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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:
2011-11-06 to 2011-11-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive and done to a valid guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Dark Brown Liquid
Specific details on test material used for the study:
EC Number: 939-946-2

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals:4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 9 weeks old (age-matched, within one week)
Body weight range at starting: 20.6-21.9 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 6 days
Note: In the preliminary experiment, mice of 11-12 weeks of age (21.3-24.6 grams) were used.
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.2 – 24.7 °C
Relative humidity: 31 - 70 %
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Animals received ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice – breeding and maintenance"
Animals received tap water from the municipal supply from 500 mL bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly.
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100, 50, 25 and 10% w/v
No. of animals per dose:
Preliminary Test: 2 mice per dose level
Main Study: 4 mice per dose level
Details on study design:
The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI).
Groups of four female CBA/J Rj mice were treated with:
- four groups received 100, 50, 25 and 10 % (w/v) test substance (formulated in AOO);
- the negative (vehicle) control group received the vehicle (AOO);
- the positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA) in AOO.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Results and discussion

Positive control results:
Positive control
Measured DPM/Group 15601
DPM 15543.0
No. of Lymph Nodes = 8
DPN 1942.9
Stimulation Index = 14.5

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
>= 33.9 - <= 59.5
Variability:
The SI range was between SI 33.9 up to 59.5 for the test item concentration range applied
Test group / Remarks:
4 test groups with 4 animals each were tested for the test item in the range of 10 (w/v)% to 100 (w/v)%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: The appearance of the lymph nodes was normal in the negative (vehicle) control group. Larger than normal lymph nodes were observed in all test item treated groups, furthermore in the positive control group.
DETAILS ON STIMULATION INDEX CALCULATION: DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 (w/v) % TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

CLINICAL OBSERVATIONS:No mortality was observed during the study. Hyperactivity and intermittent tremors were observed after treatment on Day 2 for some animals in the 100 and 50 % (w/v) dose groups, increased aggressiveness was also observed for one animal. Similarly, hyperactivity, increased aggressiveness and intermittent tremors were observed after treatment on Day 3 for all animals in the 100 and 50 % (w/v) dose groups. Due to the observed effects, animals were repeatedly observed after treatment on these days; 4 hours after treatment all the animals became symptom-free on both days.
Alopecia was observed on Day 6 in three animals of the 100 and 50 % (w/v) dose groups, and in one animal of the 25 % (w/v) % dose group.
Very slight erythema (score 1) was detected after treatments at the site of application in the 100 % (w/v) dose group on Days 1-3, furthermore in the 50 % (w/v) dose group on Days 2-3. Quantification of the ear thickness was performed in the main experiment by ear punch weight determination after the euthanasia of the experimental animals. The revealing ear punch weights were in the historical control range; although in the highest dose group slightly higher values compared to the negative control group were observed (the increase was less than 25 %).

BODY WEIGHTS: No treatment related effects were observed on body weight.

Any other information on results incl. tables

 Test Group  DPM  No. Lymph Nodes  DPN  Stimulation Index
 Negative (Vehicle) Control (AOO)  1074  8  134.3  1.0
 100% Test Item  55381  8  6922.6  51.6
 50% Test Item  63871  8  7983.9  59.5
 25% Test Item  56950  8  7118.8  53.0
 10% Test Item  36449  8  4556.1  33.9
 Positive Control (25% HCA)  15543  8  1942.9  14.5

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Remarks:
Migrated information
Conclusions:
Under the conditions of the present assay the test substance, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.
The study is considered to be relevant, reliable, adequate for risk assessment, and adequate for classification purposes.
Executive summary:

Study Design:

The aim of this study was to determine the skin sensitisation potential of the test substance following dermal exposure to CBA/J Rj mice. For this purpose, the LLNA method was used. The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for 3 consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI). Groups of four female CBA/J Rj mice were treated with: - four groups received 100, 50, 25 and 10 % (w/v) test substance (formulated in AOO); - the negative (vehicle) control group received the vehicle (AOO); - the positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA) in AOO.

Results:

No mortality was observed during the study. No treatment related effects were observed on animal body weights in any treated groups. Hyperactivity, intermittent tremors or increased aggressiveness were observed after treatment on Day 2 for some animals in the 100 and 50 % (w/v) dose groups. Similarly, hyperactivity, increased aggressiveness and intermittent tremors were observed after treatment on Day 3 for all animals in the 100 and 50 % (w/v) dose groups. Due to the observed effects, animals were repeatedly observed after treatment on these days; 4 hours after treatment all the animals became symptom-free on both days. Alopecia was observed on Day 6 in some animals of the 100, 50 and 25 % (w/v) dose groups. Very slight erythema (score 1) was detected after treatments at the site of application in the 100 % (w/v) dose group on Days 1-3, furthermore in the 50 % (w/v) dose group on Days 2-3. Quantification of the ear thickness was performed in the main experiment by ear punch weight determination. The revealing ear punch weights were in the historical control range; although in the highest dose group slightly higher values were observed when compared to the negative control group. Larger than normal lymph nodes were observed in all test item treated groups. The observed stimulation index values were 51.6, 59.5, 53.0 and 33.9 at concentrations of 100, 50, 25 and 10 % (w/v), respectively.

In conclusion, under the conditions of the present assay, tested in a suitable vehicle, the test substance was shown to have skin sensitisation potential (sensitiser) in the Local Lymph Node Assay.