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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Buehler test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 02, 2005 to October 04, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The study was conducted before the formal requirement for LLNAs was implemented.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
This is formulation of 49.2% of the test substance in water.
Specific details on test material used for the study:
- Physical state: Clear yellow liquid
- Analytical purity: 80.9% w/w active substance
- Purity test date: 16 September, 2004
- Lot/batch No.: J2220459
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Elm Hill Breeding Labs, Chelmsford, MA, USA
- Age at study initiation: Young adult animals
- Weight at study initiation: 327-399 g
- Housing: The animals were group housed in suspended stainless steel caging with mesh floors or plastic perforated bottom caging which conform to the size recommendations according to ‘Guide for the care and use of laboratory animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times/wk.
- Diet: Pelleted Purina guinea pig chow # 5025, ad libitum
- Water: Filtered tap water, ad libitum
- Acclimation period: 3-17d

Environmental conditions
- Temperature: 19-22°C
- Humidity: 31-58%
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

In-life dates: From: May 2, 2005 to: June 10, 2005

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction: 1% w/w mixture of the test substance in distilled water (0.4 mL)
Challenge: 0.25% w/w mixture of the test substance in distilled water (0.4 mL)
Day(s)/duration:
6h exposure, observations 24 and 48h after exposure
Challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction: 1% w/w mixture of the test substance in distilled water (0.4 mL)
Challenge: 0.25% w/w mixture of the test substance in distilled water (0.4 mL)
Day(s)/duration:
6h exposure, observations 24 and 48h after exposure
No. of animals per dose:
20 animals in the test substance group and 10 animals in the control group
Details on study design:
Range finding tests:
A preliminary irritation study was performed to determine the highest non-irritating concentration. 8 guinea pigs (5 males and 3 females) were exposed for 6 h period to various concentrations of the test substance as follows: 100, 75, 50, 25, 1.25, 1.0, 0.75, 0.25 and 0.10%.w/w in distilled water. The fur was removed by clipping the dorsal area and flanks of each guinea pig. The area was divided into two or four test sites on each animal. Two to four concentrations of the test substance were applied to the sites. 0.4 mL of each concentration was applied to a test site using a 25 mm Hill Top Chamber. The test sites were wrapped with non-allergenic adhesive tape. All animals were evaluated at approximately 24 hours, 2 days and one animal on Day 5. Each site was evaluated for erythema.

Main study
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6h
- Test groups: One test group, including 20 guinea pigs
- Control group: No control animals were employed in induction phase
- Site: Left side of each animal
- Frequency of applications: Once a week for three consecutive weeks
- Duration: 6 h/application
- Concentrations: 1 % w/w mixture of the test substance in distilled water (0.4 mL)
- Procedure for application: Once a week for three weeks, 0.4 mL of a 1% w/w mixture of the test substance in distilled water was applied to the left side of each animal. Due to residual irritation from the previous doses, the dose site was moved each induction. Approximately 24 and 48h after each induction exposure, the skin was evaluated for local reactions (erythema).

B. CHALLENGE EXPOSURE
- No. of exposures: One
- Day(s) of challenge: Twenty-seven days after the first induction dose
- Exposure period: 6h
- Test groups: One test group (20 animals) exposed with 0.25% w/w mixture of the test substance in distilled water
- Control group: One naive control group (10 animals) exposed with 0.25% w/w mixture of the test substance in distilled water
- Site: The test substance was applied to a naïve site on the right side of each test animal.
- Concentrations: 0.25% w/w mixture of the test substance in distilled water
- Evaluation (hr after challenge): These sites were evaluated for a sensitisation response approximately 24 and 48h after the challenge exposure.

Other: Historical Positive Control Validation Study: The procedures used in this study were validated using alpha-Hexylcinnamaldehyde Technical (HCA) grade as a positive control substance. A positive control validation study is run at no less than six months intervals at the testing laboratory. The results from the most recent positive control validation study performed on March 2005 are provided in the study report.
Challenge controls:
10 naive animals exposed with 0.25% w/w mixture of the test substance in distilled water
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:
Induction: Very faint to faint erythema (0.5-1.0) was noted for all of the positive control sites during the induction phase.
Challenge: Four of nine positive control animals exhibited signs of skin sensitisation response 24 and 48h after exposure. Very faint erythema was observed for four other sites after challenge. Very faint erythema was observed in three of five positive control naive test sites after 24h. Irritation persisted at one of these sites through 48h.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.25% w/w mixture of the test substance in distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No animals with positive response
Remarks on result:
other: see Remark
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.25% w/w mixture of the test substance in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No animals with positive response
Remarks on result:
other: see Remark
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.25% w/w mixture of the test substance in distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No animals with positive response
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.25% w/w mixture of the test substance in distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No animals with positive response .
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.25% w/w mixture of the test substance in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No animals with positive response
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.25% w/w mixture of the test substance in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No animals with positive response .

Any other information on results incl. tables

Summary of results in the main study:

- Induction Phase: Very faint to faint erythema (0.5-1.0) was observed for all sites during the induction phase.

- Challenge Phase: 

Test group: None of the test animals exhibited a positive skin sensitisation response (score greater than 0.5) at 24 or 48h after challenge. Very faint erythema (0.5) was noted in 9 out of 20 test animals at 24h after challenge. Similar irritation persisted at two sites through 48h.

Naive Control group: Very faint erythema (0.5) was observed in 5 out of 10 test animals at 24h after challenge. Similar irritation persisted at two sites through 48h.

Table 1. The severity index of test and control group

Treatment

Severity

24 h

48 h

Test

0.23

0.05

Control

0.25

0.10

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was determined to be non-sensitising in a Buehler test when applied topically to albino Hartley guinea pigs.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 406 and US EPA OPPTS 870.2600 (Buehler test), in compliance with GLP. The study was conducted in two phases, with induction and challenge exposures. To determine the highest non-irritating concentration (HNIC) for the challenge phase, a preliminary irritation study was performed with 8 guinea pigs. The concentrations used were 100, 75, 50, 25, 1.25, 1.0, 0.75, 0.25 and 0.10%. w/w in distilled water. After treatment, the application sites were evaluated and scored. During the induction phase of the main study, 0.4 mL of a 1% w/w mixture of the test substance in distilled water was applied to the left side of each animal, once per week for three weeks. Twenty-seven days after the first induction dose, 0.4 mL of a 0.25% w/w (HNIC) mixture of the test substance in distilled water was applied to a naïve site on the right side of each test animal for challenge exposure. Ten additional animals exposed with 0.25% w/w mixture of the test substance in distilled water served as naive control group in the challenge phase only. Approximately 24 and 48 h after each induction and challenge dose, the animals were scored for erythema. Very faint to faint erythema (0.5 to 1) was observed in all test animals during induction. None of the test animals exhibited a positive skin sensitisation response (score greater than 0.5) at 24 or 48 h after challenge. Under the test conditions, the test substance was determined to be non-sensitising when applied topically to albino Hartley guinea pigs (Durando, 2005).