Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Endpoint summary

Description of key information

Skin Sensitisation

Under the conditions of the study, the test material is classified as a non-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:

7 December 1992 to 15 January 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The skin sensitization potential of the test material was tested at a time that predates the LLNA method.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately eight to twelve weeks old.
- Weight at study initiation: The bodyweights of the animals ranged from 301 to 361 g.
- Housing: The animals were housed in groups of up to three in solid-floor polypropylene cages furnished with softwood shavings.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: A minimum acclimatisation period of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22 °C
- Humidity (%): Relative humidity of 32 - 59 %
- Air changes (per hr): approximately 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

5 % (w/v) in arachis oil B.P.

Day(s)/duration:

7 days

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, open

Vehicle:

arachis oil

Concentration / amount:

25 % (w/w) in arachis oil B.P.

Day(s)/duration:

2 days

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, open

Vehicle:

arachis oil

Concentration / amount:

25 % and 10 % (w/w) in arachis oil B.P.

Day(s)/duration:

1 day

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 animals were treated with test material and 10 animals were treated with the positive control.

Details on study design:

RANGE FINDING TESTS:
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:

- Selection of Concentration for Intradermal Induction (Sighting Test):
Three animals were intradermally injected with preparations of test material (1 %, 5 % or 10 % w/v in arachis oil B.P.). The highest concentration that did not cause local necrosis, ulceration or systemic toxicity, was selected for the intradermal induction stage of the main study.

- Selection of Concentration for Topical Induction:
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant fifteen days earlier) were treated with four preparations of the test material (25 %, 10 %, 5 % and 2 % w/w in arach is oil B.P.).

- Selection of Concentration for Topical Challenge:
The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure, was selected for the topical induction stage of the main study.
Four preparations of the test material (25 %, 10 %, 5 % and 2 % w/w in arachis oil B.P.) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

MAIN STUDY
A group of thirty guinea pigs was used for the main study, twenty test and ten control. The bodyweight of each animal was recorded at the start and end of the study.

Two main procedures were involved in the maximisation test; (a) an induction of a response and (b) a challenge of that response.

A. INDUCTION EXPOSURE
Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the mid-line. The injections were:

i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
ii) A 5 % (w/v) dilution of test material in arachis oil B.P.
iii) A 5 % (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant plus arachis oil B.P.

One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation (25 % w/w in arachis oil B.P.). The test material formulation (0.2 - 0.3 mL) was applied on filter paper (Whatman No. 4, approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (Blenderm, approximate size 60 mm x 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

Erythematous reactions were quantified one and twenty-four hours following removal of the patches.

INDUCTION OF CONTROL ANIMALS
Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:

i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
ii) Arachis oil B.P.
iii) Freund's Complete Adjuvant plus arachis oil B.P. in the ratio 1:1.

The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area, approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.

A quantity of 0.1 - 0.2 mL of the test material formulation (25 % w/w in arachis oil B.P.) was applied to the shorn right flank of each animal on a square of filter paper (Whatman No. 4, approximate size 20 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (Blenderm, approximate size 40 mm x 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 10 % (w/w) in arachis oil B.P. was also similarly applied to a separate skin site on the right shorn flank. The vehicle alone was similarly applied to the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive bandage (approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.

After 24 hours, the dressing was carefully cut using blunt tipped scissors, removed and discarded. The challenge sites were swabbed with cotton wool soaked in diethyl ether to remove residual material. The vehicle sites were similarly swabbed. The position of the treatment sites was identified by using a black indelible marker-pen.

Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.

EVALUATION OF SKIN REACTIONS
Approximately 24 and 48 hours after challenge dressing removal erythematous reactions were quantified using the four-point scale shown below:
Scale:
0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling

Challenge controls:

The vehicle alone was applied to the left shorn flank during the challenge phase.

Positive control substance(s):

yes

Remarks:

2,4-Dinitrochlorobenzene

Positive control results:

The known contact sensitiser, 2,4-Dinitrochlorobenzene (DNCB), produced a 90 % (9/10) sensitisation rate. This was considered to be a satisfactory sensitisation response for this material under the conditions of the test.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.25% (w/v) in absolute ethanol

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

None reported

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.25% (w/v) in absolute ethanol

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

None reported

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Vehicle only

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None reported

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Vehicle only

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None reported

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

1

Total no. in group:

18

Clinical observations:

Black-coloured staining observed on all the surviving animals. There was not data available for two of the animals as one was killed and the other one was found dead.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

1

Total no. in group:

18

Clinical observations:

Black-coloured staining observed on all the surviving animals. There was not data available for two of the animals as one was killed and the other one was found dead.

