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

Start of the experiment: 9 March 1994; End of the experiment: 8 June 1994; Study completion date: 08 June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

See Principles of method if other than Guideline part.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

DEVIATION:
The application described under the point 3 in the control and test group is in accordance with the OECD 406 "Skin Sensitisation", July 17, 1992. However it is a deviation of the Directive 92/69 EEC, B.6. "Acute Toxicity-Skin Sensitization", July 31, 1992 in which the vehicle is mixed with FCA only in the control group and liposoluble or insoluble substances are mixed with FCA only in the test group. A mixture of FCA:physiological saline was chosen instead of FCA only in order to decrease the site effects of FCA when applied alone.

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

274-660-5

EC Name:

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

Cas Number:

70528-90-4

Molecular formula:

C15H12ClN5O4

IUPAC Name:

5-[(4-chloro-2-nitrophenyl)diazenyl]-1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Identification: TERASIL GELB 4G ROH FEUCHT LABORGETROCKNET (FAT 36091/G)
Description: yellow powder
Batch Number: 9300010
Purity/ Formulation: 98.9 % a.i
Stability of test article: stable; expiration date: 01/99
Storage Conditions: at room temperature (approx. 20 °C), away from direct sunlight
Safety precautions: Gloves, goggles and face mask were obligatory to assure personnel health and safety.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

-Test system: Albino Dunkin Hartley Guinea Pigs, SPF delivered by WIGA
- Rationale: Recognized by the international guidelines as the recommended test system (e.g. OECD, EEC).
- Source: CHARLES RIVER Wiga GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Number of animals for main study / pretest: 30 males/6 males
- Age at beginning of Acclimatization period: approx. 7 - 9 weeks
- Body Weight at beginning of Acclimatization period: Control and Test Group: 355 - 469 g and Pretest: 357 - 398 g
- Identification: By unique cage number and corresponding ear tags.
- Randomization: Randomly selected at time of delivery.
- Acclimatization: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visual signs of illness were used for the study.

Conditions:
Standard Laboratory Conditions
Air-conditioned with 10-15 air changes per hour and continuously monitored environment with a temperature between 20-24 °C, a relative humidity between 42-68 %, 12 hours artificial fluorescent light (approx. 100 Lux) /12 hours dark, music during the light period. The duration of the cleaning process was approximately 30 minutes.

Accommodation:
Individually in Makrolon type-3 cages (size: 22x37x15 cm) with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).

Diet:
Pelleted standard Kliba 342, Batch nos. 61/94 (at delivery of the animals to 07-APR-1994) and 62/94 (from 08-APR-1994 to termination of test) guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.

Water:
Community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/L) via the drinking water.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

1%

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:

other: Vaselinum album

Concentration / amount:

25%

Day(s)/duration:

Day 8

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: vaseline album

Concentration / amount:

5 %

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 males were used as control group in the main test.
20 males were used as test group in the main test.
6 males in the pretest.

Details on study design:

PRETEST / performed during the acclimatization period.
INTRADERMAL INJECTIONS:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1 % of the test article in paraffin oil. The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 1 % test article dilution was selected.

EPIDERMAL APPLICATIONS :
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches* of filter paper ( 2 x 2 cm) were saturated with the test article, at A = 25 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 15 %, C = 10 % and D = 5 % of the test article in vaselinum album and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours. 21.5 hours after removing of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the application site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time. The depilatory was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize.

For the epidermal induction the test article at 25 % and for the challenge procedure the test article at 5 % was selected.

MAIN STUDY
INDUCTION
Intradermal injections / performed on test day 1:
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:

Test group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 1 % with paraffin oil.
3)* The test article diluted to 1 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline. (*) deviation

Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Paraffin oil.
3)* 1:1 (w/w) mixture of paraffin oil in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline (*) deviation


Epidermal applications / performed on test day 8:
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test article (25 % in vaselinum album) and placed over the injection sites of the test animals. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal, application procedure described ensured intensive contact of the test article. The guinea-pigs of the control group were treated as described above with vaselinum album only. Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

CHALLENGE / performed on test day 22
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. The test and control guinea-pigs were treated in the same way. Fur was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig just prior to the application. Two patches ( 2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 5 % (left flank) and the vehicle only (vaselinum album, applied to the right flank) using the same method as for the epidermal application. The dressing were left in place for 24 hours. 21 hours after removing of the dressing the test sites were depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil). The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed the animals were dried with a disposable paper towel and returned to their cages. 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

Challenge controls:

None

Positive control substance(s):

yes

Remarks:

4-Aminobenzoic acid ethyl ester

Results and discussion

Positive control results:

