N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyphenyl]acetamide

Endpoint summary

Description of key information

Based on the data presented, the test substance Disperse Blue 291:1 (Br) seems to react as a skin sensitiser at concentration of ≥ 25% if the animals were pre-treated with an adjuvant.

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:

March/April 1987

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 (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Method was not available

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
albino Dunkin-Hartley guinea pigs
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, U.K.
- Weight at study initiation: 330 - 411g
- Age: six to ten weeks
- Housing: groups of up to 4
- Diet: Guinea Pig FD1 Diet, Special Diet Services Limited, Witham, Essex, U.K. ad libitum
- Water: tap water ad libitum
- Acclimatisation period: at least five days,

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 25°C
- Humidity (%): 45 to 60%
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 17. March To: 1 May 1987

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

5% / 2 x 0.1 mL
5% / 2 x 0.1 mL in arachis oild : Freund's Complete Adjuvant (FCA) 1:1

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:

petrolatum

Concentration / amount:

50% (w/w) in petroleum jelly B.P. / 0.1 to 0.2 mL

Day(s)/duration:

on Day 8 for 48 hours

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50% (w/w) in petroleum jelly B.P. / 0.1 to 0.2 mL

Day(s)/duration:

on Day 22 for 24 hours

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

25% (w/w) in petroleum jelly B.P.
10% (w/w) in petroleum jelly B.P.

Day(s)/duration:

Day 29 for 24 hours

No. of animals per dose:

Determination of primary not irritating concentration: 4
Determination of intradermal tolerability: 2
Control group: 10
Re-challenge control group; 10
Treatment group: 20

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal + 1 epicutaneous
- Exposure period: intradermal: 24 hours; epicutaneous: 48 hours
- Test groups: 1
- Control group: 2
- Site: shoulder
- Frequency of applications: Day 1; Day 8
- Duration: 24 and 48 hours
- Concentrations: 5 and 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: Day 22 and Day 29
- Exposure period: 24 hours
- Test groups: 1
- Control group: 1 + 1
- Site: flank
- Concentrations: 50%, 25%, 10%
- Evaluation (hr after challenge): Challenge 1: 24, 48, 72 hours Challenge 2: 24 and 48 hours after removal of patches

Positive control substance(s):

yes

Positive control results:

valid

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

14

Total no. in group:

20

Clinical observations:

yellow/green coloured staining of skin

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

11

Total no. in group:

20

Clinical observations:

yellow/green coloured staining of skin; one animal not evaluable due to skin alterations was counted as positive

Remarks on result:

positive indication of skin sensitisation

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

yellow/green coloured staining of skin; one animal not evaluable due to skin alterations was counted as positive

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

yellow/green coloured staining of skin

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

yellow/green coloured staining of skin

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

yellow/green coloured staining of skin

Yellow/green coloured staining of the skin was noted at all test sample sites but did not prevent assessment of the skin responses.

 Initial Challenge - 50% (w/w)

Positive results were also observed in the negative control group. The sensitisation potential of the test sample could not be evaluated at this concentration. A re-challenge was performed at two lower concentrations.

Re-challenge - 25% and 10% (w/w)

Moderate and diffuse redness (score 2) and scattered mild redness (score 1) were noted at the 25% and 10% test sample re-challenge sites of test animalsat the 24-hour observation and at the 25% test sample re-challenge sites of test animals at the 48-hour observation. Scattered mild redness (score 1) continued to be noted at the 10% test sample re-challenge sites of the test animals at the 48-hour observation. Severe desquamation was noted at a number of 25% test sample re-challenge sites and at one 10% test sample re-challenge site at the 48-hour observation. The 25% and 10% test sample sites of control animals showed no erythema at the 24 or 48-hour observations.

The test sample, therefore, at re-challenge concentrations of 25% and 10%, produced a skin reaction in 85% (17/20) and 80% (16/20) animals, respectively after 24 hours. After 48 hours, a skin reaction was observed in 45% (9/20) and 15% (3/20) at concentrations of 25% and 10%, respectively.

Bodyweight gains of surviving 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:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test substance caused positive reactions in 85% and 45% of animals after 24 and 48 hours after removal of the test substance at 25%; it is hence considered to be a skin sensitiser.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test sample in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 "skin Sensitisation" - Magnusson and Kligman Maximisation Test.

 

Twenty test and ten control animals were used for the main study.

Following sighting studies, the following concentrations were used in the induction and challenge phases:

Intradermal Induction       5% (w/v) in arachis oil

Topical Induction             50% (w/w) in petroleum jelly

Topical Challenge             50% (w/w) in petroleum jelly; re-challenge at 25% and 10%

Yellow/green coloured staining of the skin was noted at all test sample sites but did not prevent assessment of the skin responses.

 Initial Challenge - 50% (w/w)

Positive results were also observed in the negative control group. The sensitisation potential of the test sample could not be evaluated at this concentration. A re-challenge was performed at two lower concentrations.

