N-(dodecylphenyl)naphthalen-1-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 1999 to 27 June 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guidelines for the Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Method B6 of Commission Directive 96/54/EC (which constitutes Annex V of Council Directive 67/548/EEC).

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study data available from study performed in 1999.

Test material

Test material information

1

Reference substance name:

-

EC Number:

437-450-6

EC Name:

-

Cas Number:

64654-05-3

Molecular formula:

Hill formula: C28 H37 N

IUPAC Name:

N-(dodecylphenyl)naphthalen-1-amine

Test material form:

liquid: viscous

Details on test material:

Sponsor's identification: APAN
Description: redbrown viscous liquid
Lot number: EL01010B01/KZ8911.5
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

No further details specified in the study report.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

Forty male albino Dunkin Hartley guinea pigs supplied by David Hall Limited, Burton-on-Trent, Staffordshire, UK were used. At the start of the main study the animals weighed 352 to 4468, and were approximately eight to twelve weeks old. After an acclimatisation period of at least five days, each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible markerpen.
The animals were housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes. Free access to mains tap water and food (Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

arachis oil

Concentration / amount:

25% w/v

Day(s)/duration:

21

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

75%

Day(s)/duration:

2

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:

Selection of Concentrations for Main Study (Sighting 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 lntradermal Induction
Four concentrations of test material were investigated (1 %, 5%, 10% and 25% w/v in arachis oil BP). A total of four guinea pigs were used, each guinea pig receiving four 0.1 ml injections of only one concentration of test material. The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage of the main study.

Selection of Concentration for Topical lnduction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant ten days earlier) were treated with the undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in arachis oil BP). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately
1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.

Selection of Concentration for Topical Challenge
The undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs had been intradermally injected with Freund's Complete Adjuvant ten days earlier. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. 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 phases were involved in the main study; (a) an induction of a response and (b) a challenge of that response.

Induction
Induction of the Test Animals: 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:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1 :1
b) a 25% w/v formulation of the test material in arachis oil BP
C) a 25% w/v formulation of the test material in a 1 :1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (ie. Injection site b) was evaluated.
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 undiluted test material. A filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), saturated with the undiluted test material was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal.
This occlusive dressing was kept in place for 48 hours.
The degree of erythema and oedema was quantified one and twenty four hours following removal of the patches.
Any other reactions were also recorded.

Induction of the Control Animals: lntradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) arachis oil BP
C) 50% w/v formulation of arachis oil BP in a 1:1 mixture of Freund's Complete Adjuvant/distilled water
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the vehicle injection sites (ie injection site b) was evaluated.
The topical applications followed the same procedure as for the test animals except that nothing was applied to the filter paper. Skin reactions were quantified as for the test animals.

Challenge
Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
A square filter paper patch (WHATMAN No.4: approximate size 20 mm x 20 mm), saturated with the undiluted test material was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 75% v/v in arachis oil BP was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage 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 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, the degree of erythema and oedema was quantified.
Any other reactions were also recorded.

Challenge controls:

Not specified

Positive control substance(s):

yes

Remarks:

historical control data

Results and discussion

Positive control results:

Reported in table form - See "Any other information" for details.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75% v/v

No. with + reactions:

4

Total no. in group:

20

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

5

Total no. in group:

20

Results 3

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

4

Total no. in group:

20

Results 4

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75%

No. with + reactions:

2

Total no. in group:

20

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

75% & 100%

No. with + reactions:

0

Total no. in group:

10

Results 6

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

75% & 100%

No. with + reactions:

0

Total no. in group:

10

Results 7

Key result

Reading:

1st reading

Group:

positive control

Remarks on result:

other: Positive control data for the Magnusson & Kligman Maximisation study in the guinea pig were based on laboratory historical control data for the strain tested

Any other information on results incl. tables

INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS AT CHALLENGE

CHALLENGE CONCENTRATIONS: UNDILUTED AS SUPPLIED AND 75% v/v                             VEHICLE: ARACHIS OIL BP

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 Hours						48 Hours
	100%			75%			100%			75%
	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
1	2	1	-	1	0	-	1	1	-	0	0	-
2	0	0	-	0	0	-	0	0	-	0	0	-
3	0	0	-	0	0	-	0	0	-	0	0	-
4	0	0	-	0	0	-	0	0	-	0	0	-
5	0	0	-	0	0	-	0	0	-	0	0	-
6	0	0	-	0	0	-	0	0	-	0	0	-
7	0	0	-	0	0	-	0	0	-	0	0	-
8	0	0	-	0	0	-	0	0	-	0	0	-
9	0	0	-	0	0	-	0	0	-	0	0	-
10	1	1	-	1	1	-	1	0	-	1	0	-
11	0	0	-	0	0	-	0	0	-	0	0	-
12	0	0	-	0	0	-	0	0	-	0	0	-
13	1	0	-	2	1	-	1	0	-	1	1	-
14	2	1	-	0	0	-	1	0	-	0	0	-
15	0	0	-	0	0	-	0	0	-	0	0	-
16	0	0	-	0	0	-	0	0	-	0	0	-
17	0	0	-	0	0	-	0	0	-	0	0	-
18	0	0	-	0	0	-	0	0	-	0	0	-
19	0	0	-	0	0	-	0	0	-	0	0	-
20	1	0	-	1	0	-	0	0	-	0	0	-

