Benzyl 4-[(1-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxopyridin-3-yl)azo]benzoate

Endpoint summary

Description of key information

Skin sensitiser

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

29 April to 6 June, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read-across approach used. Reliability of source study is R1.

Justification for type of information:

Justification for the use of a read-across approach is provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted on July 17, 1992

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1996

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Test conducted on 2002

Species:

guinea pig

Strain:

Himalayan

Remarks:

lbm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wölferstrasse 4, 4414 Füllinsdorf, CH
- Number of animals: preliminary test: 3 females; main study: 15 females (10 treatment + 5 control animals)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 to 7 weeks
- Weight at study initiation: preliminary test: 400 to 437 g; main study: 408 to 448 g
- Housing: individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, 4132 Muttenz, CH)
- Diet: pelleted standard Provimi Kliba 3418, batch nos. 116/01 and 25/02, Guinea Pig Breeding / Maintenance Diet, containing Vitamin C (Provimi Kliba AG, 4303 Kaiseraugst, CH), ad libitum. Results of analyses for contaminants are archived at the test laboratory
- Water: community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at the test laboratory
- Acclimation period: preliminary test: none; main study: one week
- Indication of any skin lesions: none at check at beginning of study period

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12/12 (music was played during the daytime light period)

Route:

intradermal

Vehicle:

polyethylene glycol

Concentration / amount:

5 % test item

Day(s)/duration:

test day 1

Adequacy of induction:

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

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

25 % test item

Day(s)/duration:

test day 8

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

5 % test item in PEG 300 to the left flank; PEG 300 alone to the right flank

Day(s)/duration:

test day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 per test group
5 per control goup

Details on study design:

RANGE FINDING TESTS
A. Intrademal injections: 4 intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant / physiological saline were made into the shaved neck of one guinea pig. One week later, intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of A = 5 %, B = 3 %, C = 1 % test item in PEG 300. Dermal reactions were assessed 24 hours later.

B. Epidermal applications: 4 intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later, both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Then, 4 patches of filter paper (3 × 3 cm) were saturated with the test item at D = 25 % (technically the highest possible concentration to be applied sutficiently), E = 15 %, F = 10 % and G = 5 % in PEG 300 and applied to the clipped and shaved flanks. The amount of test item preparation applied was approximately 0.2 g for the test item 25 % and a volume of approximately 0.2 ml was applied for the remaining concentrations. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with imperuious adhesive tape. This procedure ensured the intensive contact of the test item. The dressings were removed after an exposure period of 24 hours. 21 h after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) in order to visualise any resulting erythema. The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. lt was then thoroughly washed off with a stream of warm, running water. Then, the animals were 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 according to the method of Magnusson and Kligman. The allocation of the different test item dilutions to the sites (D, E, F, G) on the two animals was alternated in order to minimize site-to-site variation in responsiveness.

Based on the results obtained the concentration selected for induction and challenge in the main study was 25 % and 5 %, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
(I) Intradermal injections (day 1)
- No. of exposures: 3 pair of injections per animal (0.1 ml/site)
- Site: injections were made along the border of an area of dorsal, scapular skin measuring 4 × 6 cm
- Preparation of site: fur was clipped (6 × 8 cm area)
- Concentrations: 5 % (w/w)
- Injections to treatment animals: (1) 1:1 (v/v) Freund's Complete Adjuvant:physiological saline; (2) 5 % test item in PEG 300; (3) 5 % test item in 1:1 (v/v) Freund's Complete Adjuvant:physiological saline
- Injections to control animals: (1) 1:1 (v/v) Freund's Complete Adjuvant:physiological saline; (2) PEG 300; (3) 1:1 (w/w) mixture of PEG 300 in 1:1 (v/v) Freund's Complete Adjuvant:physiological saline
- Frequency of injections: once
- Evaluation: skin response after 24 and 48 hours

(II) Epidermal applications (day 8)
- No. of exposures: 1
- Site: an area of dorsal, scapular skin measuring 4 × 6 cm, over the injection sites
- Preparation of site: fur was clipped and shaved
- Application: 2 × 4 cm patch of saturated filter paper and covered with aluminium foil, then firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape
- Concentrations: 25 % (w/w)
- Exposure period: 48 hours
- Frequency of applications: once
- Test groups: ca. 0.3 g test item as 25 % (w/w) test item in PEG 300
- Control group: 0.3 ml PEG 300
- Evaluation: skin response after 24 and 48 hours

