Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 28 November 1994 and 31 December 1994
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 the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Version / remarks:
FIFRA, Pesticide Assessment Guidelines Subdivision F; Hazard Evaluation Human and Domestic Animals Section 81-6 "Dermal Sensitisation Study". Revised Edition Nov 1984
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.4100 (Skin Sensitisation)
Version / remarks:
TSCA. Health Effects Testing Guidelines; Subpart E, Section 798.4100 "Dermal Sensitisation".
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 17 July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The work described was performed in compliance with the UK Principles of Good Laboratory Practice (The United Kingdom Compliance Programme, Department of Health 1989).
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
David Hall Limited, Burton-on-Trent, Staffordshire, UK.

- Age at study initiation:
At the start of the main study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the main study the animals weighed 345 to 450g

- Housing:
The animals were housed singly or in pairs in grid bottom polypropylene cages.

- Diet:
Free access to food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the test.

- Water (e.g. ad libitum):
Free access to mains tap water was allowed throughout the test.

- Acclimation period:
A minimum acclimation period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
The animal room was maintained at a temperature of 20 to 22 deg C

- Humidity (%):
Relative humidity of 50 to 67%

- Air changes (per hr):
The rate of air exchange was approximately 15 changes per hour

- Photoperiod (hrs dark / hrs light):
Lighting was controlled by a time switch to give 12 hours continuous lighting and 12 hours darkness.


IN-LIFE DATES: From: Day one To: Day 24

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
Intradermal Induction:
25% w/v in dried arachis oil B.P
25% w/v in a mixture of Freund's Complete Adjuvant plus distilled water (1:1)
Complete Adjuvant plus distilled water (1:1)

Topical Induction: Undiluted as supplied

Topical Challenge: Undiluted as supplied
75% w/v in dried arachis oil B.P
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Intradermal Induction:
25% w/v in dried arachis oil B.P
25% w/v in a mixture of Freund's Complete Adjuvant plus distilled water (1:1)
Complete Adjuvant plus distilled water (1:1)

Topical Induction: Undiluted as supplied

Topical Challenge: Undiluted as supplied
75% w/v in dried arachis oil B.P
No. of animals per dose:
A group of thirty guinea pigs was used for the main study, twenty test and ten control.
Details on study design:
RANGE FINDING TESTS:

The concentrations of test material to be used at each stage of the main study was determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:
- Selection of concentration for Intradermal Induction:
Four animals were intradermally injected with preparations of the material (25%, 10%, 5% or 1% w/v in dried arachis oil B.P). The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection according to the Draize scale shown in Attachment 2. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation and was well tolerated systemically, was selected for the intradermal induction stage of the main study.

- Selection of Concentration for Topical Induction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant nine days earlier) were treated with the undiluted test material and three preparations of the test material (75%, 50% and 25% w/v in dried arachis oil B.P).

The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure was selected for the topical induction stage of the main study.

- Selection of Concentrations for Topical Challenge
The undiluted test material and three preparations of the test material (75%, 50% and 25% w/v in dried arachis oil B.P) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to day 14. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:
2

- Exposure period:
1st exposure: 7 days
2nd exposure: 14 days

- Test groups:
Immediately before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 ml each) was made on each side of the mid-line.

Approximately 24 and 48 hours after intradermal injection the degree of erthema at the test material injection sites (ie injection site ii) was evaluated according to the scale shown in Attachment 2.

One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the undiluted test material. The undiluted test material was applied to saturation on filter paper (WHATMAN No. 4: approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25 mm) and covered wit an overlapping length of aluminium foil. The patch and foil were further secured by a strip of surgical adhesive bandage (ELASTOPLAT: approximate size 250 mm x 35 mm) wound in double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours

The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches using the scale shown in Attachment 2.

- Control group:
Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:

i) Freund's Complete Adjuvant plus distilled water in the ratio of 1:1
ii) Dried arachis oil B.P
iii) 50% w/v formulation of dried arachis oil B.P in a 1:1 mixture v/v of Freund's Complete Adjuvant/distilled water.

The topical applications followed the same procedure as for the test animals except nothing was applied to the filter paper. Skin reactions were quantified as for the test animals.