Er = erythema        Oe = oedema         - = no other reactions noted

 

INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS AT CHALLENGE

CHALLENGE CONCENTRATIONS: UNDILUTED AS SUPPLIED AND 75% v/v                             VEHICLE: ARACHIS OIL BP

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 Hours						48 Hours
	100%			75%			100%			75%
	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
21	0	0	-	0	0	-	0	0	-	0	0	-
22	0	0	-	0	0	-	0	0	-	0	0	-
23	0	0	-	0	0	-	0	0	-	0	0	-
24	0	0	-	0	0	-	0	0	-	0	0	-
25	0	0	-	0	0	-	0	0	-	0	0	-
26	0	0	-	0	0	-	0	0	-	0	0	-
27	0	0	-	0	0	-	0	0	-	0	0	-
28	0	0	-	0	0	-	0	0	-	0	0	-
29	0	0	-	0	0	-	0	0	-	0	0	-
30	0	0	-	0	0	-	0	0	-	0	0	-

Er = erythema        Oe = oedema         - = no other reactions noted

 

INTRADERMAL SIGHTING TEST – SUMMARY OF RESULTS

VEHICLE: ARACHIS OIL BP

Animal Identification	Time of Observation	Concentration of Test Material (% w/v)	Grade of Erythema at Injection Sites	Evidence of Systemic Toxicity
A	24 Hours 48 Hours 72 Hours 7 Days	1	1 1 1 1	None None None None
B	24 Hours 48 Hours 72 Hours 7 Days	5	2 2 2 1	None None None None
C	24 Hours 48 Hours 72 Hours 7 Days	10	2 2 2 2	None None None None
D	24 Hours 48 Hours 72 Hours 7 Days	25	2 2 2 2	None None None None

The concentration of the test material selected for the intradermal induction stage of the main study was 25% w/v in arachis oil BP.

 

TOPICAL SIGHTING TEST FOR INDUCTION APPLICATION (48-HOUR EXPOSURE) – INDIVIDUAL SKIN REACTIONS

VEHICLE: ARACHIS OIL BP

Animal Identification	Concentration of Test Material (% v/v)	Skin Reactions (Hours After Removal of Patches)
		1			24			48
		Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
E	100 75 50 25	1 2 1 1	0 0 0 0	- - - -	1 1 1 1	0 0 0 0	- - - -	1 1 1 1	0 0 0 0	- - - -
F	100 75 50 25	2 2 2 2	0 0 0 0	- - - -	1 1 1 1	0 0 0 0	- - - -	1 1 1 1	0 0 0 0	- - - -

Er = erythema        Oe = oedema         - = no other reactions noted

The undiluted test material was selected for the main study topical induction

 

TOPICAL SIGHTING TEST FOR CHALLENGE APPLICATION (24-HOUR EXPOSURE) – INDIVIDUAL SKIN REACTIONS

VEHICLE: ARACHIS OIL BP

Animal Identification	Concentration of Test Material (% v/v)	Skin Reactions (Hours After Removal of Patches)
		1			24			48
		Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
I	100 75 50 25	1 1 0 0	0 0 0 0	- - - -	0 0 0 0	0 0 0 0	- - - -	0 0 0 0	0 0 0 0	- - - -
J	100 75 50 25	2 1 0 0	0 0 0 0	- - - -	0 0 0 0	0 0 0 0	- - - -	0 0 0 0	0 0 0 0	- - - -

Er = erythema        Oe = oedema         - = no other reactions noted

The concentrations of the test material selected for the main study topical challenge were undiluted as supplied and 75% v/v in arachis oil BP

 

INTRADERMAL INDUCTION – INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS

INDUCTION CONCENTRATION: 25% w/v                      VEHICLE: ARACHIS OIL BP

Animal Number	Grade of Erythema at Observation Time
	24 Hours		48 Hours
	Left Side	Right Side	Left Side	Right Side
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20	2 2 2 2 2 2 2 2 2 2 1 2 2 1 2 2 2 2 2 2	2 2 2 2 2 2 2 2 2 2 1 2 2 1 2 2 2 2 2 2	2 2 2 2 2 2 2 2 2 2 1 1 2 1 2 2 2 2 2 2	2 2 2 2 2 2 2 2 2 2 1 1 2 1 2 2 2 2 2 2

 

INTRADERMAL INDUCTION – INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS

VEHICLE: ARACHIS OIL BP

Animal Number	Grade of Erythema at Observation Time
	24 Hours		48 Hours
	Left Side	Right Side	Left Side	Right Side
21 22 23 24 25 26 27 28 29 30	1 1 1 1 1 1 1 1 1 1	1 1 1 1 1 1 1 1 1 1	1 1 1 1 1 1 1 1 1 1	1 1 1 1 1 1 1 1 1 1

 