For validation of the sensitivity of test method and test system used, a known (4-AMIN0BENZ0IC ACID ETHYL ESTER) was selected as a positive control. This was performed in accordance with the recommendation of the OECD for testing of chemicals number 406 "Skin Sensitization Test", adopted by the council on July 17, 1992 (reported Paris, April 29, 1993). According to the procedures used in this experiment (run from 15-NOV to 28-DEC- 1993) positive results were observed in the treated animals after the epidermal challenge application. The intradermal induction was performed with a dilution at 1% in mineral oil. For the induction period and challenge procedure a 25 % dilution of 4-AMINOBE ZOIC ACID ETHYL ESTER in mineral oil was used. This concentration was found in a pretest to be the highest primary not-irritant concentration. The test article at 10 % and 5 % in mineral oil was additionally applied in the second challenge to compare with the literature. From the results described above moderate allergenic potency (grade 3) of the test article 4-AMINOBENZOIC ACID ETHYL ESTER was concluded. The results were interpreted according to the rating of Magnusson and Kligman (1969). The response of at least 30 % positive animals is considered positive following the "Commission Directive 93/21/EEC, April 27, 1993 adapting to technical progress for the 18th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. In this study 30-50 % of the animals were positive after treatment with a nonirritant test substance concentration of 25 % and with concentrations of 10 % and 5 % in mineral oil. Therefore, according to EEC (European Economic Community) classification criteria described in guidelines 93/21/EEC (EEC Official Journal Nr. L 110A, May 04, 1993), this test article is considered to be a sensitizer.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Intradermal Induction: 1%; Epidermal induction: 25%; Epidermal Challenge: 5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Intradermal Induction: 1%; Epidermal induction: 25%; Epidermal Challenge: 5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal Induction: 1%; Epidermal induction: 25%; Epidermal Challenge: 5%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Intradermal Induction: 1%; Epidermal induction: 25%; Epidermal Challenge: 5%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25% in Mineral oil

No. with + reactions:

10

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25% in Mineral oil

No. with + reactions:

9

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

RESULTS

Main Study

SKIN EFFECTS AFTER INTRADERMAL INDUCTION PERFORMED ON TEST DAY 1

CONTROL GROUP

- Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from test day 2 to 6 followed by necrosis, encrustation and exfoliation of the encrustation up to the termination of test (test day 29).

Injection site 2 (paraffin oil)

The area around the injection site was oedematous and erythematous from test day 2 to 6 followed by necrosis, encrustation and exfoliation of the encrustation up to the test day 23.

Injection site 3 (1:1 (w/w) mixture of paraffin oil in a 1:1 (v/v) mixture of FCA and physiological saline)

The reactions observed were identical to those obtained at injection site 1 with the mixture of FCA and physiological saline. As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

TEST GROUP

- Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The reactions observed were identical to those obtained in the control group with the mixture of FCA and physiological saline, at injection site 1.

- Injection site 2 (1 % solution of test article in paraffin oil)

The area around the injection site was oedematous and erythematous from test day 2 to 6 and yellow discoloured from test day 2 to 8, became necrotic from test day 7 to 13 followed by encrustation and exfoliation of encrustation up to test day 23.

- Injection site 3 (1 % solution of test article in a 1:1 (v/v) mixture of FCA and physiological saline)

The reactions observed were identical to those obtained at injection site 2 with the 1 % solution of test article in paraffin oil. However the exfoliation of encrustation was prolonged up to the termination of test. As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION PERFORMED ON TEST DAY 8

CONTROL GROUP:

No erythematous or oedematous reaction was observed in the animals treated with vaselinum album only.

TEST GROUP:

As the test article stained the skin yellow, it was not possible to determine whether erythema was present. However, no oedema was observed. Yellow discolouration was noted from test day 10 to 27.

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

CONTROL AND TEST GROUP:

No positive reactions were observed in the animals either when treated with vaselinum album alone or when treated with the test article at 5 % in vaselinum album. Yellow discolouration was noted from test day 23 (after removal of the dressing)m to 24 (prior to the depilation).

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

As there were no deaths during the course of the treatment period no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

The body weight of the animals was within the normal range of variability.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study 0 % of the animals were positive after treatment with the highest non-irritant test substance concentration of 5 % in vaselinum album. Therefore, the test article (FAT 36091/G) is considered to be a non-sensitizer.

Executive summary:

The allergenic potential of the test item in albino guinea pigs the test was evaluated according to the OECD Guidelines for Testing of Chemicals, Section 4, number 406, "Skin Sensitization", adopted July 17, 1992 (reported Paris 29 April 1993).

Ten males were used as control group and 20 males were used as test group. The highest non-irritating test article concentration used for challenge application was 5% in vaselinum album.

POSITIVE ERYTHEMA REACTIONS AFTER THE CHALLENGE PROCEDURE

	After 24 hours	After 48 hours
	Positive / total	Positive / total
	% positive of total	% positive of total
Control group
(FAT 36'091/G), 5% (left flank)	0/10 ----0	0/10 ----0
Vaseline album only (right flank)	0/10 ---- 0	0/10 -----0
Test group
(FAT 36'091/G), 5% (left flank)	0/20 ----0	0.20 ----0
Vaseline album only (right flank)	0/20 ----0	0/20 ----0

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred. For positive control ( 4-AMINOBENZOIC ACID ETHYL ESTER); 50 % and 45 % of animals showed positive response after 24 h and 48 h in first challenge, respectively. 30 % of animals showed positive response after 24h and 48h in second challenge.

CONCLUSION

In this study 0 % of the animals were positive after treatment with the highest non-irritant test substance concentration of 5 % in vaselinum album. Therefore the test article is considered to be a non sensitizer.