2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 November 1987 to 20 December 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guidelines for Testing of Chemicals (1981) No. 406 "Skin Sensitisation" -Magnusson and Kligman Maximisation Test.

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study data over 12 years old.

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

EC Number:

284-395-7

EC Name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Cas Number:

84870-65-5

Molecular formula:

C19H18N6O2

IUPAC Name:

2-[[4-(diethylamino)-2-methylphenyl]azo]-5-nitrobenzene-1,3-dicarbonitrile

Test material form:

solid

Specific details on test material used for the study:

No further details sepcified in the study report.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Thirty-eight female, albino Dunkin-Hartley guinea pigs were supplied by Interfauna (UK) Limited, Wyton, Huntingdon, Cambridgeshire. At the start of the main study the animals weighed 308 - 405g, and were approximately six to ten weeks old. After a minimum acclimatisation period of five days, each animal was selected at random and given a number unique within the study which was written both on a small area of clipped rump using a black waterproof marker-pen, and on the cage card.
The animals were housed in groups of up to four in solid-floor polypropylene cages furnished with softwood shavings. Free access to mains tap water and food (Guinea Pig FD1 Diet, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
The animal room was maintained at a temperature of 17 – 23 °C and relative humidity of 40 - 65%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

10% (w/v)

Day(s)/duration:

one administration on day 1

Adequacy of induction:

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

Induction 2

Route:

intradermal

Vehicle:

other: 1:1 preparation of Freund’s Complete Adjuvant plus arachis oil B.P.

Concentration / amount:

10% (w/v)

Day(s)/duration:

one administration on day 1

Adequacy of induction:

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

Induction 3

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50% w/w

Day(s)/duration:

day 8/48 hours

Adequacy of induction:

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

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

25% w/w

Day(s)/duration:

day 22/24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

A group of thirty guinea pigs was used for the main study, twenty test and ten control.

Details on study design:

Procedure
The sensitising properties of the test material were assessed using the maximisation test of Magnusson B. & Kligman A.M., J. Invest. Dermatol. (1969) 52: 268 - 276.

The dose levels for each of the three stages of the main study were determined by a ‘sighting study’ in which groups of one or two guinea pigs were used and up to two dose levels were tested on each animal. The procedure was as follows:
i) Intradermal Injection (Induction):
Dilutions of the test material in arachis oil B.P. were tested to determine the highest practical level that could be intradermally injected and well tolerated both locally and systemically.
ii) Topical Application (Induction):
Dilutions of the test material in petroleum jelly B.P. were tested to determine the highest practical level which did not produce excessive inflammation on the flank of animals injected with Freund’s Complete Adjuvant (Difco Laboratories, Michigan, U.S.A.) at least seven days previously.
iii) Topical Application (Challenge):
Dilutions of the test material in petroleum jelly B.P. were tested to determine the highest practical level which did not produce inflammation or irritation on the flank of animals injected with Freund’s
Complete Adjuvant at least seven days previously.

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
Induction of the Test Animals: The hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers and 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 arachis oil B.P. in the ratio 1:1.
ii) a 10% (w/v) dilution of test material in arachis oil B.P.
iii) a 10% (w/v) dilution of test material in a 1:1 preparation of Freund’s Complete Adjuvant plus arachis oil B.P.

One week later, 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 (50% w/w in petroleum jelly 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 (ELASTOPLAST: 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 immediately following removal of the patches using the 0 - 3 scale shown in Section b.

Induction of the 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 arachis oil B.P. 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
Two weeks after the topical inductions, an area, approximately 50 - 70 mm x 50 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 petroleum jelly B.P.) was applied to the shorn right flank of each animal on a 20 mm x 20 mm square of filter paper (WHATMAN No. 4) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). 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 (ELASTOPLAST: 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 position of the treatment sites was identified by using a black indelible marker-pen. After a further 24 and 48 hours, any erythematous reactions were quantified using the four-point scale shown and the number of positive responses recorded.
Scale
0 - no reaction
1 - scattered mild redness
2 - moderate and diffuse redness
3 - intense redness and swelling

To classify the sensitisation response, the percentage of test animals that showed a more severe reaction at the test material challenge site than the most severe reaction seen in the control animals, was compared with the following scale:
%of animals sensitised Classification of sensitisation potential
0 non-sensitiser
> 0 8 weak sensitiser
> 8 – 28 mild sensitiser
> 28 – 64 moderate sensitiser
> 64 – 80 strong sensitiser
> 80 – 100 extreme sensitiser

Challenge controls:

The vehicle alone was applied to the left shorn flank.

Positive control substance(s):

yes

Remarks:

FORMALDEHYDE (40% AQUEOUS SOLUTION)

Results and discussion

Positive control results:

The positive test material Formaldehyde (40% aqueous solution), produced a 95% (19/20) sensitisation rate and was classified as an extreme sensitiser to guinea pig skin.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Heavy blue staining of the test material sites precluded evaluation of skin reactions.

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Heavy blue staining of the test material sites precluded evaluation of skin reactions.

