2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1H-pyrrolo[1,2-b][1,2,4] triazole-7-carboxylic acid 2,6-di-tert-butyl-4-methylcyclohexylester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Aug - 19 Sep 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study with acceptable restrictions (The epicutaneous induction and challenge were performed under semi-occlusive conditions.)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

The epicutaneous induction and challenge were performed under semi-occlusive conditions.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

The epicutaneous induction and challenge were performed under semi-occlusive conditions.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

The epicutaneous induction and challenge were performed under semi-occlusive conditions.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agricuiture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No. 8147, November 2000, including the most recent partial revisions

Deviations:

yes

Remarks:

The epicutaneous induction and challenge were performed under semi-occlusive conditions.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-050-6

EC Name:

-

Cas Number:

444065-11-6

Molecular formula:

C41 H57 N5 O8

IUPAC Name:

2,6-di-tert-butyl-4-methylcyclohexyl 5-{[bis(2-ethoxy-2-oxoethyl)carbamoyl]oxy}-2-(4-tert-butylphenyl)-6-cyano-1H-pyrrolo[1,2-b][1,2,4]triazole-7-carboxylate

Details on test material:

Name of test material (as cited in study report): UC-141
- Physical state: White powder
- Analytical purity: 99.7% (area %), 98.2% (mass %)
- Lot/batch No.: CE-203
- Expiration date of the lot/batch: 1 Jan 2005
- Storage condition of test material: At room temperature in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Approximately 4 weeks
- Weight at study initiation: 345-409 g (treatment group) and 369-415 g (control group)
- Housing: Group housing of maximally 5 animals per labelled cage (74 cm x 54 cm x 25 cm height) containing purified sawdust as bedding material (SAWI, Jelu Werk, Rosenberg, Germany)
- Diet: standard guinea pig diet, including ascorbic acid (1000 mg/kg); (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Lage, Germany), ad libitum. Pressed hay (B.M.l., Helmond, the Netherlands) was provided twice a week.
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-22.7
- Humidity (%): 45-89
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Induction 2

Concentration / amount:

Intradermal induction: 0.5%
Epicutaneous induction: 50%
Challenge: 50%

Challenge

Challenge 1

Route:

epicutaneous, semiocclusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

Intradermal induction: 0.5%
Epicutaneous induction: 50%
Challenge: 50%

No. of animals per dose:

10 in treatment group
5 in control group

Details on study design:

RANGE FINDING TESTS:
Prior to the start of the main study, the intradermal and epidermal irritancy of UC-141 was investigated to select test substance concentrations suitable for the induction and challenge phase of the main study. The selection was based on the absence of toxicity and on the following criteria for each route and/or study phase:
Induction (intradermal and epidermal): The highest possible concentration that produced moderate irritation (the intradermal reactions may include slight necrosis (< 3 mm in diameter)).
Challenge: The maximum non-irritant concentration.

A series of test substance concentrations were tested. The feasibility of administration determined the highest concentration for each route. The first and subsequent concentrations were selected from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps. The test system and procedures were identical to those used during the main study, unless otherwise specified. The four animals were 4-9 weeks old. No body weights were determined.

Intradermal Injections:
A series of four test substance concentrations was used; the highest concentration was the maximum concentration that could technically be
injected (5%). One animal received 2 and 5%, and the second animal received 0.5 and 1%, respectively, in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment. Slight erythema was observed at all 4 injection sites, 24 and 48 h after exposure. Injection of a 5%, 2% or 1% concentration was difficult and the reliability of the injected volume could not be established. Therefore, a 0.5% test substance concentration was considered the highest concentration that could reproducibly be injected.

