1-amino-4-hydroxy-2-phenoxyanthraquinone

Endpoint summary

Description of key information

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:

from December 6th, 1999 to January 28th, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Guinea pig maximisation test was available.

Species:

guinea pig

Strain:

other: Ibm: GOHI; SPF-quality guinea pigs

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 4 - 6 weeks
- Body weight at pretest start: 348 - 385 g (pretest group)
- Body weight at beginning of acclimatization: 283 - 387 g (control and test group)
- No. of animals for intradermal pretest: 1
- No. of animals for epidermal pretest: 2
- No. of animals for main study: 15 males (test group: 10, control group: 5)
- Housing: individual housing in Makrolon type-4 cages, with standard softwood bedding, "Lignocel," Schill AG, CH-4132 Muttenz
- Diet: Pelleted standard Nafag Ecosan 845 25W4, batch nos. 85199 and 96199, guinea pig breeding / maintenance diet, containing Vitamin C ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 1 week for control and test animals. No acclimatization for the animals of the pretest.
- Identification: unique cage no. and corresponding ear tags
- Randomization: random selection at time of delivery
- Selection: only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %, although values > 70 % during the cleaning process was possible
- Air changes: 10 - 15 per h
- Photoperiod: 12 h light/ 12 h dark, with music during the light period

Route:

intradermal and epicutaneous

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Concentration / amount:

50 %

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Concentration / amount:

25 %

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS: Yes.

A. 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 one intradermal injection (0.1 ml/site) was made into the clipped flank of the same guinea pig at the concentration of 1 % of the test article in PEG 400. The test article concentrations of A = 10 %, B = 5 % and C = 3 % were (barely) technically not applicable. The positions of application are shown in the illustration.

Dermal reactions were assessed 24 hours later. Based on the results, the test article concentration of 1 % was selected for intradermal induction in the main study.

B. 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 (3 × 3 cm) were saturated with the test article at D = 50 % (this concentration was found to be the maximum possible under the conditions of the procedure), E = 25 %, F = 15 % and G = 10 % in PEG 400 and applied to the clipped and shaved flanks. The amount of test article preparation applied was approximately 0.2 g for the test article at 50 % and a volume of approximately 0.2 ml was applied for the remaining test article concentrations. 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 h.
21 h after removal of the dressing, the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colmar AG, CH-4123 Allschwil) in order to visualize any resulting erythema.

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. Thereafter, the animals were 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 according to the method of Magnusson and Kligman.

The position of epidermal applications is shown in the attached illustration. The allocation of the different test dilutions to the sites (D, E, F, G) on the two animals was alternated in order to minimize site-to-site variation in responsiveness. Based on the results obtained, the concentration selected for induction and challenge in the main study was 50 % and 25 %, respectively.

MAIN STUDY
1. INDUCTION EXPOSURE

A. INTRADERMAL INJECTIONS - PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 cm × 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 cm × 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, at 1 % in PEG 400.
3) The test article at 1 % 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) PEG 400
3) 1:1 (w/w) mixture of PEG 400 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

The position of intradermal injections is shown in the attached illustration.

B. EPIDERMAL APPLICATIONS - PERFORMED ON TEST DAY 8
On test day 7 and approximately 23 h prior to the epidermal application the scapular area (approximately 6 cm × 8 cm) of the animals of the control and test group was clipped, shaved free of hair and the test area was pretreated with 0.5 ml of 10 % 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).

On test day 8, a 2 cm × 4 cm patch of filter paper was saturated with the test article (50 % in PEG 400) and placed over the injection sites of the test animals. The amount of test article preparation 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 occlusive dressings were left in place for 48 h. The epidermal application procedure described ensures intensive contact of the test article.
The guinea pigs of the control group were treated as described above with PEG 400 only, applied at a volume of approximately 0.3 ml.
The reaction sites were assessed 24 h and 48 h after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

2. CHALLENGE EXPOSURE - PERFORMED ON TEST DAY 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.
Hair was clipped and shaved from a 5 × 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3 × 3 cm) of filter paper were saturated with the test article at the highest non-irritating concentration of 25 % (left flank) and the vehicle only (PEG 400 applied to the right flank) using the same method as for the epidermal application. The volume of test article preparation and vehicle applied was approximately 0.2 ml. The dressings were left in place for 24 hours.

