1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-5-[(2-nitrophenyl)azo]-2-oxonicotinonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08. August to 10 September 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

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:

Test method was not available

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-310-7

EC Name:

-

Cas Number:

77889-90-8

Molecular formula:

C14H10ClN5O4

IUPAC Name:

5-[2-(4-chloro-2-nitrophenyl)diazen-1-yl]-6-hydroxy-1,4-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

female

Details on test animals and environmental conditions:

Species / sex: Pirbright-White guinea pig / female
Strain: HsdPoc:DH
Origin: HARLAN WINKELMANN Gartenstr, 27 D-33178 Borchen SPF breeding colony
Body weight of the main test group at start of study: mean: 352 g; min: 328 g; max:383 g
Randomisation: yes
Animal maintenance: in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals
Room temperature: 20±3°C
Relative humidity: 50±20%
Lighting time: 12 hours daily
Acclimatisation: at least 7 days
Food: ssnlff Ms-H (V2233), ad libitum
Water: tap water in plastic bottles, ad libitum
Animal identification: fur marking with KMnO4 and cage numbering

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

paraffin oil

Remarks:

semi-liquid paraffin [percentages w/v]

Concentration / amount:

2 x 0.1 ml 50 % Freund's Adjuvant
2 x 0.1 ml 1 % substance in semi-liquid paraffin
2 x 0.1 ml 1 % substance in 50 % Freund's Adjuvant

The following vehicles were used:
- Freund's Complete Adjuvant (Behringwerke Aktiengesellschaft)
- isotonic saline (sterile, pyrogen-free; Fresenius Aktiengesellschaft)
- semi-liquid paraffin, DAB (Riedel-de Haen Aktiengesellschaft)

Freund's Complete Adjuvant was mixed immediately before use with an equal volume of physiological saline. This 50 % adjuvant solution was administered to the animals by intradermal injection.
For the intradermal injections, Dispersionsgelb FN-68826 was diluted with semi-liquid paraffin [percentages w/v].
For the intradermal injections of the test substance in 50 % Freund's adjuvant, Dispersionsgelb FN-68826 was diluted with Freund's Original Adjuvant and then mixed with an equal volume of isotonic saline [percentages w/v].

Day(s)/duration:

Day 1

Adequacy of induction:

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

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Remarks:

DAB (Riedel-de Haen Aktiengesellschaft)

Concentration / amount:

25 %

For the dermal treatments, Dispersionsgelb FN-68826 was applied in petrolatum [percentages w/w]

Day(s)/duration:

Day 8 for 48 hours

Challenge

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

25 %

Day(s)/duration:

Day 22 for 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Determination of the primary non-irritant concentration – 2 animals
Determination of the tolerance of the intradermal injections – 3 animals
Control group – 5 animals
Treatment group -10 animals
Challenge treatment - escort group - 5 animals

Details on study design:

Determination of the tolerance of the intradermal injections
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 3 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders.

Site appl.vol. conc. Vehicle
1 2 x 0.1ml 5.0% semi-liquid paraffin
2 2 x 0.1ml 1.0% semi-liquid paraffin
3 2 x 0.1ml 0.2% semi-liquid paraffin

24, 48, 72 and 96 hours after administration the injection sites were examined tor local tolerance.

The intradermal injections with the 5.0 %, 1.0 % and 0.2 % preparation caused severe erythema, slight oedema as well as indurations The application sites were discoloured yellow.
Based on this preliminary test, a 1 % preparation was selected for the intradermal injections in the main test.


Determination of the primary non-Irritant concentration
Prior to the determination of the primary non-irritation concentration in a dermal-occlusive test the animals received 4 intradermal injections of a 50% Freund's Complete Adjuvant emulsion (4 x 0.1 ml) into the dorsal area, since Freund's Complete Adjuvant may lower the threshold of primary irritation. Thereafter, each of the following test concentrations was administered to the flanks of two guinea pigs:
25.0 % in petrolatum
5.0 % in petrolatum
1.0 % in petrolatum

The hair on the flanks of the animals was removed mechanically. 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema.



Main test for the sensitizing properties
The following preparations were used for the intradermal injections:

Control group
50 % Freund's Complete Adjuvant: Freund's Complete Adjuvant was mixed immediately before use with an equal volume of physiological saline.
semi-liquid paraffin
50 % Freund's Complete Adjuvant

Treatment group
50 % Freund's Complete Adjuvant
1 % test substance in semi-liquid paraffin
1 % test substance in 50 % Freund's Complete Adjuvant

For the dermal treatments, the test substance was applied in petrolatum [percentages w/w]

Chronological description of the test procedure indicating the day, at which procedure was carried out, on the left margin of the page.


