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Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 17 September 2004 and 16 December 2004 (in-life phase 30 September to 05 November)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid and conclusive guideline study under GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Remarks:
Except analytical investigations for characterisation, stability and homogeneity of the test item
Type of study:
Buehler test
Justification for non-LLNA method:
Not relevant

Test material

Constituent 1
Chemical structure
Reference substance name:
Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.
EC Number:
300-326-6
EC Name:
Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.
Cas Number:
93925-25-8
Molecular formula:
Complex
IUPAC Name:
Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.
Test material form:
liquid: viscous
Details on test material:
- Substance type: Organic
- Physical state: Pale yellow liquid with white specks
- Storage condition of test material: At room temperature
- Other: Date received at the test laboratory: 17 September 2004

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop Lab Animals, Inc., Scottdale, Pennsylvania, U.S.A.
- Age at study initiation: Male animals approximately 7 weeks, female animals approximately 8 weeks
- Weight at study initiation: Male animals 309 to 358 g, female animals 299 to 357 g
- Housing: The animals were housed individually in suspended stainless steel cages. All housing and care were based on the standards recommended by the Guide for the Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals, DHHS Publication No. (NIH) 96-03, 1996)
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Minimum 5 d; upon receipt, the animals were removed randomly from the shipping cartons, examined by qualified personnel, identified with plastic ear tags and then acclimated to the laboratory conditions. The animals were observed daily for overt physical or behavioural abnormalities, general health/moribundity and mortality.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 22 °C
- Humidity: 35 to 63 % relative humidity
- Air changes: 15/h (at least)
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: Test item administration on 30 September 2004 (day 0), final scoring on 05 November 2004

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
Mineral oil USP, CAS 8012-95-1; in positive Control Ethanol used for induction and acetone for challenge
Concentration / amount:
Induction:
25 % w/w test item dissolved in mineral oil USP (treatment)
5 % w/v HCA dissolved in ethanol (positive control treatment)

Challenge:
15 % w/w test item dissolved in mineral oil USP (treatment and test item challenge control)
2.5 and 1 % w/v HCA dissolved in acetone (positive control treatment and challenge control)

Rechallenge:
10 and 5 % w/w test item dissolved in mineral oil USP and challenge control for the test item
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
Mineral oil USP, CAS 8012-95-1; in positive Control Ethanol used for induction and acetone for challenge
Concentration / amount:
Induction:
25 % w/w test item dissolved in mineral oil USP (treatment)
5 % w/v HCA dissolved in ethanol (positive control treatment)

Challenge:
15 % w/w test item dissolved in mineral oil USP (treatment and test item challenge control)
2.5 and 1 % w/v HCA dissolved in acetone (positive control treatment and challenge control)

Rechallenge:
10 and 5 % w/w test item dissolved in mineral oil USP and challenge control for the test item
No. of animals per dose:
As required in the guidance (OECD TG 406, paragraph 26) 20 animals were used in the treatment group and 10 animals in the control group.
Topical Range-finding:
- First test: 4 (2 males and 2 females)
- Second test: 4 (2 males and 2 females)
Test item:
- Treatment: 20 (10 males and 10 females)
- Challenge control: 10 (5 males and 5 females)
- Rechallenge control: 10 (5 males and 5 females)
Positive control (HCA):
- Treatment: 10 (5 males and 5 females)
- Challenge control: 10 (5 males and 5 females)
Details on study design:
RANGE FINDING TESTS:
Prior to initiation of the main sensitization study, topical range-finding studies were conducted in guinea pigs (2 males 2 females) to aid in the selection of dosage levels. The in-life phase of the range-finding studies was initiated with test item administration on 24 September 2004 and concluded on 30 September 2004.
After 6 h exposure of 100 % w/v (pure as supplied) test item at 24 and 48 h slight, but confluent or moderate patchy erythema, graded 1 were reported in all 4 test animals and 1 female showed additionally a very slight edema (barely perceptible), graded ED-1, at both observation times.
The results of the range-finding studies indicated that a test item concentration of 25 % w/w in mineral oil USP was considered appropriate for induction as it produced mild irritation, respectively. A test item concentration of 15 % w/w in mineral oil USP, was considered appropriate for challenge as it produced minimal irritation.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: 1 test item treatment (10 male and 10 female test animals)
- Control groups: 1 positive control (5 male and 5 female test animals), no vehicle control
- Site: Left side, no. 1
- Frequency of applications: Weekly, topical treatment with the test item or HCA (positive control) once per week for three consecutive weeks
- Duration: 27 d (day 0 to day 26); the induction procedure was repeated on study day 6 and on study day 13 (so that a total of three consecutive induction exposures were made to the test item and HCA test animals), followed by a 2-week rest period
- Concentrations:
25 % w/w test item dissolved in mineral oil USP
5 % w/v HCA dissolved in ethanol (positive control)

