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Diss Factsheets

Administrative data

Description of key information

FAT 20003 is not a skin sensitiser.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
not applicable
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 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.
Specific details on test material used for the study:
Identification : FAT 20003/I
Description : Red powder
Batch number : VERS.NR 9-5-97
Purity : approx 90%
Stability of test article : Stable under storage conditions; expiration date : FEB 2001.
Stability of test article dilution :Stable in bi-distilled water and in a 1:1 (v/v) mixture of FCA/physiological saline for at least 24 hours.
Storage conditions : In the original container at room temperature (approx. 20 °C), away from direct sunlight.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland B.V. Postbus 167, NL-3700 AD Zeist / The Netherlands; Woundenbergseweg 55 NL-3707 HW Zeist / The Netherlands.
- Age at beginning of pretest/acclimatization period: 4-6 weeks/5-7 weeks
- Weight at study initiation: 323 - 360 g (pretest groups), 304 - 384 g (Control and test group at beginning of acclimatization period)
- 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. 69/97 and 80/97 guinea pig breeding / maintenance diet ("Nafag", Nahr- und Futtermittel AG, CH-9202 Gossau), (ad libitum)
- Water: Community tap water (ad libitum). Once weekly additional supply of ascorbic acid (approx. 1 g/L) via the drinking water was provided
- Acclimation period: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.
-Identification: By unique cage number and corresponding ear tags.
-Randomization: Randomly selected at time of delivery.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 % (values above 70 % during cleaning process possible)
- Air changes: 10-15 air changes/h
- Photoperiod: 12 h/12 h (music was played during the light period)

IN-LIFE DATES: From 15 October, 1997 to 17 November, 1997
Route:
intradermal
Vehicle:
other: bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline
Concentration / amount:
5 %
Day(s)/duration:
day 1
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
other: bi-distilled 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
Route:
epicutaneous, occlusive
Vehicle:
other: Intradermal (id) and epidermal pretests : Bi-distilled water, 1:1 mixture (v/v) of Freund's Complete Adjuvant (FCA): physiological saline (PS); main study: Bi-distilled water (id, epidermal induction and the challenge), 1:1 (v/v) FCA: PS (id induction)
Concentration / amount:
15 %
Day(s)/duration:
day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary study:
Intradermal injections group: 1 animal
Epicutaneous applications group: 2 animals
Main study:
Control group: 5 animals
Test group: 10 treated animals
Details on study design:
PRETEST

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 substance in bidistilled water. The resulting dermal reactions were assessed 24 h later. For intradermal induction application in the main study a 5 % test substance 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 substance at 50 % (this concentration was found to be the most qualified to assure an optimum technical application procedure), 25 %, 15 % and 10 % in bi-distilled water and applied to the clipped and shaved flanks. The volume of test substance applied was approximately 0.2 g for the concentration of 50% and 0.2 ml for the remaining concentrations. The patches were covered by a strip of aluminium 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 substance. The dressings were removed after an exposure period of 24 h.

Approx. 21 h after removal of the dressing the application site was depilated with an approved depilatory cream to clean the application site from dark red staining produced by the test substance, 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 h after removal of the bandage for erythema and oedema on a numerical basis according to Draize.
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 15%, respectively.

MAIN STUDY

INDUCTION:
An area of dorsal skin from the scapular region (approx. 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 substance, diluted to 5% with bi-distilled water.
3) The test substance diluted to 5% by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bidistilled water.
3) 1:1 (w/w) mixture of bidistilled 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 8, a 2 x 4 cm patch of filter paper was saturated with the test substance (50% in bidistilled water) and placed over the injection sites of the test animals. The volume of test substance applied was approx. 0.3 g. The patch was covered with aluminium 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 h. The epidermal application procedure described ensured intensive contact of the test substance. The guinea pigs of the control group were treated as described above with bidistilled water only. The volume applied was approximately 0.3 mL. Reaction sites were assessed for erythema and oedema 24 and 48 h after removal of the dressing, using the numerical grading system according to Draize.

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 15% (left flank) and the vehicle only (bidistilled water applied to the right flank) using the same method as for the epidermal application. The volume of test substance applied was approximately 0.2 mL. The dressings were left in place for 24 h.
Approx. 21 h after removal of the dressing the test sites treated with the test substance were depilated as described in the epidermal pretest.
Approx. 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 score.
Challenge controls:
Test substance (15 % (left flank); vehicle only (bi-distilled water applied to the right flank)
Positive control substance(s):
yes
Remarks:
(2-mercaptobenzothiazole tested in another study)
Positive control results:
In this study 100 % and 90 % of the animals of the test group were observed with positive skin reactions at the 24- and 48-hour reading, respectively after treatment with a non-irritant test article concentration of 25 % in mineral oil.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
15 % epidermal application
No. with + reactions:
0
Total no. in group:
9
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
15 % epidermal application
No. with + reactions:
0
Total no. in group:
9
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25 %
No. with + reactions:
10
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25 %
No. with + reactions:
9
Total no. in group:
10

Main Study:

Skin effects after intradermal induction - performed on test Day 1

The expected local symptoms were observed in the animals of the control and test group after the intradermal injections.

Skin effects after epidermal induction - performed on test Day 8

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

Test group: As the test substance stained the skin dark red, it was not possible to determine whether erythema was present or not. However no oedema was observed when treated with the test substance at 50 % in bi-distilled water.

Skin effects after the challenge - performed on test Day 22

Control and test group : No positive reactions were observed in the animals either when treated with bi-distilled water only or when treated with the test substance at 15 % in bi-distilled water. A dark red discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approx. 3 h prior to challenge reading.

Viability / mortality / macroscopic findings : One animal of the test group was found dead on test 9 d. At necropsy, several dark red foci (D= 5 mm) were observed in the lungs.

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

Body weights: The body weight of the animals was within the range of physiological variability known for guinea pigs of this strain and age.

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was not considered to induce delayed contact hypersensitivity in treated animals.
Executive summary:

A guinea pig maximization study was conducted to evaluate the skin sensitization potential of the test substance (of 90% purity) according to OECD Guideline 406 and EU Method B.6 and in compliance with GLP.

 

The intradermal induction of sensitization was carried out with a 5 % dilution of the test substance in bidistilled water and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion with the test substance at 50 % in bi-distilled water. Two weeks after the epidermal induction application the challenge was completed by epidermal application of the test substance at 15 % in bidistilled water under occlusive dressing. The animals of the control group were induced with bidistilled water and FCA/physiological saline and challenged similarly to 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.

None 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 bidistilled water. No skin reactions were observed in the control group.

                     

Under the conditions of the study, the test substance was not considered to induce delayed contact hypersensitivity in treated animals.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a Guinea pig maximization test none of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 15 % in bi-distilled water during the challenge stage. No skin reactions were observed in the control group.

Therefore, the test article applied at a concentration of 15 % in bi-distilled water is considered not to be a sensitizer when used under the described test conditions.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the above stated assessment of the skin sensitisation potential, the substance does not need to be classified as a skin sensitizer according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and 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:

As no data on respiratory sensitization are available for the substance a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.