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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:
2012-03-07 to 2012-04-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
(22 July, 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
EC No.440/2008 (May 30, 2008)
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:
solid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks (beginning of treatment)
- Weight at study initiation: 17.9- 22.7 g
- Housing: Group in cages: Makrolon Type II / III, with wire mesh top
- Diet: Rodent 2019C Teklad Global certified diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 days prior to the start of dosing under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 45-65 %
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
other: Tetrahydrofuran
Concentration:
Test item concentration:
- Pre test: 25, 50 %
- Main test: 12.5, 25, 50 %
No. of animals per dose:
Test item: 5 animals/dose
Control THF: 5 animals
Details on study design:
RANGE FINDING TEST
Pre- test was performed to determine the highest non-irritant test concentrations that did at the same time not induce signs of systemic toxicity. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% (w/v) once daily each on three consecutive days.
- Compound solubility: At 50 % solution in tetrahydrofuran
- Irritation: Yes up to 50 % of test item
- Lymph node proliferation response: The proliferative response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation background DPM was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION
The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Application volume: 25 µL/ear/day.

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6). In the main experiment, prior to the first application (day 1), on study day 3 and prior to treatment with 3HTdR (day 6).
Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis( One-Way-Analysis-of-Variance) was conducted on the DPM values. For all statistical calculations SigmaStat for Windows (Version 2.0) was used.Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice. The periodic positive control experiment was performed with α-Hexylcinnamaldehyde in acetone:olive oil (4+1 v/v) using CBA/CaOlaHsd mice in December 2011.
Stimulation Index: 1, 1.7, 1.81, 5.9 respectively to the concentrations 0, 5, 10, 25 %
The Estimated concentration for a S.I. of 3 (EC3) resulted 14.4% (w/v).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
4.36
Variability:
3.2-5
Test group / Remarks:
12.5 %
Key result
Parameter:
SI
Value:
4.41
Variability:
2.0- 9.5
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
4.34
Variability:
2.8- 4.9
Test group / Remarks:
50 %
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 1 394 - <= 2 319
Test group / Remarks:
12.5 %
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 839 - <= 4 085
Test group / Remarks:
25 %
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 1 216 - <= 2 126
Test group / Remarks:
50 %

Any other information on results incl. tables

Viability / Mortality: No deaths occurred during the study period

Clinical Signs: No symptoms of systemic findings were observed during the study period. On day 3 and day 4, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). From day 3 to 6 the animals treated with a test item concentration of 50% showed an erythema of the ear skin as well (day 3 and day 6: Score 1, day 4 and day 5: Score 2). Animals treated with 12.5% test item concentration did not show any signs of local skin irritation.

Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights: The measured lymph node weights of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node weight was observed in the all dose groups in comparison to the vehicle control group.

Ear Thickness: The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to necropsy (day 6). A statistical relevant increase in ear thickness was not observed.

Ear Weight: The measured ear weight of all animals treated was recorded on day 6 after necropsy. A relevant increase in ear thickness was observed in all dose groups.

The EC3 value could not be calculated, since all S.I.´s are above the threshold value of 3. Also, an extrapolated EC3 could not be calculated, since a clear dose response could not be observed.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
According to the results of the LLNA test, the test item Dimyristylperoxydicarbonate is considered to be a skin sensitiser.
Executive summary:

The LLNA test was conducted according to the OECD guideline 429 adopted 22 July 2012.

In the study the test item Dimyristylperoxydicarbonate dissolved in tetrahydrofuran was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 12.5, 25, and 50% (w/v). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

A control group of five mice was treated with the vehicle (tetrahydrofuran) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3-H-methyl thymidine measured in a β-scintillation counter.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3 and day 4, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). From day 3 to 6 the animals treated with a test item concentration of 50% showed an erythema of the ear skin as well (day 3 and day 6: Score 1, day 4 and day 5: Score 2). Animals treated with 12.5% test item concentration did not show any signs of local skin irritation. A statistically significant relevant increase in ear weights was observed for all tested dose groups. A statistically relevant increase in ear thickness was not observed in any treated group in comparison to the vehicle control group.

In this study Stimulation Indices (S.I.) of 4.36, 4.41, and 4.34 were determined with the test item at concentrations of 12.5, 25, and 50% (w/v) in tetrahydrofuran, respectively, all exceeding the trigger value of 3 for a biologically relevant S.I. increase. A statistically significant increase in DPM value was observed in all dose groups in comparison to the vehicle control group. A statistically significant and biologically relevant increase in lymph node weight was observed in all dose groups in comparison to the vehicle control group. The EC3 value could not be calculated, since all obtained SI´s were above the threshold value of 3. Also, an extrapolated EC3 could not be calculated, since a clear dose response could not be observed.

The test item Dimyristylperoxydicarbonate was found to be a skin sensitiser.