4-Decenal, 9-hydroxy-5,9-dimethyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2013-05-16 to 2013-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 429 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Dated May 30, 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Compliance certificate (signed on 11 April 2013)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

9-hydroxy-5,9-dimethyldec-4-enal

EC Number:

940-437-2

Cas Number:

926-50-1

Molecular formula:

C12H22O2

IUPAC Name:

9-hydroxy-5,9-dimethyldec-4-enal

Test material form:

liquid

Details on test material:

Appearance: colourless to pale yellow liquid
Expiry date: 18 March 2014, extended to 19 April 2014, and then 22 May 2015

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

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 the beginning of treatment:
1st pre-test: 10 - 11 weeks
2nd pre-test: 9 - 10 weeks
Main study: 8 - 9 weeks
- Weight at the beginning of treatment:
1st pre-test: 19.7-20.9 g
2nd pre-test: 19.9-23.3 g
Main study: 17.7-22.0 g
- Housing: In group, housed, in Makrolon Type II (pre-test) / III (main study) cages, with wire
mesh top with granulated soft wood bedding
- Diet: : ad libitum, 2018C Teklad Global 18% protein rodent diet (certified)
- Water: tap water ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): ca. 45-75%
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5, 10 and 25 % (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
PRE-TEST 1
- Procedure: 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.
- Results: At the tested concentrations the animals did not show any signs of systemic toxicity. From day 3 to 6, both animals showed an erythema of the ear skin and the threshold for earweight of 25% (recommended by the OECD guideline) was exceeded. Therefore, a second pre-test was performed using test item concentrations of 10 and 25%.

PRE-TEST 2
- Procedure: The same procedure that for the first pre-test was used with test item concentrations of 10 and 25%.
- Results: At the tested concentrations the animals did not show signs of systemic toxicity. From day 3 to 6, both animals showed an erythema of the ear skin. In none of the two animals ear weights (compared to historical vehicle data) or ear thickness values were increased by ≥ 25%, as recommended by OECD 429.
Thus, the test item in the main study was assayed at 5, 10, and 25% (w/w). 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
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: individual approach, using tritiated (3H)-methyl thymidine, according to the OECD 429 test guideline.
- Measurement of ear thickness: Prior to the first and third application of the test item (study days 1 and 3) and prior to treatment with 3HTdR (study day 6), the ear thickness was determined using a micrometer. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.
- Determination of ear weights: After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) in the pre-test and main experiment after sacrifice. For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Criteria used to consider a positive response: The decision process with regard to identification of a positive response will include a SI of ≥ 3, together with consideration of dose-response, and where appropriate, statistical significance. Any test item failing to produce a SI of ≥ 3 at all test concentrations will not be regarded as a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test Item Preparation: The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1, v/v) was added. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. The concentration, homogeneity or stability of the formulations was not determined by analysis.
- Administration: Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1, v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.6 µCi of 3H-methyl thymidine (equivalent to 78.4 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
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 µm mesh 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 scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mLaliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

OBSERVATIONS
- Mortality / Viability: yes, at least once daily from experimental start to necropsy.
- Body weights: yes, in the pre-test, prior to the first application and prior to sacrifice and in the main experiment, prior to the first application and prior to treatment with 3HTdR.
- Ear thickness: yes, in the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) and 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: yes, in the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
- Clinical signs (local / systemic): yes, 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 and for the DPM values (group mean DPM ± standard deviation).
The Dean-Dixon-Test and was used for identification of possible outliers (performed with Microsoft Excel 2007).
Also the ANOVA (Dunnett-test) was conducted on the ear weights to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group. Furthermore, a statistical analysis was conducted on the ear thickness values to assess whether a statistically significant increase in ear thickness could be observed when comparing the values measured on day 1 prior to application with the values measured on days 3 or 6 in the respective test item groups or within the vehicle control group. 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). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

Four groups were treated with the positive control α-Hexylcinnamaldehyde as a solution in acetone/olive oil 4:1 at a concentration of 0, 5, 10 and 25% v/v. With a SI = 5.9 at the concentration of 25%, resulting an EC3 value of 12.6% (w/w), α-Hexylcinnamaldehyde was considered to be a sensitiser under the conditions of the test at this dose concentration.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.8

Test group / Remarks:

5%

Results 2

Parameter:

SI

Value:

1.2

Test group / Remarks:

10%

Results 3

Parameter:

SI

Value:

1.6

Test group / Remarks:

25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
0% : mean DPM / animal = 345.3 (SD = 171.0)
5%: mean DPM / animal = 287.9 (SD = 70.4)
10%: mean DPM / animal = 428.1 (SD = 120.7)
25%: mean DPM / animal = 553.5 (SD = 96.7)

Stimulation Indices of 0.8, 1.2, and 1.6 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1, v/v). A dose response was observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. 
No signs of systemic toxicity were observed during the study period. From day 3 to 5, the animals treated with the test item concentrations of 10 and 25% showed an erythema of the ear skin (10%: day 3 and 4 Score 1. 25%: from day 3 to 5 Score 1). Animals treated with 5% test item concentration did not show any signs of local skin irritation.

EARS MEASUREMENTS:
- Ear thickness: The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to treatment with 3HTdR (day 6). A relevant increase in ear thickness was not observed.
- Ear weights: The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase in ear weight (p < 0.05) was observed in all dose groups in comparison to the values of the vehicle control group. However, only in the high dose group (animals treated with 25% test item) the threshold, recommended by the OECD guideline, was slightly exceeded (25.5% in comparison to the vehicle control group).

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:

GHS criteria not met

Conclusions:

Under the test conditions, the test item is not classified as a skin sensitizer in the Local Lymph Node Assay according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following two preliminary screening tests, the test item in the main study was assayed at 5, 10, and 25% (w/w). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation.

Three groups of five 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. A control group of five 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 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 3H-methyl thymidine measured in a β-scintillation counter.

 

All treated animals survived the scheduled study period and no signs of systemic toxicity, body weights changes or local skin irritation were observed. From days 3 to 5, the animals treated with the test item at 10 and 25% showed an erythema of the ear skin (10%: day 3 and 4 Score 1. 25%: from day 3 to 5 Score 1). Animals treated with the test item at 5% did not show any signs of local skin irritation. A statistically significant increase in ear weight (p < 0.05) was observed in all dose groups in comparison to the values of the vehicle control group. However, only in the high dose group (animals treated with 25% test item) the threshold recommended by the OECD guideline was slightly exceeded (25.5% in comparison to the vehicle control group). A statistically increase in ear thickness was not observed in any dose group.

The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration	Mean dpm/animal	SD	S.I.
Vehicle	345.3	171.0	1.0
5%	287.9	70.4	0.8
10%	428.1	120.7	1.2
25%	553.5	96.7	1.6

In this study, Stimulation Indices were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1, v/v). A dose response was observed, but the EC3 value could not be calculated since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 5.9 when tested at 25 % v/v. The test system was therefore considered to be valid.

 

Under the test conditions, the test item is not classified as a skin sensitizer in the Local Lymph Node Assay according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS. This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.