B. CHALLENGE EXPOSURE
- No. of exposures: 2 (challenge and rechallenge)
- Days of challenge: Day 27 (challenge) and day 34 (rechallenge)
- Exposure period: 6 h
- Test groups: 1 test item treatment (10 male and 10 female test animals, previously induced)
- Control groups: 4 controls (each consisting of 5 male and 5 female test animals); test item challenge & rechallenge control (previously untreated animals), HCA positive control (animals previously treated with HCA ) and HCA challenge control (previously untreated animals), no vehicle control
- Sites: Right side no. 2, 4 and 6
no. 2 for test item challenge treatment, test item challenge control and the higher concentration in HCA positive control and HCA challenge control
no. 4 for the lower concentrations in test item rechallenge treatment, test item rechallenge control, HCA positive control challenge treatment and HCA challenge control
no. 6 for higher concentrations in test item rechallenge treatment and test item rechallenge control
- Concentrations:
15 % w/v test item dissolved in mineral oil USP at site no. 2 (test item challenge and challenge control)
10 and 5 % w/w test item dissolved in mineral oil USP at sites no. 6 and 4, respectively (test item rechallenge and rechallenge control)
2.5 and 1.0 % w/v HCA dissolved in acetone (positive control) at sites no. 2 and 4, respectively (HCA positive control and HCA challenge control)
- Evaluation: 24 and 48 h after challenge

OTHER:
- At challenge, 3/10 challenge control animals had scores of 1 indicating that the concentration was too irritating, therefore a rechallenge was conducted at lower concentrations (10 and 5 % w/w test item in mineral oil USP).
- Approximately 6 h after chamber application, the binding materials were removed. The test sites were wiped with gauze moistened in deionized water, followed by dry gauze, to remove test article residue.
- Test item and control substance HCA were applied in a volume of 0.3 mL
Challenge controls:
Included for the test item and positive control
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde (HCA), CAS 101-86-0

Results and discussion

Positive control results:
1st reading (24 h following challenge with 2.5 % w/v HCA in acetone):
- Dermal scores of 1 were noted in 5 of 10 test animals, while 5 test animals were graded ± (counted 0.5) and 0 animals showed no reactions and were graded 0. Therefore the mean score value is (5∙1 + 5∙0.5)/10 = 7.5/10 = 0.75.
- Control animals showed no reactions. Therefore the mean control score value is 0.

1st reading (24 h following challenge with 1 % w/v HCA in acetone):
- Dermal scores of 1 were noted in 3 of 10 test animals, while 3 test animals were graded ± (counted 0.5) and 4 animals showed no reactions and were graded 0. Therefore the mean score value is (3∙1 + 4∙0.5)/10 = 5/10 = 0.5.
- Control animals showed no reactions. Therefore the mean control score value is 0.

2nd reading (48 h following challenge with 2.5 % w/v HCA in acetone):
- Dermal scores of 1 were noted in 1 of 10 test animals, while 4 test animals were graded ± (counted 0.5) and 5 animals showed no reactions and were graded 0. Therefore the mean score value is (1∙1 + 4∙0.5)/10 = 3/10 = 0.3.
- Control animals showed no reactions. Therefore the mean control score value is 0.

