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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Test system: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF
- Rational: Recognized by the international guidelines as a recommended test system (e.g. OECD, EEC).
- Source: Harlan Nederland B.V.
Postbus 167
NL-3700 AD Zeist / The Netherlands
Woundenbergseweg 55
NL-3707 HW Zeist / The Netherlands
- Age at delivery: 5-7 weeks
- Age at beginning of pre-test/acclimatization period: 6-8 weeks
- Body weight at pretest start: Pretest group: 406-448 gr
- Body weight at beginning of acclimatization period: Control and test group: 425-510 gr.
- Identification: By unique cage number and corresponding ear tags.
- Randomization: Randomly selected at time of delivery.
- Diet (e.g. ad libitum): Pelleted standard Nafag Ecosan 845 25W4, batch nos. 37/97 and 58/97 guinea pig breeding / maintenance diet ("Nafag",
Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/1) via the drinking water was provided.
- Acclimation period: One week for the control and test group under test conditions after health examination. One week for the animals of the pretest. Only animals without any visible signs of illness were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 40-70%
- Air changes (per hr): Air-conditioned with 10-15 air chages per hour
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hours dark cycle. Music was played during the light period.
Route:
intradermal and epicutaneous
Vehicle:
other: Bi-distilled water
Concentration / amount:
*Pre-test (Intradermal injections):
5%, 3%, 1%

*Pre-test (epidermal applications):
50%, 25%, 15%, 10%

*Main study (induction, intradermal injections):
Test group: diluted to 5% with bidistilled water.

*Main study (induction, epidermal applications):
50% of test article in bi-distilled water.

*Main study (Challenge):
25 % of test article
Route:
epicutaneous, occlusive
Vehicle:
other: Bi-distilled water
Concentration / amount:
*Pre-test (Intradermal injections):
5%, 3%, 1%

*Pre-test (epidermal applications):
50%, 25%, 15%, 10%

*Main study (induction, intradermal injections):
Test group: diluted to 5% with bidistilled water.

*Main study (induction, epidermal applications):
50% of test article in bi-distilled water.

*Main study (Challenge):
25 % of test article
No. of animals per dose:
Number of animals for main study / pretest: 15 females / 3 females , nulliparous and non-pregnant.
- Control group: 5 animals (main study; animal number 535-539)
- Test group: 10 animals (main study; animal number 540-549)
- Intradermal pretest: 1 animal; animal number 550
- Epidermal pretest: 2 animals; animal number 551-552
Details on study design:
PRETEST/PERFORMED DURING ACCLIMATIZATION PERIOD:
The procedure employed for these investigations was as follows:

INTRADERMAL INJECTIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved
neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig 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 in the main study a 5 % test article
concentration was selected

EPIDERMAL APPLICATIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved
neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4
patches of filter paper (2x2 cm) were saturated with the test article at A = 50 % (this concentration was found to be the most qualified to assure an optimum technical apphcation procedure), B = 25 %, C = 15 % and D = 10 % in bi-distilled water and applied to the clipped and shaved flanks. The volume of test article applied at 50 % was approximately 0.2 g. The volume of the remaining test article concentrations was 0.2 ml. The
patches were covered by a strip of aluminum foil and firmly secured by elastic plaster 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.
Approximately 21 hours after removal of the dressing the apphcation site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the apphcation site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time.

The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to
Draize described above.
The position of the epidermal applications is shown below:

Cranial
-----------
A C
left right
B D
------------
Caudal


Cranial
-----------
D B
left right
A C
------------
Caudal
The allocation of the different test dilutions to the sites (A, B, C, D) on the animals was alternated in order to minimize site-to-site variation in responsiveness. The concentration selected for the induction period and challenge procedure was 50 % and 25 %, respectively.


MAIN STUDY
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:

TEST GROUP:
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.

CONTRAOL GROUP:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bi-distilled water.
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

EPIDERMAL APPLICATIONS / PERFORMED ON TEST DAY 8
On test day 7 and approximately 20 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) of the animals of the control and test group was clipped, shaved free of hair and the test area was pretreated with a 10 % dilution of Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10 % concentration of SLS enhances sensitization by provoking a mild inflammatory reaction (Magnusson and Kligman 1970).
One week after the injections, the scapular area (approximately 6 x 8 cm) was again cupped 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 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 bi-distilled 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.


B. CHALLENGE EXPOSURE/PERFORMED ON TEST DAY

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 as described in the epidermal pretest.
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.

