methyl-bis[2-(4-methylalicyclyl)propyl]dihydro-heteropolycyle

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - June 2014

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- female mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-test: 9 - 10 weeks (beginning of treatment)
2nd pre-test: 8 - 9 weeks (beginning of treatment)
Main study: 10 - 11 weeks (beginning of treatment)
- Weight at study initiation: 19.3 - 23.4 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5, 10 and 25%

No. of animals per dose:

4

Details on study design:

- VEHICLE AND DOSE SELECTION-
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone/olive oil (4+1, v/v) as vehicle. Vortexing was used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of systemic toxicity. Both animals showed an erythema of the ear skin (Score 1) during the observation period. Furthermore, both animals showed an ear weight increase of 32.0 and 37.4%, exceeding the threshold of 25%.
Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. Both animals showed a very slight erythema of the ear skin (Score 1) during the observation period but no excessive increase in ear weights or ear thickness values during the course of the study.
Thus, the test item in the main study was assayed at 5, 10 and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

- MAIN STUDY-
TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10 and 25% in acetone/olive oil (4+1, v/v). The application volume, 25 µL was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A futher group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE:
Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a scintillation counter.

INTERPRETATION OF RAW DATA:
The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-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.

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).
- Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
- Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) 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.

Results and discussion

Positive control results:

Experiment performed in April 2014 (Harlan study number 1620700) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 2.51, 1.76, and 6.79, respectively.
The EC3 value calculated was 13.7 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

8.1

Test group / Remarks:

test item

Results 2

Parameter:

SI

Value:

1.45

Test group / Remarks:

5 % test item

Results 3

Parameter:

SI

Value:

3.95

Test group / Remarks:

10 % test item

Results 4

Parameter:

SI

Value:

3.41

Test group / Remarks:

25 % test item

Any other information on results incl. tables

CALCULATION AND RESULTS OF INDIVIDUAL DATA

Vehicle: acetone/olive oil (4:1 v/v)

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	68	---	---	---	---
---	BG II	22	---	---	---	---
0	1	5187	5142.0	8	642.8	1.00
5	2	7525	7480.0	8	935.0	1.45
10	3	20359	20314.0	8	2539.3	3.95
25	4	17595	17550.0	8	2193.8	3.41

1 = Control Group

2 -4 = Test Group

a) The mean value was taken from the figures BG I and BG II

b) Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

CALCULATION OF EC3 VALUE

	Test item concentration %	S.I.
Test Group 2	(a)	(b)
Test Group 3	(c)	(d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 8.1%(w/w)

(a,b,c,d) Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No signs of systemic toxicity were observed during the study period. On day 3 and 4, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). Animals treated with 5 and 10% 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.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item test item was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of test item, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10 and 25% (w/w) in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments. A control group of four mice was treated with the vehicle (acetone/olive oil (4+1, v/v)) only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radiolabelIed thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. 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 ³H-methyl thymidine measured in ab-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 4, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). Animals treated with 5 and 10% test item concentration did not show any signs of local skin irritation.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.l. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 1.45, 3.95 and 3.41 were determined with the test item at concentrations of 5, 10 and 25% in acetone/olive oil (4+1, v/v), respectively. The EC3 value calculated was 8.1 % (w/w).