- Site:
Approximately 40 mm x 60 mm on the shoulder region of each animal

- Frequency of applications:
Once for each exposure

- Duration:
Duration of Induction exposure: 21 days

- Concentrations:
i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
ii) a 25% w/v formulation of test material in dried arachis oil B.P
iii) a 25% w/v formulation of test material in a 1:1 preparation of Freud's Complete Adjuvant plus distilled water.

B. CHALLENGE EXPOSURE
- No. of exposures:
One

- Day(s) of challenge:
Two

- Exposure period:
24 hous

- Test groups:
Shortly before treatment on Day 21, an area approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.

The undiluted test material was applied to saturation to the shorn right flank of each animal on a square of filter paper (WHATMAN No. 4: approximate size 20 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). To ensure that the maximum non-irritant concentration was used at challenge the test material at a concentration of 75% w/v in dried arachis oil B.P. was also similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive banadage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.

After 24 hours the dressing was carefully cut using blunt tipped scissors, removed and discarded. The challenge sites were swabbed with cotton wool soaked in diethyl ether in order to remove residual test material. The position of the treatment skin was identified by using a black inedible marker pen.

Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.

- Control group:
None

- Site:
An area approximately 50 mm x 70 mm on both flanks of each animal

- Concentrations:
75% w/v in dried arachis oil B.P
Undiluted

- Evaluation:
Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified using the scale shown in Attachment 2.

Any other reactions were also recorded.

OTHER:
The percentage of test group animals that showed a more severe reaction at the test material challenge sites than the most severe reaction seen at the corresponding sites in the control group animals was determined.
Challenge controls:
Not applicable
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde Tech. 85%, 2-mercaptobenzothiazole, Ethyl 4-aminobenzoate and 2,4-Dinitrochlorobenzene.

Results and discussion

Positive control results:
Please see Attachment 1 for a table of results.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Undiluted
No. with + reactions:
0
Total no. in group:
30
Clinical observations:
No skin reactions were noted at the challenge sites of the test group animals. Very slight erythema was noted at the challenge site of one control group animals
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Undiluted. No with. + reactions: 0.0. Total no. in groups: 30.0. Clinical observations: No skin reactions were noted at the challenge sites of the test group animals. Very slight erythema was noted at the challenge site of one control group animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Undiluted
No. with + reactions:
0
Total no. in group:
30
Clinical observations:
No skin reactions were noted at the challenge sites of the test group animals. No skin reactions were noted in the control group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Undiluted. No with. + reactions: 0.0. Total no. in groups: 30.0. Clinical observations: No skin reactions were noted at the challenge sites of the test group animals. No skin reactions were noted in the control group animals. .

Any other information on results incl. tables

Skin Reactions Observed After Intradermal Induction:

Very slight erythema was noted at the intradermal induction sites of seven control group animals at the 24 -hour observation and persisted at the intradermal induction sites of sex control group animals at the 48 -hour observation.

Skin Reactions Observed After Topical Induction:

Well-defined erythema was noted at the induction sites of three test group animals with very slight erythema noted at the induction sites of seventeen test group animals at the 1 -hour observation. Bleeding at the intradermal injection sites was noted in three test group animals. Very slight erythema was noted at the induction sites of six test group animals at the 24 -hour observation.

No skin reactions were noted at the treatment sites of the control group animals at the 1 and 24 -hour observations.

TABLES 1 AND 2 ARE ATTACHED

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test material, Alkane 2, produced a 0% (0/20) sensitisation rate.
Executive summary:

1. A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig.

The study was designed to comply the the requirements of the U.S. Environmental Protection Agency (EPA) Guidelines, the OECD Guidelines for Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992) and Method B1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

2. Twenty test and ten control animals were used for the main study.

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal Induction:

25% w/v in dried arachis oil B.P

25% w/v in a mixture of Freund's Complete Adjuvant plus distilled water (1:1)

Complete Adjuvant plus distilled water (1:1)

Topical Induction: Undiluted as supplied

Topical Challenge: Undiluted as supplied

75% w/v in dried arachis oil B.P

3. The test material produced a 0% (0/20) sensitisation rate.