Re-challenge - 25% and 10% (w/w)

Moderate and diffuse redness (score 2) and scattered mild redness (score 1) were noted at the 25% and 10% test sample re-challenge sites of test animalsat the 24-hour observation and at the 25% test sample re-challenge sites of test animals at the 48-hour observation. Scattered mild redness (score 1) continued to be noted at the 10% test sample re-challenge sites of the test animals at the 48-hour observation. Severe desquamation was noted at a number of 25% test sample re-challenge sites and at one 10% test sample re-challenge site at the 48-hour observation. The 25% and 10% test sample sites of control animals showed no erythema at the 24 or 48-hour observations.

The test sample, therefore, at re-challenge concentrations of 25% and 10%, produced a skin reaction in 85% (17/20) and 80% (16/20) animals, respectively after 24 hours. After 48 hours, a skin reaction was observed in 45% (9/20) and 15% (3/20) atconcentrations of 25% and 10%, respectively.

Bodyweight gains of surviving 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.

The test substance caused positive reactions in more than 30% of animals after 24 hours after removal of the test substance at 10% and 25% test substance contentration. After 48 hours, only the 25% concentration caused skin reddening in more that 30% of test animals.

The test substance is hence considered to be a skin sensitiser at concentrations of 25% and above.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Two studies for skin sensitising properties were conducted with Disperse Blue 291, a structural analogue of Disperse Blue 291:1. One of those studies were negative and one of these studies were positive.

The first skin sensitisation study performed in 1978 with Disperse Blue 291 (Br) with a purity of ca. 95% was conducted according to the ear/flank test method established by Stevens (Stevens MA. Use of the Albino Guinea-pig to Detect the Skin-sensitizing Ability of Chemicals. British journal of industrial medicine 1967;24(3):189-202) in albino guinea pigs. For this purpose, the test article (0.1 mL of a 10% w/v solution in dimethylformamide) was applied daily by means of a glass syringe to the outer surface of the ears of 6 guinea pigs for three days. On Day 8, 0.2 mL of the challenge solutions (5% w/v, 0.1% w/v and 0.01% w/v in dimethylformamide) was applied topically to 1 cm diameter circular areas on the clipped flanks of each of the same 6 animals. Solutions of test article were also applied in the same way on Day 8 to the clipped flanks of 4 control animals which had no previous treatment on the ears. The applications were made on both flanks of all 10 guinea pigs, with each concentration being applied to each flank. The treated skin at each site was assessed 24 hours later (Day 9). No erythema was noted in any of the test or control animals. The test article was thus not considered to be a strong sensitiser.

 

The second study performed in 1987 with bulk material out of the Disperse Blue 291 (Br) synthesis without information of its purity, was performed in the guinea pig maximisation test according to Magnusson & Kligman (OECD Guidelines for Testing of Chemicals (1981) No. 406 "skin Sensitisation"). Twenty test and ten plus 10 control animals were used for the main study. Following sighting studies, the intradermal induction was carried out with 5% (w/v) in arachis oil and in Freund’s Adjuvant. The topical induction and challenge was carried out with a concentration of 50% (w/w) in petroleum jelly. The skin was assessed 24, 48, and 72 hours after end of the challenge exposure. As also control animals showed skin reddening, a further challenge was done at 10% and 25% (w/w) using the same treatment and a new control group.

Yellow/green coloured staining of the skin was noted at all test sample sites but did not prevent assessment of the skin responses.

At the first challenge with 50% (w/w) positive results were also observed in the negative control group. The sensitisation potential of the test sample could not be evaluated at this concentration.

At re-challenge with 25% and 10% (w/w), moderate and diffuse redness (score 2) and scattered mild redness (score 1) were noted at the 25% and 10% test sample re-challenge sites of test animals at the 24-hour observation and at the 25% test sample re-challenge sites of test animals at the 48-hour observation. Scattered mild redness (score 1) continued to be noted at the 10% test sample re-challenge sites of the test animals at the 48-hour observation. Severe desquamation was noted at a number of 25% test sample re-challenge sites and at one 10% test sample re-challenge site at the 48-hour observation. The 25% and 10% test sample sites of control animals showed no erythema at the 24 or 48-hour observations.

The test sample, therefore, at re-challenge concentrations of 25% and 10%, produced a skin reaction in 85% (17/20) and 80% (16/20) animals, respectively after 24 hours. After 48 hours, a skin reaction was observed in 45% (9/20) and 15% (3/20) at concentrations of 25% and 10%, respectively. Of these animals, only 25% (5/20) and 10% (2/20) were identified positive for reddening, the remaining 4 and 1 animals could not be assessed due to desquamation of the skin at concentrations of 25% and 10%, respectively.

Bodyweight gains of surviving 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.

 

The test substance caused positive reactions in more than 30% of animals after 24 hours after removal of the test substance at 10% and 25% test substance concentration. After 48 hours, only the 25% concentration caused skin reddening in more than 30% of test animals.

The test substance is hence considered to be a skin sensitiser at concentrations of 25% and above.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data presented, the test substance Disperse Blue 291:1 (Br) seems to react as a skin sensitiser at concentration of ≥ 25% if the animals were pre-treated with an adjuvant.