2-hexyldecanoic acid [4-(6-tert-butyl-7-chloro-1H-pyrazolo[1,5-b][1,2,4]triazol-2-yl)phenylcarbamoyl]methylester

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July - 10 Oct 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

adopted 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

adopted 2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), november 2000, including the most recent partial revisions

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

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 5 weeks old
- Weight at study initiation: control group I (mean) - 377 g ± 12 g, control group II (mean) - 342 g ± 6 g, experimental group I (mean) - 373 g ± 19 g, experimental group II (mean) - 320 g ± 8 g
- Housing: group housing of maximal 5 animals per cage containing purified sawdust bedding material
- Diet: standard guinea pig diet including asorbic acid (1000 mg/kg) (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin), ad libitum and pressed hay twice a week
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 - 23.9 (actual range)
- Humidity (%): 38 - 96 (actual range)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

Induction:
2% intradermal, 50% epicutaneous
Challenge:
50% epicutaneous

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

Induction:
2% intradermal, 50% epicutaneous
Challenge:
50% epicutaneous

No. of animals per dose:

10 (control), 20 (in test group)

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted, the selection of concentrations was based on the following criteria:
- the concentrations are well-tolerated systemically
- for induction exposure: the highest possible concentration that produced mild to moderate irritation (grades 2-3)
- for challenge exposure: the maximum not-irritant exposure
Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting concentration for each route. The starting and subsequent concentrations were taken from a series of dilutions: 100, 50, 20, 10, 5, 2, 1% and if needed further lower concentrations. The system and procedure were the same as used in the main study. The 4 animals used were between 4 and 9 weeks old.

Intradermal injections:
A series of test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. Assessment for irritation was done 24 and 48 hours after treatment.
Epidermal application:
A series of test substance concentrations was used, the highest being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank. The animals receiving intradermal injections were treated with the lowest concentrations and two additional animals with the highest concentrations. After 24 hours the dressing was removed and the skin cleaned of residual test substance using water. The treated skin area was assessed for irritation 24 and 48 hours after exposure ended.

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

- Test groups:
Intradermal (pairs of 3 injections):
Injection 1) 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection
Injection 2) test substance at 2 % concentration in vehicle
Injection 3) 1:1 w/w mixture of Freunds' Complete Adjuvant and the test substance at 4% concentration
Epicutaneous:
- Test substance in vehicle (50%)

- Control group:
Intradermal (3 injections):
Injection 1) 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection
Injection 2) vehicle
Injection 3) 1:1 w/w mixture of Freunds' Complete Adjuvant and vehicle
Epicutaneous:
- vehicle

- Site: scapular region (intradermal and epicutaneous)
- Frequency of applications: on Day 1 and Day 8
- Duration: Days 0-8
- Concentrations: intradermal 2%, epicutaneous 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 1 challenge
- Day(s) of challenge: Day 21
- Exposure period: 24 h
- Test groups: test substance in vehicle only
- Control group: test substance in vehicle only
- Site: flank
- Concentrations: 50%
- Evaluation (hr after challenge): 48 and 72 h

OTHER:
To clarify the results of the main study, the study was extended using another set of 10 experimental and 5 control animals that were treated using the procedures described for the first set of animals. On Day 7 10% SDS was applied to one topical application site to induce skin irritation.

Challenge controls:

The control group is actually a challenge control.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamicaldehyde (tested in a reliability check which was conducted regularly, last check 6 months before start of test with test substance, induction: intradermal 20%, epicutaneous undiluted; challenge: 20%)

Positive control results:

The skin reactions in 6 of 10 experimental animals (at 24 h reading) were considered indicative of sensitisation, based on the absence of any response in control animals. These result lead to a sensitisation rate of 60%.

Reading:

1st reading

Hours after challenge:

48

Group:

other: test group I

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: test group I. Dose level: induction: 2%; challenge: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

48

Group:

other: control I (negative)

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: control I (negative). Dose level: induction: 2%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

other: test group I

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: other: test group I. Dose level: induction: 2%; challenge: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

other: control I (negative)

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: other: control I (negative). Dose level: induction: 2%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

1st reading

Hours after challenge:

48

Group:

other: test group II

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: test group II. Dose level: induction: 2%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

48

Group:

other: control II (negative)

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: control II (negative). Dose level: induction: 2%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

other: test group II

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: other: test group II. Dose level: induction: 2%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

other: control II (negative)

Dose level:

induction: 2%; challenge: 50%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: other: control II (negative). Dose level: induction: 2%; challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

The erythema seen after the topical induction exposure was considered to be enhanced by the SDS treatment. The first test with 5 control and 10 test animals showed two animals with positive reactions at the 48 and 72 h reading time points. This result was not sufficient to exclude a skin sensitising potential. Therefore, the study was extended using another 15 animals.