Epidermal application:
A series of four test substance concentrations (5, 10, 20 and 50%) was used; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 ml each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape , which were held in place with Micropore tape and subsequently Coban elastic bandage (all semi-occlusive covering). The animals receiving intradermal injections were treated with the lowest concentrations (5 and 10%) and two further animals with the highest concentrations (20 and 50%). After 24 hours, the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 hours after exposure. No skin irritation was observed at any of the treated sites at any of the reading time points. As the epidermal induction using the test substance did not cause any skin irritation, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction. A 50% solution was selected for the challenge phase in the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal and epicutaneous
- Exposure period: single injection (intradermal) and 48 hours (epicutaneous)

Intradermal, day 1 (3 pairs of injections, 0.1 mL/site):
Injection 1: a 1:1 mixture (w/w) Freunds Complete Adjuvant (FCA)/water for injection
Injection 2: 0.5% (w/w) test substance in corn oil
Injection 3: 1% (w/w) test substance in a 1:1 mixture (w/w) with FCA (final concentration is 0.5% test substance)
48 h after intradermal injection (day 3), the degree of erythema and edema was evaluated.

On day 7, the scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium dodecyl sulfate (SDS, Boom, Meppel, the Netherlands) in vaseline using a spatula. This concentration of SDS provoked a mild inflammatory reaction.

Epicutaneous, day 8:
0.5 mL 50% (w/w) test substance in corn oil
The semi-occlusive dressing was kept in place for 48 h. The degree of erythema and edema was evaluated directly after cleaning the skin area with water (day 10).

- Control group:
Intradermal, day 1 (3 pairs of injections, 0.1 mL/site):
Injection 1: a 1:1 mixture (w/w) FCA/water
Injection 2: corn oil
Injection 3: corn oil (w/v) in a 1:1 mixture (w/w) FCA (final concentration is 50% in corn oil)

On day 7, the scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, the Netherlands) in vaseline using a spatula. This concentration of SDS provoked a mild inflammatory reaction.

Epicutaneous, day 8: 0.5 mL corn oil

- Site: the shoulder region
- Frequency of applications: once (intradermal on day 1 and epicutaneous on day 8)
- Duration: day 1 (intradermal), day 8-10 (epicutaneous)
- Concentrations: 0.5% (intradermal), 50% (epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: 2 (challenge and rechallenge)
- Day(s) of challenge: 22 (challenge) and 29 (rechallenge)
- Exposure period: 24 hours
- Test groups: 0.1 mL test substance in corn oil
- Control group: 0.1 mL test substance in corn oil
- Site: approximately 20 mm x 30 mm, on one flank of the animals
- Concentration: 50%
- Evaluation (hr after challenge): 24 and 48 hours after the challenge ended

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamicaldehyde, tech. 85%

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals with alpha-hexylcinnamic aldehyde to check the sensitivity of the test system and the reliability of the experimental methods used by the test laboratory. In an independent study performed in 2003, alpha-hexylcinnamic aldehyde induced sensitisation in 60% (6/10) of the Dunkin-Hartley guinea pigs challenged with a 20% solution. A 20% solution was used for intradermal induction and undiluted test substance was used for topical induction.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 5

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 6

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 7

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 8

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

There was no mortality, no signs of toxicity and no treatment-related effects on body weight.

24 hours after intradermal induction, slight to moderate erythema was noted at all of the injection sites of 10/10 treated and 5/5 control animals. Following the topical induction, slight erythema was noted at the test site in 4/10 treated animals and 2/5 control animals.

48 and 72 hours after the first challenge (day 22), no sensitisation was observed in the treated animals. At the 72 -hour reading time point, scaling was observed in 4/10 animals exposed to the test substance. This may have been and effect of the irritation caused by the skin irritation induced on day 1 and 8 of the study period. The animals were re-challenged one week after the first challenge (day 29). No sensitisation reactions were observed in the treated or control animals, 48 and 72 hours after re-challenge.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classifed

24 h after intradermal induction, all test animals showed erythema on the injection sites. The concentration tested was 5% UC-141. However, the same effect was observed in all controls. Therefore, it is unclear whether the skin irritation was caused by the test substance itself or was a non-specific response to the injections. During the topical induction, 4/10 treated animals and 2/5 control animals had slight erythema on the test sites. Again, it is unclear whether the skin irritation was caused by the test substance or was a non-soecific response to the treatment. As no control animals showed sensitisation during the challenge and rechallenge, and no treated animals showed sensitisation reactions during the rechallenge, the test substance is not classified as sentitising.