2nd reading (48 h following challenge with 1 % w/v HCA in acetone):
- Dermal scores of 1 were noted in 0 of 10 test animals, while 5 test animals were graded ± (counted 0.5) and 5 animals showed no reactions and were graded 0. Therefore the mean score value is (0∙1 + 5∙0.5)/10 = 2.5/10 = 0.25.
- Control animals showed no reactions. Therefore the mean control score value is 0.

Thus group mean dermal scores were noted to be slightly higher in the HCA test animals as compared with the HCA challenge control animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
15 % w/w test item
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
0.7 (mean score), one open lesion (right pinna, day 33) occurred in the ± scored female G0936/F
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15 % w/w test item. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: 0.7 (mean score), one open lesion (right pinna, day 33) occurred in the ± scored female G0936/F .
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15 % w/w test item
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
0.55 (mean score)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 15 % w/w test item. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: 0.55 (mean score) .
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
2.5 % w/v HCA
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
0.75 (mean score)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 2.5 % w/v HCA . No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: 0.75 (mean score).
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2.5 % w/v HCA
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 2.5 % w/v HCA . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1 % w/v HCA
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
0.5 (mean score)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 1 % w/v HCA. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: 0.5 (mean score).
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1 % w/v HCA
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1 % w/v HCA. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
15 % w/w test item
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
0.325 (mean score), scabbing (right pinna, days 34-36) was observed in the 0 scored female G0936/F
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15 % w/w test item. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: 0.325 (mean score), scabbing (right pinna, days 34-36) was observed in the 0 scored female G0936/F .
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15 % w/w test item
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
0.2 (mean score)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 15 % w/w test item. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: 0.2 (mean score).
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
2.5 % w/v HCA
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
0.75 (mean score)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 2.5 % w/v HCA. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: 0.75 (mean score).
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2.5 % w/v HCA
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2.5 % w/v HCA. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1 % w/v HCA
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
0.25 (mean score)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 1 % w/v HCA. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 0.25 (mean score).
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1 % w/v HCA
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1 % w/v HCA. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10 % w/w test item
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
0.2 (mean score)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 10 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 0.2 (mean score).
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
10 % w/w test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
0.05 (mean score)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 0.05 (mean score).
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
5 % w/w test item
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
0.15 (mean score)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 5 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 0.15 (mean score).
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
5 % w/w test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
0.05 (mean score)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 5 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 0.05 (mean score).
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10 % w/w test item
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
0.05 (mean score)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 10 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 0.05 (mean score).
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
10 % w/w test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
0.1 (mean score)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 0.1 (mean score).
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
5 % w/w test item
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
0.05 (mean score)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 5 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: 0.05 (mean score).
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
5 % w/w test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 5 % w/w test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Any other information on results incl. tables

Challenge

1st reading (24 h following challenge with 15 % w/w test item in mineral oil USP):

- Dermal scores of 1 were noted in 10 of 20 test animals, while 8 test animals were graded ± (counted 0.5) and 2 animals showed no reactions and were graded 0. Therefore the mean score value is (10∙1 + 8∙0.5)/20 = 14/20 = 0.7.

- Control dermal scores of 1 were noted 3 of 10 challenge control animals, while 5 test animals were graded ± and 2 animals showed no reactions. Therefore the mean control score value is (3∙1 + 5∙0.5)/10 = 5.5/10 = 0.55.

2nd reading (48 h following challenge with 15 % w/w test item in mineral oil USP):

- Dermal scores of 1 were noted in 2 of 20 test animals, while 9 test animals were graded ± (counted 0.5) and 9 animals showed no reactions and were graded 0. Therefore the mean score value is (2∙1 + 9∙0.5) = 6.5/20 = 0.325

- Control dermal scores of 1 were noted 1 of 10 challenge control animals, while 2 test animals were graded ± and 7 animals showed no reactions. Therefore the mean control score value is (1∙1 + 2∙0.5)/10 = 2/10 = 0.2.

