Hexane, 1,6-diisocyanato-, homopolymer, 3,5-dimethyl-1H-pyrazole-blocked

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug-Oct 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

(1992)

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

This in vivo study on skin sensitization according to Buehler and OECD test guidelines (OECD TG 406) was conducted in 1998, which was prior to the adoption of the initial OECD guideline on the Local Lymph Node Assay in 2002. By that time, the method according to Buehler was one of two preferred test protocols for studying a skin sensitisation potential and is still considered adequate and sufficient for the assessment of that endpoint.

Test material

Test material information

Constituent 1

Reference substance name:

N-(6-{3-[6-(3-{6-[3,5-bis({6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-5-{6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-5-{6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl}hexyl)-3,5-dimethyl-1H-pyrazole-1-carboxamide; N-[6-(3-{6-[3,5-bis({6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-5-{6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-3,5-dimethyl-1H-pyrazole-1-carboxamide; N-{6-[3,5-bis({6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-3,5-dimethyl-1H-pyrazole-1-carboxamide

EC Number:

605-318-9

Cas Number:

163206-31-3

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

N-(6-{3-[6-(3-{6-[3,5-bis({6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-5-{6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-5-{6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl}hexyl)-3,5-dimethyl-1H-pyrazole-1-carboxamide; N-[6-(3-{6-[3,5-bis({6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-5-{6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl}-2,4,6-trioxo-1,3,5-triazinan-1-yl)hexyl]-3,5-dimethyl-1H-pyrazole-1-carboxamide; N-{6-[3,5-bis({6-[(3,5-dimethyl-1H-pyrazole-1-carbonyl)amino]hexyl})-2,4,6-trioxo-1,3,5-triazinan-1-yl]hexyl}-3,5-dimethyl-1H-pyrazole-1-carboxamide

Details on test material:

- Homogeneity and stability under test conditions: The formulations were visually described as emulsion. The emulsions were continuously homogenized on a magnetic stirrer during the treatments. No data concerning the stability of the test item in the vehicle.
- Content: 75 % in solvent

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Hsd Poc:DH

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: Hsd Poc:DH, SPF-bred
- Source: Harlan Winkelmann, 33176 Borchen, Germany
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: mean weight 359-363 g
- Fasting period before study: yes, from 16 hours (over night) prior to at least 4 hours after application
- Housing:in groups of two or three per cage throughout the study period; cages: Makrolon® Type-IV cages
- Diet and Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-60
- Air changes (per hr): >/= 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Induction 2

Concentration / amount:

1st to 3rd epicutaneous induction: 50 % test substance formulation
challenge: 25 % test substance formulation

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Challenge 2

Concentration / amount:

1st to 3rd epicutaneous induction: 50 % test substance formulation
challenge: 25 % test substance formulation

No. of animals per dose:

experimental group: 20
control group: 10

Details on study design:

RANGE FINDING TESTS:
induction: For range-finding three concentrations and the vehicle were tested in each case on five guinea pigs (25, 50 and 100 %). Based on the results of the range-finding a 50 % concentration was chosen for the inductions in the main study, as this concentration was the highest to cause mild irritation.
challenge: The challenge concentration was determined on 2 guinea pigs which were treated in the same manner as the control animals during the inductions of the main study. The challenge concentrations tested were 6, 12 and 25 %. Based on the results of the range-finding a 25 % concentration was chosen for the challenge in the main study, as this concentration was the highest non-irritating concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
The animals were dermally treated with the test item three times at intervals of seven days. The suitable areas of the body were shaved one day (24 hours) before each treatment. The animals in the test item group were treated with a hypoallergic patch loaded with the test item formulation applied to the left flank for the first and second induction. Due to the local effects after the first two inductions, the third induction was applied on the right flank. The patches were held in place on the skin with "Fermoflex" adhesive plaster (Transatlantic GmbH, Schwarzenbach). The volume applied per animal was 0.5 mL. In the case of the control group animals, a hypoallergenic patch loaded with the vehicle was applied to the left flank for the first and second induction and on the right flank for the third induction. The patches were removed after an exposure period of six hours, and any remaining test item was washed off the skin with physiological saline solution.

