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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:
13 February 1996 to 13 May 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
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

Constituent 1
Reference substance name:
FAT 40'549/A
IUPAC Name:
FAT 40'549/A
Test material form:
other: solid
Details on test material:
Identity: FAT 40'549/A
Batch No.: TV 1
Expiration date: January 01, 2001
Stability of Test Article: Stable under storage conditions
Stability of Test Article in Vehicle: Stable in bi-distilled water for 48 hours and in a 1:1 (v/v) mixture of FCA/physiological saline for at least 2 days.
Description: yellow-brown solid
Storage conditions: In the original container at room temperature (approx. 20 °C), away from direct sunlight.
Safety Precautions: Gloves, goggles and face mask were obligatory to ensure the health and safety of the personnel.
Specific details on test material used for the study:
Identification: FAT 40549/A
Description: Yellow brown solid
Batch Number: TV 1
Stability of Test Article: Stable under storage conditions; Expiration date: 01 Jan 2001
Stability of Test Article in Vehicle: Stable in bi-distilled water for 48 hours and in a 1:1 (v/v) mixture of FCA/physiological saline for at least 2 days.
Storage Conditions: In the original container at room temperature (approx. 20°C), away from direct sunlight.
Safety Precautions: Gloves, goggles and face mask were obligatory to ensure the health and safety of the personnel.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Test system: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)
Rationale: Recognized by the international guidelines as a recommended test system (e.g. OECD, EEC).
Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
Age at beginning of acclimatization period: 5 - 7 weeks
Number of animals for main study / pretest: 30 females/6 females, nulliparous and non-pregnant
Body Weight at beginning of acclimatization period: Control and Test Group 354 - 417 g, Pretest 373 - 412 g
Identification: By unique cage number and corresponding ear tags
Randomization: Randomly selected at time of delivery.
Acclimatization: One week for the control and test group under test conditions after health examination. No Acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Standard Laboratory Conditions:
Air-conditioned with 10 - 15 air changes per hour and continuously monitored environment with a temperature between 21 ± 3 °C and a relative humidity between 40 - 70 % (values above 70 % during cleaning process possible). The animals were provided with a 12-hour light, 12-hour dark cycle. Music was played during the daytime light period.
Accommodation: 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. 118/95 and 09/96 guinea pig breeding/ maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum.
Water: Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid (1 g/L) via the drinking water.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Remarks:
Bi-distilled
Concentration / amount:
5%
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
Bi-distilled
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
Concentration / amount:
25%
Day(s)/duration:
Day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 animals in control group
20 animals in test group
Details on study design:
PRETEST
INTRADERMAL INJECTIONS: Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in bi-distilled water. The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 5 % test article dilution was selected.
EPIDERMAL APPLICATIONS: Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper ( 2 x 2 cm) were saturated with the test article at A = 50 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 25 %, C = 15 % and D = 10 % in bi-distilled water and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminum foil and firmly secured by elastic piaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours.

MAIN TEST
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 as follows:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5 % with bi-distilled water.
3) The test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Epidermal applications/ performed on test day 8:
One week after the injections, the scapular area (approximately 6 x 8 cm) was again 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 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 48 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 bidistilled water only.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

CHALLENGE
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 ( 2 x 2 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 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 (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil). 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 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize

Erythema and eschar formation:
No erythema - 0
Very slight erythema (barely perceptible) - 1
Well-defined erythema - 2
Moderate to severe erythema - 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) - 4

Oedema formation:
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 millimeter) - 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) - 4
Challenge controls:
Bi-distilled water
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE (RCC project 900731) and ALPHA-HEXYLCINNAMALDEHYDE (RCC project 900742)

Results and discussion

Positive control results:
Positive control test with 2-MERCAPTOBENZOTHIAZOLE:
In this study 95 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 15 % in mineral oil. No skin reactions were observed in the control group.


Positive control test with ALPHA-HEXYLCINNAMALDEHYDE:
In this study 75 % and 45 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 3% in polyethyleneglycol (PEG 400). No skin reactions were observed in the control group.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5% Intradermal, 50% epidermal induction. 25% epidermal challlenge.
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5% Intradermal, 50% epidermal induction. 25% challlenge.
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5% Intradermal, 10% epidermal induction. 3% epidermal challenge in PEG 400
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
none
Remarks on result:
positive indication of skin sensitisation
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
5% Intradermal, 10% epidermal induction. 3% epidermal challenge in PEG 400
No. with + reactions:
9
Total no. in group:
20
Clinical observations:
none
Remarks on result:
positive indication of skin sensitisation
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE

Any other information on results incl. tables

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
FAT 40549/A was found to be a non-sensitizer.
Executive summary:

The purpose of this skin sensitizing study was to assess the possible allergenic potential of FAT 40549/A to albino guinea pigs. The maximization-test of B. Magnusson and A.M. Kligman (1969) was conducted according to OECD test guideline 406 and EU method B.6 in a GLP certified laboratory.

Twenty female animals of the test group were induced intradermally once in the first week and epidermally once in the second week with FAT 40549/A at 5 % and 50 % in bi-distilled water, respectively.

Two weeks after the epidermal induction application the animals were challenged with the vehicle bi-distilled water and the same test substance used for induction at the highest non-irritating concentration of 25 % in bi-distilled water.

Ten animals of control group were induced with bi-distilled water (vehicle) only and treated once at challenge with bi-distilled water and FAT 40'549/A at 25 % in bidistilled water.

In this study 0 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 25 % in bi-distilled water. No skin reactions were observed in the control group. No toxic symptoms were evident in the guinea pigs of either the control and test group. No death occurred.

Positive control Alpha- hexyl cinnamaldehyde showed 75% and 45% animals at 24 and 48 hours respectively.

Therefore, FAT 40549/A was found to be a non-sensitizer.and shall not be classified in accordance with CLP (Regulation EC No.1272/2008).

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