Rechallenge

1st reading (24 h following rechallenge with 10 % w/w test item in mineral oil USP):

- Dermal scores of 1 were noted in 0 of 20 test animals, while 8 test animals were graded ± (counted 0.5) and 12 animals showed no reactions and were graded 0. Therefore the mean score value is (0∙1 + 8∙0.5)/20 = 4/10 = 0.2.

- Control dermal scores of 1 were noted 0 of 10 challenge control animals, while 1 test animal was graded ± and 9 animals showed no reactions. Therefore the mean control score value is (0∙1 + 1∙0.5)/10 = 0.5/10 = 0.05.

1st reading (24 h following rechallenge with 5 % w/w test item in mineral oil USP):

- Dermal scores of 1 were noted in 0 of 20 test animals, while 6 test animals were graded ± (counted 0.5) and 14 animals showed no reactions and were graded 0. Therefore the mean score value is (0∙1 + 6∙0.5)/20 = 3/20 = 0.15.

- Control dermal scores of 1 were noted 0 of 10 challenge control animals, while 1 test animal was graded ± and 9 animals showed no reactions. Therefore the mean control score value is (0∙1 + 1∙0.5)/10 = 0.5/10 = 0.05.

2nd reading (48 h following rechallenge with 10 % w/w test item in mineral oil USP):

- Dermal scores of 1 were noted in 0 of 20 test animals, while 2 test animals were graded ± (counted 0.5) and 18 animals showed no reactions and were graded 0. Therefore the mean score value is (0∙1 + 2∙0.5)/20 = 1/20 = 0.05.

- Control dermal scores of 1 were noted 0 of 10 challenge control animals, while 2 test animal was graded ± and 8 animals showed no reactions. Therefore the mean control score value is (0∙1 + 2∙0.5)/10 = 1/10 = 0.1.

2nd reading (48 h following rechallenge with 5 % w/w test item in mineral oil USP):

- Dermal scores of 1 were noted in 0 of 20 test animals, while 2 test animals were graded ± (counted 0.5) and 18 animals showed no reactions and were graded 0. Therefore the mean score value is (0∙1 + 2∙0.5)/20 = 1/20 = 0.05.

- Control animals showed no reactions. Therefore the mean control score value is 0.

Weight gain

The sensitization study animals gained weight during the test period and the remaining study animals generally appeared in good health.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Absence of sensitizing potential in vivo evidenced
Executive summary:

The sensitisation potential of the test item was investigated in a GLP-compliant study using skin exposure of Harley guinea pigs according to the EU B.6 (1992), OECD TG 406 (1992) and OPPTS 870.2600 (2003) protocols. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

A pre-test was conducted to determine the appropriate doses for the study. Concentrations of 25 and 15 % w/w of the test item in mineral oil USP for the induction and challenge phases, respectively, were considered appropriate because they produced mild and minimal irritation, respectively. A volume of 0.3 mL was used for both the test item and positive control, α-Hexylcinnamaldehyde (HCA). Approximately 6 h after chamber application, the binding materials were removed. The test sites were wiped with gauze moistened in deionised water, followed by dry gauze, to remove any residue. Sites were scored at 24 and 48 h following the induction, challenge and rechallenge phases. During the induction phase animals were topically treated with the test item and positive control once per week for three consecutive weeks. After a two week rest period, a challenge was performed with the test item, the test item challenge control, the HCA positive control and the HCA challenge control. The concentration used were considered too irritating. Therefore, after a one week rest period, a rechallenge was performed with the test item at 10 and 5 % w/w in mineral oil USP and its rechallenge control.

The results of the positive control indicated that the study was valid and able to detect a potential contact sensitizer. Scores of 1 (considered a positive, “+”, signal) were observed following challenge with 15 % w/w test item in 10 of 20 animals at the 24 h grading and in 2 of 20 animals at 48 h. However, no positive scores were observed following rechallenge with 10 or 5 % w/w. Therefore the absence of sensitising properties in the test item can be concluded and classification e.g. in accordance with EU CLP Regulation No. 1272/2008 is not required on the basis of this study.