Challenge controls:

Yes. The right flank was treated with the vehicle alone

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

Positive control results:

See other information on results below

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

10

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

10

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50%

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

PRETEST RESULTS

Test concentrations used in the pretest were 10 %, 5 %, 3 % and 1 % on 1 animal in intradermal injection, and 50 %, 25 %, 15 % and 10 % on two other animals in epidermal application. According to Magnusson - Kligman and to the observations in the study, the test article at 50 % was considered to be the maximally tolerated concentration to stimulate a state of immune hypersensitivity and 25 % as the highest non-irritating concentration to be used for the challenge.

SKIN REACTION RESULTS

SKIN EFFECTS AFTER INTRADERMAL INDUCTION - PERFORMED ON TEST DAY 1

The expected and common findings were observed in the control and test group after the different applications using FCA intradermally and consisted of erythema, oedema, necrotizing dermatitis, encrustation, exfoliation of encrustation and red discoloration.

No description of the results is given in the report as these FCA effects are well-known.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION - PERFORMED ON TEST DAY 8

Control group

Discrete/patchy erythema was observed at the 24 h reading in three out of 5 animals treated with PEG 400 only. Animals in this group were pretreated with 10 % SLS in paraffinum perliquidum.

Test group

As the test article at 50 % stained the skin red, it was not possible to determine whether erythema was present or not. However, no oedema was observed. Animals in this group were pretreated with 10 % SLS in paraffinum perliquidum.

SKIN EFFECTS AFTER THE CHALLENGE - PERFORMED ON TEST DAY 22

Control group

No skin reactions were observed in the animals when treated with either PEG 400 only or when treated with the test article at 25 % in PEG 400.

Red discoloration produced by the test article was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 h prior to challenge reading.

Test group

Moderate/confluent to intense erythema and swelling were observed in all animals at the 24 h and 48 h reading after treatment with the test article at 25 % in PEG 400. No skin reactions were observed in the animals when treated with PEG 400 only.

Red discoloration produced by the test article was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 h prior to challenge reading.

Table 1: Skin reactions in the induction and challenge procedures, after 24 h and 48 h, in control and test animals

Animal number	INDUCTION		CHALLENGE
			RIGHT FLANK		LEFT FLANK
	After epidermal application of 50 % test substance in PEG 400		After epidermal application of PEG 400		After epidermal application of 50 % test substance in PEG 400
	24 h	48 h	24 h	48 h	24 h	48 h
Control group
1 2 3 4 5	1 0 1 1 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0
Test group
6 7 8 9 10 11 12 13 14 15	– * – – – – – – – – –	– * – – – – – – – – –	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	2 2 2 2 2 2 3 2 3 2	2 2 2 3 2 2 3 2 3 2

Key

* Due to red discolouration produced by the test substance, a possible erythema could not be determined

Viability / Mortality / Macroscopic Findings

No deaths occurred in the study, hence, no necropsies were performed.

Clinical signs (systemic)

No signs of systemic toxicity were observed in the animals

Body weights

The body weight of the animals was within the range commonly recorded for animals of this strain and age

POSITIVE CONTROL RESULTS

For validation of sensitivity of the Maximization-Test of B. Magnusson and A.M. Kligman (1969) as well as the sensitivity of the test system used, a known sensitizer 2-MERCAPTOBENZOTHIAZOLE was selected as a positive control. This was performed in accordance with the recommendation of the OECD Guideline 406 "Skin Sensitization Test", adopted on July 17, 1992, under GLP conditions.

The study was performed with 15 (10 test and 5 control) male albino guinea pigs (GOHI) sourced from RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf, Switzerland.

