main study and a maximum temperature of 24.1°C on a single day of the main study). Based on the laboratory’s extensive experience with variations in these parameters and absence of any clinical signs among the animals that could be associated to these variations, these were considered to have no effect on the outcome of the study.

IN-LIFE DATES: From: July 17, 2013 to August 5, 2013

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25, 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS
- In a pre-screen test, two test substance concentrations were tested; a 25% and 50% concentration. Two young adult animals per concentration were selected. Each animal was treated with one concentration for three consecutive days. Ear thickness measurements were conducted prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.
- Based on the results of this pre-screen test (see below), six additional animals were treated in a similar manner with 10, 25 and 50% at a later stage

MAIN STUDY
INTERPRETATION
- 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 dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels by shaking, vortexing and stirring. This resulted in a turbid formulation for the 10 and 25% concentration and in a suspension for the 50% concentration.
- Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research.

Performed according to test guidelines:

- Days 1, 2 and 3: Induction. Topical treatment of 25 µL/ear, using a pipette or spatula (highest concentration) at approx. the same time on each day
- Day 6: Injection of 20 µCi 3H-methyl thymidine. Five hours after the injection, all animals were killed and the ear lymph nodes were excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL Phosphate buffered saline (PBS).
- Day 6: Tissue processing for radioactivity. A single cell suspension of lymph node cells was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). The cells were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4°C. To precipitate the DNA, the cells were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day.
- Day 7: Radioactivity measurements using a Packard scintillation counter (2800TR). Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, US) as the scintillation fluid.

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 within 1 hour after dosing)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

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.The SI values calculated for the concentrations 5, 10 and 25% were 1.2, 1.6 and 7.7, respectively. An EC3 value of 13.4% was calculated using linear interpolation.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
Substance concentration 10%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
Substance concentration 25%
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
Substance concentration 50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The auricular lymph nodes of four animals at 50% appeared larger in size compared to the other treated groups. This increase in size did not show a clear relation to the DPM/animal values. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 410, 410 and 722 DPM respectively. The mean DPM/animal value for the vehicle control group was 367 DPM. Only four animals of the control group were available for DPM measurements as one animal died during 3H-methyl thymidine injection procedure. Variability in measured DPM values was within the expected variation for this type of study.

EC3 CALCULATION
Not applicable

CLINICAL OBSERVATIONS
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
No irritation of the ears was observed in any of the animals examined. White test substance remnants were visible on the ears on Days 1 -3, which did not hamper scoring of the skin reactions.

BODY WEIGHTS
Body weights and body weight gain of experimental animals were considered to have been unaffected by treatment. 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.

Any other information on results incl. tables

Results Pre-screen test:

In the first pre-screen test body weight loss was noted for all animals. Since no irritation and no clinical signs were noted (except for bald forepaws in one animal), toxicological relevance of the body weight loss was doubted also since most of the animals had starting body weights higher than normal. Therefore the pre-screen test was extended with three concentrations.

In this second pre-screen test, the body weight loss noted for some animals at 25 and 50% was not considered to be toxicologically relevant as the body weight loss was very slight and no other signs of systemic toxicity were noted. Redness was noted for both animals at 50% on Day 5, and scabs were noted for both animals on Day 6. No signs of systemic toxicity were observed in any of the animals examined, and no irritation was noted for any other animal. White test substance remnants were visible on the ears of all animals on Days 1-3, which did not prevent scoring of the ears. Scabbing was seen for two animals at 50% on Day 6. Variations in ear thickness during the observation period remained within the acceptable range (i.e. less than the maximum of 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 (highest technically possible concentration for a solid).

Applicant's summary and conclusion

Interpretation of results:
other: the substance does not need to be classified as skin sensitiser according to GHS and CLP
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD 429 and according to GLP principles, the substance was considered not be a skin sensitiser, as the SI was shown to be < 3 when tested up to and including 50%.
Executive summary:

An LLNA skin sensitisation study was performed according to OECD 429 and according to GLP principles with the substance. Reliable positive and negative controls were included. Based on the results of pre-screen tests, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (dimethylformamide). No irritation of the ears was observed in any of the animals examined. The auricular lymph nodes of four animals at 50% appeared larger in size as compared to the other animals. This increase in size did not show a clear relation to the DPM/animal values. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 410, 410 and 722 DPM, respectively. The mean DPM/animal value for the vehicle control group was 367 DPM. Only four animals of the control group were available for DPM measurements as one animal died during 3H-methyl thymidine injection procedure. Variability in measured DPM values was within the expected variation for this type of study. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 1.1 and 2.0, respectively. Based on these data, the substance is considered not be a skin sensitiser, as the SI was shown to be < 3 when tested up to and including 50%. The substance does not need to be classified as skin sensitiser according to GHS and CLP.