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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:
10 June - 13 July 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelnes and in accorance with GLP. All guideline criteria were met.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Due to technical error, animal #25820 (test group) was inadvertently dosed on a naive site on the right flank rather than on the left flank for the 1st, 2nd abd 3rd inductions an over all induction then dosed on the left flank for the challenge phase
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 prior to the LLNA method becoming the default approach for skin sensitisation under EU chemical control legislation. At the time of conducting this study, the Buehler method was considered the most appropriate test to assess skin sensitisation potential.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical State: Colourless liquid
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

OTHER SPECIFICS: N/A

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: Received from Elm Hill Breeding Laboratories, Chelmsford, MA
Sex/Strain//number: Male and female guinea pigs/Harley albino (n= 34)
Age/weight at study initiation: Young adults 357 - 455 grams
Housing: the animals were group housed in suspended stainless steel caging with mesh floor
Diet: pelleted purina guinea pig chow #5025
Water: Filtered tap water ad libitum
Acclimation period: 12 or 14 days

ENVIRONMENTAL CONDITIONS
Temperature: 17-23°C
Humidity: 40 - 66%
Photoperiod: 12- hour light/dark cycle

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.4 ml
Day(s)/duration:
6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Mineral oil
Concentration / amount:
0.4ml
Day(s)/duration:
6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Total Number of Animals: 34
Number of Groups: 3
Preliminary Irritation Group: 4
Test Groups: 20
Naive Control Group: 10
Details on study design:
RANGE FINDING TESTS:
A group of animals (x4) was used to determine the highest non-irritating concentration (HNIC) of the test substance prior to dosing. The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites on each animal. The test substance was applied neat (100 % w/w) and also diluted with mineral oil to yield w/w concentrations of 75 %, 50 % and 25 %. Each concentration was applied (0.4 mL) to a test site using an occlusive 25 mm Hiltop Chamber. The sites were wrappes with non-allergenic Durapore adhesive tape. After 6 hours of exposure, the chambers were removed and the test sites were cleansed of any residue substance. Approximately 24 hours after application, each site was evaluated for erythema.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: 6-hours
- Test groups: 1 (20 animals)
- Control group: 1 (10 animals)
- Site: Left flank
- Frequency of applications: Three times per week
- Duration: Three weeks
- Concentrations: 100 % (w/w)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 28
- Exposure period: 6-hours
- Test groups: 1 (20 animals)
- Control group: 1 (10 animals)
- Site: naive site on right flank
- Concentrations: 50 % (w/w)
- Evaluation (hr after challenge): 24 and 48 hours
Challenge controls:
10 guinea pigs from the same batch of animals were maintained under identical conditions and were treated with the HNIC of the test substance (50 % w/w) at challenge only.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde (HCA). Testing completed on 04 March 2005.

Results and discussion

Positive control results:
Induction Phase (HCA applied Undiluted): Very faint to faint (0.5 - 1) erythema was noted for all positive control test sites during the induction phase.
Challenge Phase Positive Control (75:25 HCA:Mineral Oil): 4/9 positive control animals exhibited signs of sensitisation response (faint erythema [1]) 24 and 48 hours after challenge. Very faint erythema (0.5) was noted for four other sites after challenge.
Challenge Phase Naive Control (75:25 HCA:Mineral Oil): Very faint erythema (0.5) was noted for three of five positive control naive test sites 24 hours after challenge. Irritation persisted at one of these sites through 48 hours.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 % (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Very faint erythema
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 % (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Very faint erythema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 % (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Very faint erythema
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 % (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None noted
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
75% w/w HCA in mineral oil
No. with + reactions:
4
Total no. in group:
9
Clinical observations:
no reaction (score = 0) in 1/9 to very faint erythema (score = 0.5) in 4/9 to faint erythema (score = 1.0) in 4/9
Remarks on result:
positive indication of skin sensitisation
Remarks:
One PC animal was excluded from evaluation as the patch was removed at 24 hours in technical error.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
75% w/w HCA in mineral oil
No. with + reactions:
4
Total no. in group:
9
Clinical observations:
no reaction (score = 0) in 1/9 to very faint erythema (score = 0.5) in 4/9 to faint erythema (score = 1.0) in 4/9
Remarks on result:
positive indication of skin sensitisation
Remarks:
One PC animal was excluded from evaluation as the patch was removed at 24 hours in technical error.

Any other information on results incl. tables

Induction Phase - Test Animals (100 % w/w test item)

Very faint to faint erythema (0.5 -1) was noted at all test sites during the induction phase. An increase in irritation was evident at all sites after each successive application with desquamation present at most sites following the third and sixth inductions. Due to the severity of irritation, the dose sites were relocated to naive sited at various intervals throughout the induction phase.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is not considered to be a skin sensitiser.
Executive summary:

A dermal sensitisation test was conducted with guinea pigs (OECD 406 - Buehler Method, 1992).  The undiluted test substance was topically applied to twenty healthy test guinea pigs, three times per week, for a three week induction period.  Twenty-eight days after the first induction dose, a challenge dose of the test item at its highest non-irritating concentration (50 % w/w) was applied to a naive site on each guinea pig.  A control group was treated with the test substance at challenge only.  Twenty four and 48 h after each induction and chellenge dose, the animals were scored for erythema.

Induction Phase (100 % test item): Very faint to faint erythema (0.5-1) was noted at all test sites during the induction phase.  Due to cumulative increase in irritation, the dose sites were relocated to other sites throughout the induction phase.

Challenge Phase (50 % test item):  Very faint erythema (0.5) was noted for five of twenty test sites after challenge.  Irritation persisted at one of these sites through 48 hours.   No positive responses evident.

Induction Phase (Control): Very faint erythema (0.5) was noted at six of the ten control sites 24 hours after challenge.  Irritation cleared from all affected sites by 48 hours. No positive responses evident.

The study met all relevant validation criteria.