B. CHALLENGE EXPOSURE
(II) Epidermal applications (day 22)
- No. of exposures: 2 (one on each flank)
- Sites: 5 × 5 cm area on both the left and right flank of each guinea pig
- Preparation of site: fur was clipped and shaved
- Application (both treatment and control animals): two 3 × 3 cm patches saturated in 0.2 ml containing either 5 % (w/w) test item in PEG 300 (left flank) or PEG 300 alone (right flank) and covered with aluminium foil, then firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape
- Concentrations: 5 % (w/w)
- Exposure period: 24 hours
- Frequency of applications: once
- Hair removal: 21 h after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) in order to visualise any resulting erythema. The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. lt was then thoroughly washed off with a stream of warm, running water. Then, the animals were 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 according to the method of Magnusson and Kligman.
- Evaluation: erythema and oedema observations were recorded at 24 h and 48 h after removal of dressing. An allergic reaction was defined by visible reddening of the challenge site. lf the dermal reactions of test animals following the challenge were more marked and/or persistent than those of the control animals, the animals were considered to show evidence of contact hypersensitivity. lf the dermal reactions of test animals following the challenge were not clearly different from the reactions seen in the control group animals, the results for the test animals were considered "inconclusive". The test animals were considered to show no evidence of contact hypersensitivity if the dermal reactions to the challenge application were identical or less marked and/or persistent than the reactions obserued in the control animals.

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde

Positive control results:

All test animals (at the 24-hour reading) and 6 out of 10 animals (at the 48-hour reading) showed discrete/patchy erythema after the challenge treatment with 0.1 % (w/w) positive control in PEG 300. No skin effect was observed in the control group.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

PEG 300 to right flank

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 % test item in PEG 300 to left flank

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

at both time points, 8/10 animals showed positive reaction

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

PEG 300 alone to the right flank

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

5 % test item in PEG 300 to the right flank

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1 % positive control in PEG 300 to left flank

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

PEG 300 to right flank

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

SKIN EFFECTS AFTER INTRADERMAL INDUCTION - PERFORMED ON TEST DAY 1

The expected and common findings were obserued in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation. No detailed description of the effects is given in the report as these FCA effects are wellknown.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION - PERFORMED ON TEST DAY 8

CONTROL GROUP

No erythematous or oedematous reaction was observed in the animals treated with PEG 300 only.

TEST GROUP

As the test item at 25 % stained the skin in yellow, it was not possible to determine whether erythema was present or not. However, no oedema was obserued. The animals were not depilated in the epidermal induction phase.

SKIN EFFECTS AFTER THE CHALLENGE - PERFORMED ON TEST DAY 22

CONTROL GROUP

No skin reactions were observed in the animals when treated with either PEG 300 only or when treated with the test item at 5 % in PEG 000. Yellow discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

TEST GROUP

Discrete/patchy erythema were observed in eight out of 10 animals at the 24- and 48-hour reading after treatment with the test item at 5 % in PEG 300. No skin reaction were obserued when treated with PEG 300. Yellow discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

There were no deaths during the course of the study, hence no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No signs of systemic toxicity were observed in the animals.

BODY WEIGHTS

3 animals of the control group and 7 animals of the test group showed a loss of body weight (1.4 % to 6.9 %) during the acclimatization period. They recovered between the treatment start and the end of the study. The body weight of the other animals was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

other: Skin Sensitiser Category 1B (H317), according to the CLP Regulation (EC 1272/2008)

Conclusions:

The test item elicited positive reactions in 8 out of 10 animals 24 and 48 hours after a challenge application in the GPMT.