Table 1: Induction readings (first experiment)

Animal	Intradermal injection (Day 3)						Epidermal exposure (Day 10)
number	A		B		C		D
Control	E	N	E	N	E	N	E	N
16	2	0	0	0	2	0	0	0
17	1	0	0	0	1	0	0	0
18	2	0	0	0	2	0	0	0
19	1	0	0	0	1	0	0	0
20	2	0	0	0	2	0	0	0
Experimental	E	N	E	N	E	N	E	N
21	3	0	3	0	3	0	3	0
22	3	0	3	0	3	0	3	0
23	3	0	3	0	3	0	3	0
24	3	0	3	0	3	0	3	0
25	3	0	3	0	3	0	3	0
26	3	0	3	0	3	0	3	0
27	3	0	3	0	3	0	3	0
28	3	0	3	0	3	0	3	0
29	3	0	3	0	3	0	3	0
30	3	0	3	0	3	0	3	0

Table 2: Induction readings (second experiment)

Animal	Intradermal injection (Day3)						Epidermal exposure (Day10)
number	A		B		C		D
Control	E	N	E	N	E	N	E	N
331	3	0	2	0	2	0	1	0
332	3	0	1	0	2	0	2	0
333	3	0	2	0	3	0	1	0
334	2	0	1	0	2	0	0	0
335	3	0	2	0	2	0	1	0
Experimental	E	N	E	N	E	N	E	N
336	3	0	3	0	3	0	1	0
337	3	0	3	0	3	0	2	0
338	3	0	3	0	3	0	2	0
339	3	0	3	0	2	0	1	0
340	3	0	3	0	3	0	1	0
341	3	0	3	0	3	0	2	0
342	3	0	3	0	3	0	2	0
343	3	0	3	0	3	0	2	0
344	3	0	2	0	2	0	3	0
345	3	0	3	0	3	0	1	0

A = 1:1 mixture of FCA and water for injection

B = 2% test substance concentration (experimental) or vehicle (control)

C = 1:1 mixture of FCA and a 4% test substance concentration (experimental) or vehicle (control)

D = 50% test substance concentration (experimental) or vehicle (control)

skin effects intradermal injections:

E = Erythema

N = signs of necrosis (mm in diameter)

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitising potential of 2-hexyldecanoic acid [4-(6-tert-butyl-7-chloro-1H-pyrazolo[1,5-b][1,2,4]triazol-2-yl)phenylcarbamoyl]methylester (UM-235) was evaluated in a Guinea Pig Maximization Test (GMPT) performed according to OECD 406 (Teunissen, 2003d). The two induction phases were done by intradermal injection and topical application, respectively. On Day 1, 10 animals were intradermally injected with a 2% solution of the test substance in corn oil. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. On Day 8, a 50% solution of the test substance in corn oil was applied and covered with an occlusive dressing for 48 hours. 5 control animals were treated according to the same protocol, with the vehicle only (corn oil). On Day 21, all the animals were challenged with 50% UM-235 in corn oil via topical application under occlusive cover for 24 hours.

Moderate to severe erythema was noted at most of the injection sites of treated and control animals 24 hours after intradermal induction. These effects were due to the injection and not to exposure to the test substance. Following the topical challenge, slight erythema was noted at the challenge site in 2/10 treated animals at the 24-hour- and 48-hour reading time points. No signs of erythema or oedema were observed in control animals following the challenge treatment.

The first study was conducted with 5 control and 10 test animals. 2 out of those 10 test group animals showed a sensitising reaction at the 48 and 72 h reading time points. Since it could not be concluded that the substance is not a skin sensitiser, the study was extended using another set of 10 experimental and 5 control animals that were treated using the procedures described for the first set of animals. Again slight to moderate erythema was noted at most of the injection sites of treated and control animals. However, no skin sensitisation reactions were evident in the additional 10 experimental and 5 control animals.

Overall, the skin reactions observed in response to a 50% test substance concentration in 2/20 experimental animals in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 10%. Therefore, under the conditions of this study, UM-235 is not considered to be a skin sensitiser.

Migrated from Short description of key information:
Skin (OECD 406): not sensitising (GPMT)

Justification for selection of skin sensitisation endpoint:
There is only one study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on the skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.