INTERPRETATION:
The results obtained from test animals following the challenge applications were compared with the results seen in control animals.
An allergic reaction was defined by visible reddening of the challenge site.
If the dermal reactions of test animals following the challenge were more marked and/or persistent than those of the control animals, the animals were considered to show evidence of contact hypersensitivity.
If the dermal reactions of test animals following the challenge were not clearly different from the reactions seen in the control group animals, the results for the test animals were considered "inconclusive".
The test animals were considered to show no evidence of contact hypersensitivity if the dermal reactions to the challenge application were identical
to or less marked and/or persistent than the reactions observed in the control animals.
By "maximizing" the exposure and enhancing allergenicity, some problems could arise, particularly in relation to specificity, especially the potential for false-positive reactions. An inflammatory response at challenge may not necessarily be due to allergenicity, but instead may be a false-positive irritant response caused by an inducing hyperirritability.
Challenge controls:
Control guinea pigs was challenged two weeks after the epidermal induction application.
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 as described in the epidermal pretest.
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.
Positive control substance(s):
yes
Remarks:
Alpha-hexylcinnalmaldehyde
Positive control results:
In this study 70 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 25 % in polyethylene glycol (PEG 400). No skin reactions were observed in the control group. A response of at least 30 % positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22nd time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances". Therefore, the test article ALPHA-HEXYLCINNAMALDEHYDE applied at a concentration of 25 % in polyethylene glycol (PEG 400) is considered to be a sensitizer when used under the described test conditions.
According to the rating of allergenicity by Magnusson and Kligman the test article is a strong sensitizer.

None

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
FAT 40061/E is considered as a sensitiser in this contact hypersensitivity test in albino guinea pigs (Maximization test).
Executive summary:

In this study 70 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 25 % in polyethylene glycol (PEG 400). No skin reactions were observed in the control group.

A response of at least 30 % positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22nd time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances".

Therefore, the test article ALPHA-HEXYLCINNAMALDEHYDE applied at a concentration of 25 % in polyethylene glycol (PEG 400) is considered to be a sensitizer when used under the described test conditions.

According to the rating of allergenicity by Magnusson and Kligman the test article is a strong sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A GLP compliant study was performed to determine the skin sensitisation potential of FAT 40061/E in guinea pigs. The test was performed according to OECD guideline No. 406, adopted May 12, 1981, adapted July 17, 1992 by the OECD council, and on Annex V, Part B of Council Directive 67/548/EEC (Commission Directive 92/69/EEC of July 31, 1992).

Male and female guinea pigs were used to perform the experiment. Under the experimental conditions employed, 70 % of the animals of the test group showed skin reactions after removing the dressings. Therefore, FAT 40061 is classified as a strong sensitiser in albino guinea pigs according to the grading published by Magnusson and Kligman.

In a second study, performed according to the maximization protocol by Magnussen & Kligman FAT 40061/B did not show any sensitising potential inin Pirbright white guinea pigs.

Based on the results observed in the key study selected, the substance will be classified as skin sensitizer according to GHS (EU). In additionliterature data report on urticaria and eczema observed in workers with a high occupational exposure to reactive dyes. Consequently, it was agreed between members of the ETAD to classify the test item as skin sensitizer.

Therefore, based on the results of the key study and the ETAD agreement FAT 40061 is classified as a skin sensitiser Cat. 1B according to GHS.

Migrated from Short description of key information:
FAT 40061/E showed skin-sensitising (contact allergenic) potential in albino guinea pigs.

Justification for selection of skin sensitisation endpoint:
GLP guideline study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

An experiment was performed according to the OECD 403, to determine the acute inhalation toxicity on 4 female guinea pigs.

FAT 40061/C was atomized under dynamic conditions in a cylindrical inhalation chamber. The pressure was adjusted to about 200 kPa, the inlet air was about 28 liters/min the outlet air about 20 liters/min. To optimize respirability of the particles, a particle separator was used separating big particles from the air stream (all particles <= 5 µm can pass to 100 %; particles >10 µm cannot pass). Temperature in the incubation chamber was about 22 °C, humidity about 36%. Under these test conditions, FAT 40061/C was found to be non-sensitizing by inhalation in guinea pigs.

A case report published by Romano, et al. (American Journal of Industrial Medicine 21:209-216, 1992) reported results of clinical investigations on a worker suffering from respiratory symptoms while weighing dyes at his workplace. Reactive Yellow 39 has been identified through chamber inhalation challenge as being responsible for the sensitization. A very short (4-minute) exposure produced a severe immediate obstructive ventilatory defect followed by arterial hypotension and urticaria. Bronchial hyperresponsiveness as tested through metacholine challenge was absent both in basal conditions and after the dye challenge. Both prick and patch test for the dye were positive in the absence of any sign of contact dermatitis.

Based on this publication and other literature data reporting on symptoms of respiratory allergy observed in workers with a high occupational exposure to reactive dyes, it was agreed between members of the ETAD to classify the test substance as respiratory sensitizer.

Migrated from Short description of key information:
The test item FAT 40061/C was found to be a non-sensitiser to guinea pigs, however a case report of occupational asthma showed that the substance should be considered as respiratory sensitiser.

Justification for classification or non-classification

- skin sensitisation:

Based on the above stated assessment of the skin sensitisation potential, the substance does need to be classified as Skin sensitiser Cat 1B according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.

- respiratory sensitisation:

Based on the existing case report the substance is classified as respiratory sensitiser Cat 1B according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.