Executive summary:

The skin sensitising potential of the test item was evaluated in an experimental study, a GPMT, according to the OECD guideline 406 (1992) and the EU method B.6 (1996). In a preliminary test, one female Himalayan spotted albino guinea pig was administered 4 intradermal injections of equal parts Freund's Complete Adjuvant and physiological saline to the shaved neck; one week later, 3 intradermal injections were made into the clipped flank containing either 5, 3, 1 % test item in PEG 300. 24 hours later, all three concentrations/sites showed positive reactions. Two additional guinea pigs were administered 4 intradermal injections of equal parts Freund's Complete Adjuvant and physiological saline to the shaved neck; one week later, 4 patches of filter paper (3 × 3 cm) saturated with test item (25, 15, 10 and 5 %) in PEG 300 were applied to clipped, shaved flanks for 24 hours under an occlusive dressing. 24 and 48 hours after removal, positive reactions were observed at concentration sites of 10 % and over. Based on the results obtained the concentration selected for induction and challenge in the main study was 25 % and 5 %, respectively.

In the main study, 10 treatment animals and 5 controls were administered three pairs of intradermal injections containing (i) equal parts Freund's Complete Adjuvant and physiological saline, (ii) 5 % test item in PEG 300 (controls: PEG 300 alone), and (iii) 5 % test item in equal parts Freund's Complete Adjuvant and physiological saline (controls: PEG 300 in place of test item). One week later, an epidermal application of 25 % test item in PEG 300 was applied to the same area for 48 hours (control animals recevied PEG 300 alone). Finally, a challenge was performed on day 22 of the study period whereby all animals received a dermal application of 5 % test item in PEG 300 to the left flank and PEG 300 alone to the right flank for 24 hours under an occlusive dressing. The hair was depilled and skin reactions were observed 24 and 48 hours after removal.

After the induction period, no control or treatment animals showed positive reactions, and no readings of erythema could be discerned from the treatment animals due to local yellow discolouration. Following the induction, no control animals showed positive reactions on either flank at either 24 or 48 hours; no positive reactions were observed among treatment animals on the right flank, however, positive reactions were observed in 8 out of 10 animals on the left flank at both 24 and 48 hours after removal of the challenge application.

Based on the positive reactions in 8 out of 10 animals following the challenge application, the test item shows great potential to elicit skin reactions and is considered a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

No data on Disperse Yellow 231 is available, thus data on a structural analogue, i.e. Similar Substance 01, was used. Details on the read across approach are available in section 13.

Two in vivo experimental studies according to OECD guideline 406 were available: a GPMT and a Buehler assay. In the GPMT, 80 % of animals gave a positive response for sensitisation at both 24 and 48 hours after challenge exposure. In the GPMT, intradermal induction was done at 5 % w/w, followed by a dermal induction at 25 %w/w and dermal challenge at 25 % w/w.

In the Buehler assay, no animals showed a positive response upon 25 % w/w dermal applications in both induction and challenge phases.

Based on available data, both studies were considered as reliable. Therefore, the GPMT was selected as key study following a precautionary approach.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), a skin sensitiser is a substance which will lead to an allergic response following skin contact. Sensitisation includes two phases: the first phase is induction of specialised immunological memory in an individual by exposure to an allergen. The second phase is elicitation, i.e. production of a cell-mediated or antibody-mediated allergic response by exposure of a sensitised individual to an allergen.

Skin sensitisation predictive tests usually follow this pattern: an induction phase, the response to which is measured by a standardised elicitation phase.

The Guinea Pig Maximisation Test (GPMT) is a two-phase, in vivo sensitisation test involving initial intradermal injection of the substance and an adjuvant and subsequent epidermal exposure to evaluate sensitisation potential in guinea pigs.

According to the CLP Regulation (EC 1272/2008):

- substances showing (a) a high frequency of occurrence in humans and/or (b) a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Specifically, a substance shall be classified as a Category 1A: Skin Sensitiser if 30 % or more of test animals respond to an intradermal induction concentration of ≤ 0.1 %, or if 60 % or more of test animals respond to a concentration > 0.1 % to ≤ 1 %, in a GPMT.

- substances showing (a) a low to moderate frequency of occurrence in humans and/or (b) a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Specifically, a substance shall be classified as a Category 1B: Skin Sensitiser if at least 30 % but < 60 % of test animals respond to an intradermal induction concentration of > 0.1 % to ≤ 1 %, or if 30 % or more test animals respond to an intradermal induction concentration of > 1 %, in a GPMT.

80 % of test animals responded to an intradermal induction concentration of 5.0 % test item in the Guinea Pig Maximisation Test (GPMT); therefore, the substance is considered sensitising, and classification as a Category 1B: Skin Sensitiser is warranted according to the CLP Regulation (EC 1272/2008).