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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-05-10 to 1999-06-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
(Topical applications in the control group did not use the vehicle in both induction or challenge)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
(Topical applications in the control group did not use the vehicle in both induction or challenge)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Available test report from study conducted from 1999.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid, colourless
- Storage condition of test material: approximately 4 °C in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Ltd., Burton-on-Trent
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 303 to 395 g
- Housing: Singly or in pairs in solid-floor polypropylene cages furnished with woodflakes.
- Diet: ad libitum (Guinea pig FD1 Diet, Special Diets Ltd., Essex)
- Water: Mains tap water ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light, 12 hours continuous darkness.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
5 % at the intradermal induction and undiluted for the topical induction and challenge
Day(s)/duration:
14 days
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
undiluted
Day(s)/duration:
24 hours
No. of animals per dose:
10 in the control group, 20 in the test group
Details on study design:
RANGE FINDING TESTS:
- Selection of Concentration for Intradermal Induction: Four concentrations of test substance were investigated, (1, 5, 10 and 25 % in arachis oil BP). A total of four guinea pigs were used, each guinea pig receiving four 0.1 mL injections of only one concentration of test substance. The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The 5 % concentration 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 15 days earlier) were treated with the undiluted test substance and three preparations of the the test substance (75, 50 and 25 % in ethanol/diethylphthalate 1:1). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage.

- Selection of Concentration for Topical Challenge: The undiluted test substance and three preparations of the test substance (75, 50 and 25 % in v.v in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. The undiluted test substance was found to be the maximum non-irritant concentration. 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 degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, one intradermal (5 %), one topical (undiluted)
- Exposure period: 14 days.
- Test groups: 1 group of 20 animals. Shortly 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 injection (0.1 mL each) was made on each side of the mid-line. The injections consisted of Freund's Complete Adjuvant plus distilled water in the ratio 1:1, a 5 % w/v formulation of the test substance in arachis oil BP and a 5 % w/v formulation of the test substance in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water. Approximately 24 and 48 hours after the intradermal injection the degree of erythema and the test substance injection sites (i.e. injection site b) was evaluated. One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped and treated with a topical application of the undiluted test substance. A filter paper patch (WHATMAN No. 4 approximate size 40 mm x 20 mm) saturated with the undiluted test substance was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. The dressing was kept in place for 48 hours. The degree of erythema and oedema was quantified one and twenty four hours following removal of patches.
- Control group: 1 group of 10 animals, treated identically to the test animals except the injections were Freund's Complete Adjuvant plus distilled water 1:1, arachis oil BP and 50 % w/v formulation of arachis oil BP in a 1:1 mixture of Freund's Complete Adjuvant/distilled water. The topical induction followed the same method as the test groups, however, nothing was applied to the patch.
- Site: 40 mm x 60 mm on the shoulder region of each animal
- Frequency of applications: Day 0 intradermal and Day 7 topical
- Duration: The topical application was occluded for 48 hours.
- Concentrations: 5 % for intradermal induction, undiluted for the topical induction

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 21
- Exposure period: 24 hours
- Test groups: All 20 animals from the induction exposure. An area of approximately 50 mm x 70 mm on both flanks of each animal was clipped. A square filter paper patch (WHATMAN No. 4: approximate size 20 mm x 20 mm) saturated with the undiluted test substance was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage wound in a double layer around the torso. After 24 hours, the dressing was removed. The challenge sites were swabbed with cotton wool soaked in diethyl ether to remove residual material. Prior to the 24 hour observation, the flanks were clipped using veterinary clippers.
- Site: 50 mm x 70 mm right flank
- Concentrations: Undiluted
- Evaluation (hr after challenge): 24 and 48 hours.

OTHER:
- Scales for Evaluation of Skin Reactions:
*Evaluation of Erythema*
No erythema = 0
Discrete or patch erythema = 1
Moderate and confluent erythema = 2
Intense erythema and swelling = 3

*Evaluation of Oedema*
No oedema = 0
Very slight oedema (barely perceptible) = 1
Slight oedema (edges of area well-defined by definite raising) = 2
Moderate oedema (raised approximately 1 mm) = 3
Severe oedema (raised more the 1 millimetre extending beyond the area of exposure) = 4

- Interpretation of reactions:

Percentage of Classification of
Animals Sensitised Sensitisation Potential
0 Non-sensitiser
> 0-8 Weak sensitiser
> 8-28 Mild sensitiser
> 28-64 Moderate sensitiser
> 64-80 Strong sensitiser
> 80-100 Extreme sensitiser
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole (historical control)

Results and discussion

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:
20
Clinical observations:
No reactions noted
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No reactions noted
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Reactions after Intradermal Induction

Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of nineteen test group animals at the 24 hour observation and all test group animals at the 48 hour observation. Discrete or patchy erythema was noted at the intradermal induction sites of seven control group animals at the 24 hour observation and eight control group animals at the 48 hour observation.