Remarks on result:

no indication of skin sensitisation

Skin Reactions Observed After Topical Induction

Black-coloured staining caused by the test material was noted at the induction sites of all test group animals at 1 and 24 hours following patch removal. The staining prevented accurate evaluation of erythema. Other skin reactions noted were bleeding, small scattered superficial scabs and hardened dark brown/black-coloured scab.

Skin Reactions Observed After Topical Challenge

One test group animal was found dead on day 13. The cause of death was not determined. One test group animal was killed on day 21 due to respiratory problems. The absence of these animals was not considered to affect the purpose or integrity of the study.

Black-coloured staining caused by the test material was noted at the challenge sites of all test and control group animals at the 24 and 48-hour observations. The staining did not prevent accurate evaluation of erythema.

- 25 % (w/w) in Arachis Oil B.P: A positive skin response (redness grade 1) was noted at the challenge site of one test group animal at the 24 and 48-hour observation. No skin reactions were noted at the challenge sites of control group animals at the 24 and 48-hour observations.

- 10 % (w/w) in Arachis Oil B.P: No skin reactions were noted at the challenge sites of test and control group animals at the 24 and 48-hour observations.

- Vehicle Control: No skin reactions were noted at the vehicle control sites of the test or control group animals at the 24 and 48-hour observations.

Bodyweight

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period.

Interpretation of results:

other: Not classified in accordance with EU criteria.

Conclusions:

Under the conditions of the study, the test material is classified as a non-sensitiser.

Executive summary:

The potential of the test material to act as a sensitiser was investigated in accordance with the standardised guideline OECD 406 under GLP conditions.

Albino guinea pigs were used to assess the skin sensitization potential of the test material with twenty test and ten control animals used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were 5 % (w/v) in arachis oil B.P for the intradermal induction, 25 % (w/w) in arachis oil B.P for the topical induction and 25 % and 10 % (w/w) in arachis oil B.P for the topical challenge. The animals were evaluated for skin reactions and any changes in weight gain were also assed. After topical induction of animals black-coloured staining caused by the test material was noted at the induction sites of all test group animals one and 24 hours following patch removal. The staining prevented accurate evaluation of erythema. Other skin reactions noted were bleeding, small scattered superficial scabs and hardened dark brown/black-coloured scab. After the topical challenge, one test group animal was found dead on day 13. The cause of death was not determined. One test group animal was killed on day 21 due to respiratory problems. The absence of these animals was not considered to affect the purpose or integrity of the study. Black-coloured staining caused by the test material was noted at the challenge sites of all test and control group animals at the 24 and 48-hour observations. The staining did not prevent accurate evaluation of erythema. A positive skin response (redness grade 1) was noted at the challenge site of one test group animal dosed with 25 % (w/w) in Arachis Oil B.P at the 24 and 48-hour observation. The rest of the control and test animals had no skin reactions noted at the challenge sites at the 24 and 48-hour observations.

Under the conditions of the study, the test material is classified as a non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of the test material to act as a sensitiser was investigated in accordance with the standardised guideline OECD 406 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Albino guinea pigs were used to assess the skin sensitization potential of the test material with twenty test and ten control animals used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were 5 % (w/v) in arachis oil B.P for the intradermal induction, 25 % (w/w) in arachis oil B.P for the topical induction and 25 % and 10 % (w/w) in arachis oil B.P for the topical challenge. The animals were evaluated for skin reactions and any changes in weight gain were also assed. After topical induction of animals black-coloured staining caused by the test material was noted at the induction sites of all test group animals one and 24 hours following patch removal. The staining prevented accurate evaluation of erythema. Other skin reactions noted were bleeding, small scattered superficial scabs and hardened dark brown/black-coloured scab. After the topical challenge, one test group animal was found dead on day 13. The cause of death was not determined. One test group animal was killed on day 21 due to respiratory problems. The absence of these animals was not considered to affect the purpose or integrity of the study. Black-coloured staining caused by the test material was noted at the challenge sites of all test and control group animals at the 24 and 48-hour observations. The staining did not prevent accurate evaluation of erythema. A positive skin response (redness grade 1) was noted at the challenge site of one test group animal dosed with 25 % (w/w) in Arachis Oil B.P at the 24 and 48-hour observation. The rest of the control and test animals had no skin reactions noted at the challenge sites at the 24 and 48-hour observations.

Under the conditions of the study, the test material is classified as a non-sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for sensitisation.