B. CHALLENGE EXPOSURE
The challenge was performed four weeks after the first (two weeks after the last) dermal induction. For the challenge the backs and right flanks of the animals were shorn one day prior to the challenge. During the challenge a hypoallergenic patch loaded with a 25 % test item formulation was applied and fixed to the right flank of each animal in the control and test item group. As a control a patch moisted with the vehicle was applied and fixed also to the right flank, cranial to the test item patch. The patches were held securely in place on the skin with a FERMOFLEX self-adhesive tape for 6 hours. The volume applied in each case was 0.5 mL. At the end of the six-hours exposure period, the patches were removed and the remaining test item was removed with physiological saline solution. Twenty-one hours later the skin of the animals was shorn in the region of the treatment sites.

The skin reactions were assessed 30 hours after initiation of the induction exposures (24 hours after challenge), and 30 and 54 hours after the beginning of the challenge (24 and 48 hours after challenge).

OTHER: The animals were observed for clinical signs at least once daily throughout the entire study period. The body weights of the animals were recorded before initiating the study, and at its conclusion on day 30 in the control group and the test item group and at day 23 in the range-finding group.

Challenge controls:

In the case of the control group animals, a hypoallergenic patch loaded with the vehicle was applied to the left flank for the first and second induction and on the right flank for the third induction.

Positive control substance(s):

yes

Remarks:

alpha-hexyl cinnamic aldehyde

Results and discussion

Positive control results:

Challenge control was conducted in a Buehler test on male guinea pigs with alpha-hexyl cinnamic aldehyde formulated in Polyethylene glycol at a 40 % test item formulation for epicutaneous inductions and a 20 % test item formulation for challenge. 65 % of the animals of the test item group exhibited dermal reactions and none of the animals of the control group exhibited dermal reactions following challenge (Report No 26864, 1997). Thus the sensitivity as well as the reliability of the experimental technique was confirmed.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % test substance formulation

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 % test substance formulation. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % test substance formulation

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 % test substance formulation. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 % test substance formulation

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25 % test substance formulation. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 % test substance formulation

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 % test substance formulation. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: none.

Any other information on results incl. tables

Appearance and behaviour of the test item group animals were not different from the control group animals. By the end of the study the mean body weight of the treatment group animals was in the same range than that of the control group.

There were no skin effects in the control group animals after the first, second and third induction treatment. In the test item group all animals showed glued fur at the treatment areas after all three induction treatments. In addition, skin effects (grade 1) were shown in 6/20 animals after the first induction, in 3/20 animals after the second induction and in 4/20 animals after the third induction.

The challenge with the 25 % test item formulation led to slight skin effects (grade 1) in the test item group in 1/20 animals after 24 hours and in 1/20 animals after 48 hours. Slight skin effects (grade 1) were also seen in 1/10 of the animals in the control group after 24 and 48 hours. The total number of reacting animals was 1/10 (10 %) in the control group and 2/20 (10 %) of the animals in the test item group.

Applicant's summary and conclusion

Executive summary:

The Buehler epicutaneous patch test was conducted on male guinea pigs according to OECD guideline 406 with a test item containing 75 % active ingredient. A 50 % test substance formulation was used for first to third induction and a 25 % test substance formulation for challenge, based on previously performed range finding tests. 24 and 48 hours after challenge exposure (30 and 54 hours after the beginning of the challenge) the treated sites were assessed.

The challenge with the 25 % test item formulation led to slight skin effects (grade 1) in the test item group in 1/20 animals after 24 hours and in 1/20 animals after 48 hours. Slight skin effects (grade 1) were also seen in 1/10 of the animals in the control group after 24 and 48 hours. The total number of reacting animals was 1/10 (10 %) in the control group and 2/20 (10 %) of the animals in the test item group.

In summary, by comparing the effects in the test item group and the control group it can be concluded that under the conditions used the test item exhibits no skin-sensitization potential.