Reactions after Topical Induction

Discrete or patchy to moderate and confluent erythema was noted at the topical induction sites of all test group animals at the 1 hour observation. Discrete or patchy erythema was noted at the topical induction sites of six test group animals at the 24 hour observation. Bleeding from the intradermal injection sites was noted in five test group animals at the 1 hour observation. No evidence of erythema or oedema was noted at the topical inductions sites of control group animals at the 1 and 24 hour observations. Bleeding from the intradermal injection site was noted in two control group animals at the 1 hour observation

Reactions after Topical Challenge

No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48 hour observations.

Table 1: Individual Skin Reactions in Test Animals at Challenge

Animal Number

Skin Reactions (Hours After Removal of Dressing)

24 Hours

48 Hours

100%

100%

Erythema

Oedema

Other

Erythema

Oedema

Other

1

0

0

-

0

0

-

2

0

0

-

0

0

-

3

0

0

-

0

0

-

4

0

0

-

0

0

-

5

0

0

-

0

0

-

6

0

0

-

0

0

-

7

0

0

-

0

0

-

8

0

0

-

0

0

-

9

0

0

-

0

0

-

10

0

0

-

0

0

-

11

0

0

-

0

0

-

12

0

0

-

0

0

-

13

0

0

-

0

0

-

14

0

0

-

0

0

-

15

0

0

-

0

0

-

16

0

0

-

0

0

-

17

0

0

-

0

0

-

18

0

0

-

0

0

-

19

0

0

-

0

0

-

20

0

0

-

0

0

-

Table 2: Individual Skin Reactions in Control Animals at Challenge

Animal Number

Skin Reactions (Hours After Removal of Dressing)

24 Hours

48 Hours

100%

100%

Erythema

Oedema

Other

Erythema

Oedema

Other

1

0

0

-

0

0

-

2

0

0

-

0

0

-

3

0

0

-

0

0

-

4

0

0

-

0

0

-

5

+

+

+

+

+

+

6

0

0

-

0

0

-

7

0

0

-

0

0

-

8

0

0

-

0

0

-

9

0

0

-

0

0

-

10

0

0

-

0

0

-

+ Animal found dead Day 20

 

Body weight

Body weights of guinea pigs in the test group between Day 0 and 24 were comparable to those observed in the control group animals over the same period.

 

Table 3: Body Weights and Body Weight Gains

Group

Animal Number

Body weight (g)

Body weight increase (g)

Day 0

Day 24

Test

1

347

488

141

2

313

478

165

3

324

502

178

4

362

632

270

5

344

555

211

6

328

589

261

7

303

589

286

8

332

532

200

9

316

482

166

10

343

531

188

11

358

588

230

12

343

532

189

13

356

609

253

14

383

578

195

15

336

574

238

16

351

550

199

17

356

560

204

18

334

576

242

19

352

550

198

20

379

633

254

Control

1

369

617

248

2

320

535

215

3

315

517

202

4

329

529

200

5

345

+

+

6

359

518

159

7

361

573

212

8

379

555

176

9

334

500

166

10

395

596

201

+ = Animal found dead Day 20

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, hexenyl-3-cis salicylate produced a 0 % (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The substance does not meet the criteria for classification as a sensitiser.
Executive summary:

In a GLP compliant sensitisation study conducted in line with OECD 406 and EU Method B.6, the sensitisation of (z)-3-hexenyl salicylate was determined using the maximisation test. Induction was initially performed intradermally at a concentration of 5%, followed by topical induction using the undiluted test substance. Animals were challenged with a topical application of undiluted test substance and observed for reaction at 24 and 48 hours post exposure. No reactions were noted at either observation period post challenge. Hexenyl-3-cis salciylate was considered to be non-sensitising.