Hatcol 1760

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 09 June 2008 and 25 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 21/08/2007. Date of signature: 01/08/2008

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

Sponsor's identification :Hatcol 1760
Description : clear colourless slightly viscous liquid
Date received : 04 February 2008
Storage conditions :room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK.
- Age at study initiation: eight to twelve weeks old.
- Weight at study initiation: weight range of 15 to 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Certified Rat and Mouse Diet, ad libitum
- Water: ad libitum
- Acclimation period: acclimatisation period of at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.


IN-LIFE DATES: From: Day 0 To: Day of sacrifice - Day 6

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: undiluted

Main test: 100, 50 and 25%

No. of animals per dose:

Preliminary screening test: 1 mouse

Main test: 5 mice per dose level

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the test material was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1
- Irritation: The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded.
- Lymph node proliferation response: not recorded for preliminary screening test.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:Local Lymph Node Assay

- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".



TREATMENT PREPARATION AND ADMINISTRATION
Preparation:
For the purpose of the study, the test material was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration.

Administration:
Groups of five mice were treated with the undiluted test material or the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:

One group of five animals was treated with 50 µl (25 µl per ear) of  Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

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

Concentration % v/v in acetone/olive oil 4:1: 15
Stimulation Index 10.91
Result Positive

Conclusion.
alpha-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Concentration (% w/w) in acetone/olive oil 4:1 Vehicle: N/A 25%: 1.49 50%: 1.28 100%: 1.72

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration (% w/w) in acetone/olive oil 4:1 Vehicle: 976.59 DPM 25%: 1453.95 DPM 50%: 1253.70 DPM 100%: 1679.04

Any other information on results incl. tables

Preliminary screening test

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 100%, 50% and 25% v/v inacetone/olive oil 4:1.

 Main Test

 Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph nodes for each individual animal and the stimulation index are given in the table below.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (100%, 50% and 25% v/v in acetone/olive oil 4:1).

Individual Disintegrations per Minute and Stimulation Indices

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	1072.01	976.59 (±196.39)	N/A	N/A
	1-2	1219.46
	1-3	758.67
	1-4	791.01
	1-5	1041.80
25	2-1	1686.81	1453.95 (±401.66)	1.49	Negative
	2-2	1738.25
	2-3	1252.50
	2-4	837.92
	2-5	1754.26
50	3-1	1530.47	1253.70 (±451.10)	1.28	Negative
	3-2	1170.77
	3-3	1003.25
	3-4	1859.14
	3-5	704.89
100	4-1	2292.24	1679.04 (±736.95)	1.72	Negative
	4-2	1689.67
	4-3	2483.37
	4-4	1219.36
	4-5	710.54

The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test groups. 

dpm= Disintegrations per minute

a= Total number of lymph nodes per animal is 2

b= Stimulation Index of 3.0 or greater indicates a positive result

N/A= Not applicable

 Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a non sensitiser under the conditions of the test.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

§        OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted)

§        Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of the undiluted test material or the test material as asolutioninacetone/olive oil 4:1at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

Results. The Stimulation Index 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 (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
25	1.49	Negative
50	1.28	Negative
100	1.72	Negative

Conclusion. The test material was considered to be a non‑sensitiser under the conditions of the test.