TOPICAL INDUCTIONS – INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS

INDUCTION CONCENTRATION: UNDILUTED AS SUPPLIED

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	1 Hour			24 Hours
	Er	Oe	Other	Er	Oe	Other
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20	2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2	0 0 0 0 1 0 1 0 1 1 1 0 1 1 0 0 0 1 1 1	Bs Bs - - - - - Bs Bs Bs Bs Bs Bs Bs - - Bs Bs Bs Bs	1 1 2 1 2 2 1 1 1 2 2 2 1 2 2 2 2 1 2 2	0 0 0 0 0 0 0 0 1 1 2 1 1 2 1 0 1 0 2 1	- - - - - - - - - - - - - - - - - - - -

Er = erythema        Oe = oedema         

Bs = bleeding from intradermal injection site      - = no other reactions noted

 

TOPICAL INDUCTIONS – INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS

VEHICLE: BLANK PATCH APPLIED

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	1 Hour			24 Hours
	Er	Oe	Other	Er	Oe	Other
21 22 23 24 25 26 27 28 29 30	0 0 0 1 0 1 1 1 1 0	0 0 0 0 0 0 0 0 0 0	Bs Bs Bs Bs Bs - Bs Bs Bs Bs	0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0	- - - - - - - - - -

Er = erythema        Oe = oedema         

Bs = bleeding from intradermal injection site      - = no other reactions noted

 

INDIVIDUAL BODYWEIGHTS AND BODYWEIGHT GAINS OF TEST ANIMALS

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	386 412 380 352 373 384 377 403 389 378 381 431 432 430 413 364 439 385 373 384	613 622 557 554 568 565 621 631 652 602 588 693 730 645 633 594 660 600 598 636	227 210 177 202 195 181 244 228 263 224 207 262 298 215 220 230 221 215 225 252

 

INDIVIDUAL BODYWEIGHTS AND BODYWEIGHT GAINS OF CONTROL ANIMALS

Animal Number	Bodyweight (g)		Bodyweight (g) Increase
	Day 0	Day 24
21 22 23 24 25 26 27 28 29 30	384 429 379 446 405 399 434 406 372 394	611 657 637 660 630 646 630 669 626 594	227 228 258 214 225 247 196 263 254 200

 

SUMMARY OF POSITIVE CONTROL DATA FOR THE MAGNUSSON AND KLIGMAN MAXIMISATION STUDY

Project Number	Date Start	Date End	Number of Animals and Sex*		Positive Control Material	Concentration			Incidence of Sensitisation
						Induction		Challenge
			Test	Control		Intradermal	Topical
039/239	11/11/96	06/12/96	10 Female	5 Female	2-Mercaptobenzothiazole	10% in arachis oil BP	50% in acetone: PEG 400 (70:30)	50% and 25% in acetone: PEG 400 (70:30)	90% (9/10)
039/249	22/05/97	15/06/97	10 Female	5 Female	2-Mercaptobenzothiazole	10% in arachis oil BP	50% in acetone: PEG 400 (70:30)	50% and 25% in acetone: PEG 400 (70:30)	70% (7/10)
039/258	17/10/97	10/11/97	10 Female	5 Female	2-Mercaptobenzothiazole	10% in arachis oil BP	50% in acetone: PEG 400 (70:30)	50% and 25% in acetone: PEG 400 (70:30)	90% (9/10)
039/284	11/05/98	04/06/98	10 Male	5 Male	2-Mercaptobenzothiazole	10% in arachis oil BP	50% in acetone: PEG 400 (70:30)	50% and 25% in acetone: PEG 400 (70:30)	100% (10/10)
039/333	22/12/98	05/02/99	10 Female	5 Female	2-Mercaptobenzothiazole	10% in arachis oil BP	50% in acetone: PEG 400 (70:30)	50% and 25% in acetone: PEG 400 (70:30)	90% (9/10)
039/370	14/06/99	17/07/99	10 Male	5 Male	2-Mercaptobenzothiazole	5% in arachis oil BP	50% in acetone: PEG 400 (70:30)	50% and 25% in acetone: PEG 400 (70:30)	100% (10/10)

* All animals supplied by David Hall Ltd.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material, APAN, produced a 25% (5/20) sensitisation rate and was classified as a MILD SENSlTlSER to guinea pig skin. The test material did not meet the criteria
for classification as a sensitiser according to EU labelling regulations. No symbol and risk phrase are required.

Executive summary:

A study was performed to assess the contact sensitisation potential of the test material in the albino guinea pig. The study was performed in compliance with the OECD Guidelines for Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992) and Method B6 of Commission Directive 96/54/EC (which constitutes Annex V of Council Directive 67/548/EEC).

 

The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/548/EEC (as adapted to technical progress by Commission Directive 93/21/EEC).

 

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

 

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal Induction: 25% w/v in arachis oil BP

Topical Induction: undiluted as supplied

Topical Challenge: undiluted as supplied and 75% v/v in arachis oil BP

 

The test material produced a 25% (5/20) sensitisation rate and was classified as a mild sensitiser to guinea pig skin. The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations. No symbol and risk phrase are required.