2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 01 To August 06, 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

Version March 2003

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 24 April 2002.

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2006-09-O2 / Signed on 2007-01-19.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-{[(2R)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate; 2-{[(2S)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate

EC Number:

700-118-9

Cas Number:

676532-44-8

Molecular formula:

C16H28O3

IUPAC Name:

2-{[(2R)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate; 2-{[(2S)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate

Test material form:

liquid

Details on test material:

- Description: Colourless liquid
- Formula: C16H28O3
- Molecular weight: 268,4 g/mol

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: 18.6-22.7 g (beginning of acclimatization)
- Housing: individually (cage type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen).
- Diet: Pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum (Gemeindewerke, D-64380 Rossdorf).
- Acclimation period: at least 5 days prior to the start of dosing under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-110 %
- Air changes: 10-15 changes/hour
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.
IN-LIFE DATES: From July 01 to August 06, 2008.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: 100% and 10%, 25% and 50% in the vehicle Acetone/olive oil
Main test (not valid): 100% and 25%, 50% in the vehicle Acetone/olive oil (4/1, v/v)
Main test (valid test): 5%, 10% and 25% in the vehicle Acetone/olive oil (4/1, v/v)

No. of animals per dose:

Preliminary screening test: 2
Main study: 5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was 100% of the undiluted test item. A 50% solution was achieved in acetone: olive oil (4+1).
- Irritation: to determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 10, 25, 50, and 100 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
- Systemic toxicity: based on the data obtained in the pre-experiment doses of 25, 50, and 100 % were selected for the first main experiment. Nevertheless, during this experiment systemic toxicity and mortality occurred in both the mid dose (50%) and the high dose (100%). Since systemic toxicity has to be avoided in the local lymph node assay, this experiment was considered not valid and a repeat experiment was performed at lower test item concentrations.
The test item in the valid main study was, therefore, assayed at 5, 10, and 25 %. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
The main experiment had to be repeated, since in the first experiment signs of clinical toxicity and mortality occurred in the mid and high dose. Therefore, the first experiment was considered not valid and was repeated using a concentration range avoiding systemic toxicity.
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item is regarded as a sensitiser 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a glass beaker on a tared Mettler balance and the vehicle (acetone:olive oil 4:1 (v/v)) was quantitatively added. A weight by weight dilution was prepared using magnetic stirrer as homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. The preparation was made shortly before each dosing.
The test item in the main study was assayed at four consecutive concentrations that were selected by the sponsor. The top dose is the highest level that could be achieved whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations. Concentrations were in terms of material as supplied unless otherwise stated by the sponsor.

- Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10 and 25% (w/v) in acetone:olive oil 4:1 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

- Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 Rl of 81.9 RCi/ml 3HTdR (corresponds to 20.5 RCi 3HTdR per mouse) by intravenous injection via a tail vein.

- Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, D-30827 Garbsen). The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 Rmmesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, D-63110 Rodgau) and thoroughly mixed. The level of 3HTdR incorporation was then measured on a Beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The Beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

other:

Statistics:

The mean values and standard deviations were calculated.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The positive control alpha-Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (6.31) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus, demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle

Results 2

Key result

Parameter:

SI

Value:

1.39

Test group / Remarks:

5% (v/v) in Acetone/Olive oil (4:1)

Results 3

Key result

Parameter:

SI

Value:

2.63

Test group / Remarks:

10% (v/v) in Acetone/Olive oil (4:1)

Results 4

Key result

Parameter:

SI

Value:

5.96

Test group / Remarks:

25% (v/v) in Acetone/Olive oil (4:1)

Results 5

Key result

Parameter:

SI

Value:

6.31

Test group / Remarks:

25% HCA

Results 6

Key result

Parameter:

EC3

Value:

11.7

Test group / Remarks:

Test item

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index of 1.39, 2.63, and 5.96 were determined with the test item at concentrations of 5, 10, and 25 % in acetone:olive oil (4+1), respectively.

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)] + c = 11.7% (w/v)
a = 10
b = 2.63
c = 25
d = 5.96

VIABILITY/MORTALITY: No deaths occurred during the study period

CLINICAL OBSERVATIONS: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period

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.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): none.

Any other information on results incl. tables

Table 7.4.1/1: Grouped Disintegrations per Minute and Stimulation Index

Test item concentration		DPM values measured	DPMBG per animal (2 lymph nodes) (a)	S.I. (b)
%(w/v)	Animal No.
---	BG I	48	---	---
---	BG II	32	---	---
---	1	1339	1299	---
---	2	1652	1612	---
---	3	939	899	---
---	4	2277	2237	---
---	5	1694	1654	---
5	6	2138	2098	1.4
5	7	2159	2119	1.4
5	8	2666	2626	1.7
5	9	2360	2320	1.5
5	10	1548	1508	1.0
10	11	4891	4851	3.1
10	12	5346	5306	3.4
10	13	2763	2723	1.8
10	14	2207	2167	1.4
10	15	5214	5174	3.4
25	16	8952	8912	5.8
25	17	8567	8527	5.5
25	18	9971	9931	6.4
25	19	8277	8237	5.3
25	20	10298	10258	6.7
25 % HCA	16	7368	7328	4.8
25 % HCA	17	9157	9117	5.9
25 % HCA	18	9333	9293	6.0
25 % HCA	19	15006	14966	9.7
25 % HCA	20	7926	7886	5.1

BG = Background (1 ml 5% trichloroacetic acid) in duplicate 1=Control Group 2-4 = Test Group S.I. = Stimulation Index a)=values corrected for mean background value (BGI and BGII). b)= Stimulation Indices relative to the mean of the control group (Group 1)HCA = alpha-hexyl cinnamic acid (positive control)

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, three groups each of five female mice were treated for three consecutive days (D1, D2, D3) with the test item at a concentrations of 5, 10, and 25 % (w/v) in acetone:olive oil (4+1) ). A further group of five animals was treated with Acetone/Olive oil (4:1). The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per individual lymph nodes and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

All treated animals survived the scheduled study period and no signs of toxicity were observed. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 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.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 1.39, 2.63, and 5.96 were determined with the test item at concentrations of 5, 10, and 25 % in acetone:olive oil (4+1), respectively. The test item was found to be a skin sensitiser and an EC3 value of 11.7 % (w/v) was derived.

The positive control alpha-hexyl cinnamic aldehyde (HCA) was tested in parallel to the test item. At a concentration of 25 % in acetone:olive oil (4+1) the positive control induced an S.I. of 6.31. Therfore, the validity of the present study is ensured.

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation EC No. 1272 /2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin sensitisation endpoint.