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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:
Nov. 27, 1996 to Feb. 12, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
German GLP
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Code number: FAT45'176/A
Batch No.: WP2/96
Purity: ca. 60 %
Appearance: orange-brown powder
Storage: room temperature
Expiration date: 11/2004
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: WP2/96
- Expiration date of the lot/batch: November 30,2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature at about 20°C; away from direct sunlight
- Stability of the test substance in the solvent/vehicle: Stable in bi-distilled water and FCA/physiological saline for at least 48 h at 20°C

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Females (if applicable) nulliparous and non-pregnant: Not applicable
- Microbiological status of animals, when known: Not specified
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 323 - 415 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz)
- Diet: Pelleted standard Nafag Ecosan 845 25W4, batch nos. 118/96 and 130/96 guinea pig breeding/ maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum.
- Water: Community tap water from Itingen, ad libitum
- Acclimation period: One week for the control and test group under test
conditions after health examination.
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h artificial fluorescent light/12 h dark
- IN-LIFE DATES: From: 11-DEC-1996 to 06-JAN-1997

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Remarks:
bi-distilled water
Concentration / amount:
5%
Day(s)/duration:
Day 1
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bidistilled water
Concentration / amount:
50 %
Day(s)/duration:
Day 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled water
Concentration / amount:
25% (0.2 ml)
Day(s)/duration:
Day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
control: 5 males
test group: 10 males
Details on study design:
INDUCTION
Intradermal injections / performed on test day 1: An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region.

Epidermal applications / performed on test day 8: One week after the injections, the scapular area was clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test article (50 % in bi-distilled water) and placed over the injection sites of the test animals. The volume of test article applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 46 hours. The epidermal application procedure described ensured intensive contact of the test article. The guinea pigs of the control group were treated as described above with bi- distilled water only.

CHALLENGE / performed on test day 22: The test and control guinea pigs were challenged two weeks after the epidermal Induction application. The test and control guinea pigs were treated in the same way. Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (2x2 cm) of filter paper were saturated with the highest non-irritating concentration of 25 % (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 ml. The dressings were left in place for 24 hours.
Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated with an approved depilatory cream. The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed the animals were dried with a disposable paper towel and returned to their cages.
Approximately 24 and 48 h after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.
Challenge controls:
vehicle: bi-distilled water
Positive control substance(s):
yes
Remarks:
2-MERCAPTO- BENZOTHIAZOLE and ALPHA-HEXYLCINNAMALDEHYDE

Results and discussion

Positive control results:
The positive control with 2-MERCAPTO- BENZOTHIAZOLE applied at concentration of 50 % in mineral oil is considered to be a sensitizer when used under the test conditions.
The positive control with ALPHA-HEXYLCINNAMALDEHYDE applied at concentration of 15 % in PEG 400 is considered to be a sensitizer when used under the test conditions.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Bi-distilled water only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Bi-distilled water only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Bi-distilled water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Bi-distilled water only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Bi-distilled water only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Bi-distilled water only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Bi-distilled water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Bi-distilled water only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
18
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
2-MERCAPT0BENZ0THIAZ0LE
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
20
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
2-MERCAPT0BENZ0THIAZ0LE
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
15%
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
15%
No. with + reactions:
12
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE

Any other information on results incl. tables

CONTROL GROUP：

No erythematous or oedematous reaction was observed in the animals treated with bi-distilled water only.

TEST GROUP：

As the test article stained the skin brownish/red, it was not possible to determine whether erythema was present or not. However no oedema was observed.

Skin effects after the challenge - performed on test day 22

CONTROL AND TEST GROUP

No positive reactions were observed in the animals neither when treated with bi- distilled water alone nor when treated with the test article at25 % in bi-distilled water.

A brownish/red discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 hours prior to challenge reading.

Viability/mortality/microscopic findings

As there were no deaths during the course of the treatment period no necropsies were performed.

Clinical signs, systemic: no symptoms of systemic toxicity were observed in the animals.

Body weights:

One animal (no. 588) of the test group lost weight during its acclimatization period. It recovered during the treatment period. One animal (no. 600) of the epidermal pretest lost weight during Its test article treatment period.

The body weight of the other animals was within the range of physiological variability known for this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
FAT45'176/A is considered to be a non-sensitizer to Rabbit skin.
Executive summary:

The Guinea Pig Maximization-Test was carried out to assess the cutaneous allergenic potential of FAT 45'176/A, in accordance with OECD 406 and EU B.6 guideline. The intradermal induction of sensitization was carried out with a 5 % dilution of the test article in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA)/physiological saline. The epicutaneous induction of sensitization was conducted under occlusion with the test article at 50 % in bidistilled water. Two weeks after the epicutaneous induction application the challenge was completed by epicutaneous application of the test article at 25 % in bi-distilled water under occlusive dressing. The animals of the control group were induced with bi-distilled water and FCA/physiological saline and challenged similarly as those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 h after removal of the dressing.

In this study no erythematous reactions after the challenge were observed in the animals of the test group. No positive skin reactions after treatment with a non-irritant test article concentration of 25 % in bi-distilled water. No skin reactions were observed in the control group. No toxic symptoms or deaths were evident in the guinea pigs of the control or test group.

Therefore, FAT45'176/A applied at a concentration of 25 % in bi-distilled water is considered to be a non-sensitizer when used under the described test conditions.