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Any other information on results incl. tables

INDIVIDUAL REACTIONS IN TEST ANIMALS AT CHALLENGE

TEST MATERIAL: DISPERSIONSBLAU F-60 768

CHALLENGE CONCENTRATION: 25% (w/w)

VEHICLE: PETROLEUM JELLY B.P.

ANIMAL NUMBER	SKIN REACTIONS (HOURS AFTER REMOVAL OF DRESSING)
	24		48
	TEST	VEHICLE	TEST	VEHICLE
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20	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 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 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 0 0

 

INDIVIDUAL REACTIONS IN CONTROL ANIMALS AT CHALLENGE

TEST MATERIAL: DISPERSIONSBLAU F-60 768

CHALLENGE CONCENTRATION: 25% (w/w)

VEHICLE: PETROLEUM JELLY B.P.

ANIMAL NUMBER	SKIN REACTIONS (HOURS AFTER REMOVAL OF DRESSING)
	24		48
	TEST	VEHICLE	TEST	VEHICLE
21 22 23 24 25 26 27 28 29 30	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 0 0 0 0	0 0 0 0 0 0 0 0 0 0

 

INTRADERMAL SIGHTING TEST – SUMMARY OF RESULTS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL IDENTIFICATION	TIME OF OBSERVATION	CONCENTRATION OF TEST MATERIAL * (% w/v)	EVIDENCE OF NECROSIS	EVIDENCE OF SYSTEMIC TOXICITY
A	24 hours 48 hours 72 hours 7 days	1	None S None S None S None S	None None None None
B	24 hours 48 hours 72 hours 7 days	5	None S None S None S None S	None None None None
C	24 hours 48 hours 72 hours 7 days	10 <	None S None S None S None S	None None None None

*Vehicle = arachis oil B.P.

< = maximum practical concentration for intradermal dosing

S = heavy blue staining precluded assessment of erythema

 

The concentration of the test material to be used for the intradermal induction stage of the main study was selected as follows:

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

 

TOPICAL SIGHTING TEST – EVALUATION OF REACTIONS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL IDENTIFICATION	CONCENTRATION OF TEST MATERIAL * (% w/v)	EVALUATION OF APPLICATION SITES (HOURS AFTER REMOVAL OF PATCHES)
		1	24	48
E	50 < 25	2 S 1 S	0 S 0 S	0 S 0 S
F	50 < 25	2 S 1 S	1 S 0 S	0 S 0 S
G	25 10	1 S 0 S	0 S 0 S	0 S 0 S
H	25 10	1 S 0 S	0 S 0 S	0 S 0 S

*Vehicle = petroleum jelly B.P.

< = maximum practical concentration for topical dosing

S = blue-coloured staining of skin/hair not affected assessment of erythema

 

The concentrations of the test material to be used for the main study were selected as follows:

Topical Induction          :          50% (w/w) in petroleum jelly B.P.

Topical Challenge         :          25% (w/w) in petroleum jelly B.P.

 

TOPICAL INDUCTION – EVALUATION OF REACTIONS IN TEST ANIMALS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

INDUCTION CONCENTRATION: 50% (w/w) IN PETROLEUM JELLY B.P.

ANIMAL NUMBER	SKIN REACTION (IMMEDIATELY AFTER REMOVAL OF DRESSINGS)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20	? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S ? S

?S = heavy blue-coloured staining precluded assessment of erythema

 

TOPICAL INDUCTION – EVALUATION OF REACTIONS IN CONTROL ANIMALS

VEHICLE: PETROLEUM JELLY B.P.

ANIMAL NUMBER	SKIN REACTION (IMMEDIATELY AFTER REMOVAL OF DRESSINGS)
21 22 23 24 25 26 27 28 29 30	0 0 0 0 0 0 0 0 0 0

 

INDIVIDUAL BODYWEIGHTS OF TEST ANIMALS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL NUMBER	BODYWEIGHT (g)		BODYWEIGHT (g) INCREASE
	DAY 0	DAY 24
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20	370 393 396 405 390 359 385 347 323 385 325 330 380 350 354 375 395 371 370 308	483 534 568 587 491 468 505 465 427 527 483 468 475 492 475 534 513 505 474 456	113 141 172 182 101 109 120 118 104 142 148 130 95 142 121 159 118 134 104 148

 

INDIVIDUAL BODYWEIGHTS OF CONTROL ANIMALS

TEST MATERIAL: DISPERSIONSBLAU F-60 768

ANIMAL NUMBER	BODYWEIGHT (g)		BODYWEIGHT (g) INCREASE
	DAY 0	DAY 24
21 22 23 24 25 26 27 28 29 30	370 352 364 399 340 380 365 350 330 365	461 470 474 532 470 540 520 493 469 499	91 118 110 133 130 160 155 143 139 134

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material, DISPERSIONSBLAU F-60 768, was found to be a non-sensitiser to guinea pig skin.
The substance is not classifiable according to CLP criteria.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test material 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 :          10% (w/v)

Topical Induction        :          50% (w/w)

Topical Challenge      :          25% (w/w)

 

The test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin.

The substance is not classified in accordance with CLP criteria.