The intradermal induction of sensitization was performed with a 5 % dilution of the test article in mineral oil and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion with the test article at 50 % in mineral oil. Two weeks after the epidermal induction application the challenge was completed by epidermal application of the test article at 10 % in mineral oil under occlusive dressing . The animals of the control group were induced with mineral oil 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 h and 48 h after removal of the dressing.

After the challenge, the following reactions were observed:

In the control group, 0 out of 5 animals showed a positive reaction 24 h and 48 h after the positive control substance treatment on the left flank. In the same group, 0 out of 5 animals showed a positive reaction 24 h and 48 h after mineral oil treatment on the right flank.

In the test group, 9 out of 10 animals and 10 out of 10 animals showed a positive reaction 24 h and 48 h after positive control substance treatment on the left flank, respectively. In the same group, 0 out of 10 animals showed a positive reaction 24 h and 48 h after mineral oil application on the right flank.

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

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Under the conditions reported, at 24 h and 48 h after challenge with the test substance, positive skin reactions were observed in 100 % of animals in the test group treated with the test substance. No positive reactions were seen in the control groups.
Therefore, the test substance is a skin sensitizer.

Executive summary:

The test article was investigated for possible allergenic potential to the skin of 15 (10 test and 5 control) male albino guinea pigs based on the Guinea-Pig Maximisation Test, of OECD Guideline 406 "Skin Sensitisation," adopted July 17th, 1992 and EEC Guideline B.6. "Acute Toxicity-Skin Sensitization," July 30th, 1996.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 1 % dilution of the test article in PEG 400 and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test article at 50 % in PEG 400 one week after the intradermal induction and following pretreatment of the test areas with 10 % Sodium-Lauryl-Sulfate (SLS) approximately 23 hours prior to application of the test article. The animals of the control group were intradermally induced with PEG 400 and FCA / physiological saline and epidermally induced with PEG 400 under occlusion following pretreatment with 10 % SLS.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test article at 25 % in PEG 400 (test article formulation) and PEG 400 alone (vehicle) under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

After the challenge, the following reactions were observed:

In the control group, 0 out of 5 animals showed a positive reaction 24 h and 48 h after test article formulation treatment on the left flank, after 24 h and 48 h. In the same group, 0 out of 5 animals showed a positive reaction 24 h and 48 h after vehicle treatment on the right flank.

In the test group, 10 out of 10 animals showed a positive reaction 24 h and 48 h after test article formulation treatment on the left flank, after 24 h and 48 h. In the same group, 0 out of 10 animals showed a positive reaction 24 h and 48 h after vehicle treatment on the right flank.

No mortality occurred. No toxic symptoms were evident in the guinea pigs of the control or test group. All test animals showed moderate/confluent to intense erythema and swelling after challenge treatment with the test article formulation.

Therefore, the test article is a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The test article was investigated for possible allergenic potential to the skin of 15 (10 test and 5 control) male albino guinea pigs based on the Guinea-Pig Maximisation Test, of OECD guideline 406 "Skin Sensitisation" (1992) and EU method B.6. "Acute Toxicity-Skin Sensitisation" (1996).

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 1 % dilution of the test article in PEG 400 and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test article at 50 % in PEG 400 one week after the intradermal induction and following pretreatment of the test areas with 10 % Sodium-Lauryl-Sulfate (SLS) approximately 23 hours prior to application of the test article. The animals of the control group were intradermally induced with PEG 400 and FCA / physiological saline and epidermally induced with PEG 400 under occlusion following pretreatment with 10 % SLS.

No mortality occurred. No toxic symptoms were evident in the guinea pigs of the control or test group. All test animals showed moderate/confluent to intense erythema and swelling after challenge treatment with the test article formulation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), Annex I, Part 3, Table 3.4.3, a test substance is assigned to Category 1A based on this criteria: "≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose," in the guinea pig maximisation test.

All animals exposed to 1 % intradermal induction dose showed response to test substance. Since this result fulfils the above criteria for classification, test substance is classified in category 1A (H317) as skin sensitiser in the CLP Regulation (EC 1272/2008).