Study day 0 The body weight of the animals was determined.
The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.
Study day 1 Intradermal induction treatment
Two intradermal injections per animal of the following preparations. The injection sites (site 1, 2 and 3) were all within a dorsal area of 2 x 4 cm. The injection sites were left uncovered.
Treatment group:
Site appl.vol. conc. Preparation
1 2 x 0.1ml - 50% Freund’s Adjuvant
2 2 x 0.1ml 1% substance in semi-liquid paraffin
3 2 x 0.1ml 1% substance in 50% Freund’s Adjuvant

Control group:
Site appl.vol. preparation
1 2 x 0.1ml 50% Freund’s Adjuvant
2 2 x 0.1ml semi-liquid paraffin
3 2 x 0.1ml 50% Freund’s Adjuvant

Study days 2-7 The administration area was examined for local tolerance. Any systemic toxic effects were recorded.

Study day 8 Dermal induction treatment
An amount of 0.5 ml of the test substance preparation (treatment group) or the vehicle (control group) was administered to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept for 48 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment group: 25.0 % test substance in petrolatum
Control group: petrolatum

Study day 10 Occlusive bandage removed, irritant effects recorded.

Study days 11-21 No treatment of control or treatment group. Test animals kept under observation.

Study days 15 - 18 Challenge treatment of escort group, carried out in the same way as the challenge treatment of control and treatment group (see days 22 - 25)

Study day 22 Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.
An amount of 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment and control group (left flank); 25.0 % in petrolatum

Study day 23 Occlusive bandage removed.

Study day 24 Examination of the skin approx. 24 hours after removal of the patches.

Study day 25 Examination of the skin approx. 48 hours after removal of the patches. Body weights of the test animals determined.

One area of approx. 5 x 5 cm on the right flank was shaved mechanically.
An amount of 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment and control group (right flank):

25.0 % T-9601 in sesame oil

30 Occlusive bandage removed. Any remnants of the test substance were carefully washed off with warm water.

31 Examination of the skin approx. 24 hours after removal of the patches.

32 Examination of the skin apprax. 48 hours after removal of the patches. Body weights of the test animals determined.

Challenge controls:

Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.

An amount of 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment and control group (left flank); 25.0 % in petrolatum

Positive control substance(s):

yes

Remarks:

alpha-hexyl cinnamic

Results and discussion

Positive control results:

Dermal induction was performed using 50 % alpha-hexyl cinnamic aldehyde in polyethylene glycol 400 (Riede! de Haen AG). The control group was exposed to polyethylene glycol 400 only. Challenge treatment was carried out with 25 % alpha-hexyl cinnamic aldehyde in polyethylene glycol 400,

Based on the results of this study alpha-butyl cinnamic aldehyde may cause sensitization by skin contact

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

other: Challenge treatment - escort group

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

other: Challenge treatment - escort group

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Any other information on results incl. tables

First challenge treatment – control and treatment group

 

25% in petrolatum (day 22)

 

Treated area: left flank

 

Scoring for dermal reactions

Time of observation: approx. 24 hours after removal of the patches (day 24)
Control animal No.	1	2	3	4	5
Value	0	0	0	0	0
Treated animal No.	6	7	8	9	10	11	12	13	14	15
Value	0	0	0	0	0	0	0	0	0	0
Time of observation: approx. 48 hours after removal of the patches (day 25)
Control animal No.	1	2	3	4	5
Value	0	0	0	0	0
Treated animal No.	6	7	8	9	10	11	12	13	14	15
Value	0	0	0	0	0	0	0	0	0	0

After the challenge treatment no animal of the treatment group showed a positive skin reaction during the observation period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, none of ten animals of the treatment group showed a positive skin response after challenge procedure.
Thus, the percentage of animals reacting positive is below the threshold of 30 %.
Based on the results of this study, the tes substance showed no evidence for sensitizing properties.

Executive summary:

Chemical substances may produce an immunological reaction (sensitization) after single or repeated dermal administration. Further administration after the elapse of a certain period may cause allergic reactions. The objective of the test procedure described here is to determine the potential sensitizing properties of a substance.

 

The present study was conducted in compliance OECD-Guideline for testing of chemicals, 406 "Skin Sensitization", adopted July 17,1992. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

Testing for sensitizing properties was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN.

 

Intradermal induction was performed using 1 % test substance in semi-liquid paraffin. Dermal induction and challenge treatment were carried out with 25 % test substance in petrolatum. The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexyl cinnamic aldehyde.

 

Based on the results of this study The test substance showed no evidence for sensitizing properties.