GASIR1

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21/2/2005-16/3/2005

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): GASIR1
- Physical state: red solid
- test material was ground using a Retsch Centrifugal Ball Mill and passed through a 250 µm sieve before use.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B&K Universal Ltd, Hull, UK (CBA/CaBkl strain mice), Charles River UK Limited, Margate Kent, UK (CBA/Ca CruBR strain mice)
- Age at study initiation:8-12 weeks old
- Weight at study initiation:15-23g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-25
- Humidity (%):30-70
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light):12hrs dark/12hrs light

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

2.5%, 5% or 10% in polypropylene glycol

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:10% in polypropylene glycol (w/w)
- Irritation:no
- Lymph node proliferation response:no data

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 the 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 (3H-methyl thymidine) incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index (SI)).
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:Groups of five mice were treated with the test material at concentrations of 2.5%, 5% or 10% w/w in propylene glycol. 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.

Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR:80μCi/ml, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total of 20 μCi to each mouse.

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for dpm and standard deviations where appropriate. Individual and group mean dpm 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
P<0.01 P<0.05 P>0.05
3.4
**
*
(not significant)
Interpretation of Results
SPL PROJECT NUMBER: 2080/006 PAGE 10
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:

α-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test:

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.58

Remarks on result:

other: concentration 2.5%

Results 2

Parameter:

SI

Value:

0.96

Remarks on result:

other: concentration 5%

Results 3

Parameter:

SI

Value:

0.94

Remarks on result:

other: concentration 10%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle: Mean DPM/animal: 1739.57 (+-452.92) Concentration: 2.5% - Mean DPM/animal: 2740.16 (+-2415.56) Concentration: 5% - Mean DPM/animal: 1668.53 (+-338.35) Concentration: 10% - Mean DPM/animal:= 1642.2 (+- 757.83)

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1. No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 2.5%, 5% and 10% w/w in propylene glycol.

Main Test

1 Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute (dpm) per lymph nodes for each individual animal and the stimulation index (SI) are given in Table 2.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (2.5%, 5% and 10% w/w in propylene glycol).

2 Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 3.

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

3 Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4.

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:

GHS criteria not met

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 24 April 2002)

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

Methods. Following a preliminary screening test, three groups, each of five animals, were treated with 50 μl (25 μl per ear) of the test material as a suspension in propylene glycol at concentrations of 2.5%, 5% or 10% w/w. A further group of five animals was treated with propylene glycol alone.

Results. The Stimulation Index (SI) 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 (%w/w) in propylene glycol	Stimulation index (SI)	Result
2.5	1.58	Negative
5	0.96	Negative
10	0.94	Negative

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