N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 July 1999 - 27 August 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japan MAFF Dermal Sensitization Study, 1985

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study conducted prior to adoption of LLNA guideline by the OECD.

Test material

Test material information

Constituent 1

Reference substance name:

1-[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

EC Number:

601-779-5

Cas Number:

121451-02-3

Molecular formula:

C17H7Cl2F9N2O3

IUPAC Name:

1-[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

Test material form:

solid: particulate/powder

Details on test material:

- Substance type: White powder
- Storage condition of test material: Room temperature.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation:
> Range finding study: Males were approximately 7 weeks old, and females approximately 8 weeks old.
> Definitive test: Males approximately 7 weeks old, females approximately 9 weeks old.
- Weight at study initiation:
> Range finding study: Males 372-398 g, females 378-398 g.
> Definitive test: Males 390-477 g, females 394-451 g.
- Housing: Individually in suspended stainless steel cages.
- Diet: Guinea Pig diet provided ad libitum.
- Water: Municipal tap water treated by reverse osmosis was available ad libitum.
- Acclimation period: Minimum of five days. Animals were observed daily for overt physical or behavioural abnormalities, general health/moribundity and mortality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-23 °C (64-74 °F)
- Humidity (%): 54-89 %
- Air changes (per hr): 12-hour light/12-hour dark cycle.
- Photoperiod (hrs dark / hrs light): 10-15 air changes/hour.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

propylene glycol

Induction 2

Concentration / amount:

Range finding Studies:
Topical study: 25, 50, 75, 100 % w/v
Intradermal study: 0.1, 1.0, 3.0 and 5.0 % w/v

Sensitisation Study
Intradermal induction: 5.0 % w/v
Topical induction and challenge: 100 % w/v
Rechallenge: 75 % w/v
Second rechallenge: 25 % w/v

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

Challenge 2

Concentration / amount:

Range finding Studies:
Topical study: 25, 50, 75, 100 % w/v
Intradermal study: 0.1, 1.0, 3.0 and 5.0 % w/v

Sensitisation Study
Intradermal induction: 5.0 % w/v
Topical induction and challenge: 100 % w/v
Rechallenge: 75 % w/v
Second rechallenge: 25 % w/v

No. of animals per dose:

Definitive test group: 10 per sex per dose
Challenge control: 5 per sex per dose

Details on study design:

RANGE FINDING TESTS

> Topical Range Finding Study:
- Animal preparation: On the day prior to dose administration, four topical range-finding guinea pigs were weighed and the hair removed from the right and left sides of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- Application: On the following day, four concentrations of the test material were prepared and each concentration was applied to the clipped area of each topical range-finding animal as indicated below:
Test site 1: 100 % (0.3 g moistened with 5 drops of propylene glycol)
Test site 2: 75 % (0.3 mL)
Test site 3: 50 % (0.3 mL)
Test site 4: 25 % (0.3 mL)
Following chamber application, the trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chambers and the animal was returned to its cage.
Approximately 24 hours after chamber application, the elastic wrap, tape and chambers were removed. The test sites were then wiped with deionized water followed by dry gauze to remove test material residue. The animals were then returned to their cages.
Note: One animal (G1745/M) was found dead prior to patch removal. The cause of death was considered to be due to binders/stress and not to be test material related. This animal was replaced (G1748/M) and the new animal was dosed as previously described.
- Observations: Dermal observations (24 and 48 hours after chamber removal), clinical observations (twice daily), body weight (day prior to dosing).
- Gross necropsy: Animals were euthanized by carbon dioxide inhalation. Gross necropsy examinations were not performed.

> Intradermal Range Finding Study:
- Animal preparation: On the day prior to dose administration, four intradermal range-finding guinea pigs were weighed and the hair removed from the right and left sides of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- Application: On the following day, four concentrations of the test material were prepared and each concentration was intradermally injected into each intradermal range-finding animal using a syringe attached to a hypodermic needle as indicated below:
Test site 1: 5.0 % (0.1 mL)
Test site 2: 3.0 % (0.1 mL)
Test site 3: 1.0 % (0.1 mL)
Test site 4: 0.1 % (0.1 mL)
- Observations: Dermal observations (24 and 48 hours after chamber removal), clinical observations (twice daily), body weight (day prior to dosing).
- Gross necropsy: Animals were euthanized by carbon dioxide inhalation. Gross necropsy examinations were not performed.

MAIN STUDY

> Intradermal Induction Exposure:
- Test site preparation: On the day prior to induction (day -1) the hair was removed from the scapular area of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures. The test site was approximately 2 x 4 cm.
- Application: Three pairs of intradermal injections were made in the clipped area of all sensitization study animals (day 0) as detailed below:
Test Group:
1) Injection Pair A: 0.1 mL of FCA emulsion
2) Injection Pair B: 0.1 mL of 5.0 % w/v (test material/propylene glycol)
3) Injection Pair C: 0.1 mL of 5.0 % w/v (test material/FCA emulsion)
Challenge and Rechallenge Control Groups:
1) Injection Pair A: 0.1 mL of FCA emulsion
2) Injection Pair B: 0.1 mL of propylene glycol
3) Injection Pair C: 0.1 mL of 5.0 % w/v propylene glycol/FCA emulsion

> Topical Induction Exposure:
- Test site preparation: On the day prior to topical induction (day 6), the guinea pigs had the hair removed with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures. Following clipping, 0.5 mL of 10 % w/w sodium lauryl sulfate in petrolatum was spread over the intradermal injection sites of all study animals.
- Application: On study day 7, any residual sodium lauryl sulfate preparation was removed with dry gauze and the appropriate material was prepared and applied to the animals as indicated below:
1) Test: 100 % test material (0.3 g, moistened with 16 drops of propylene)
2) Challenge control: 100 % propylene glycol (0.8 mL)
3) Rechallenge control: 100 % propylene glycol (0.8 mL)
The patch was applied over the intradermal injection sites. The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the patch and the animal was returned to its cage.
Approximately 48 hours after dosing, the elastic wrap, tape and patch were removed. The test sites were wiped with gauze moistened in deionized water to remove test material residue and the animals were returned to their cages.

> Challenge Exposure
- Test site preparation: On the day prior to challenge dose administration, the hair was removed from the right side of the test and challenge control animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- Application: On the following day (day 21), the appropriate concentration of the test material was prepared and applied to the animals as indicated below:
1) Test (test site No. 2): 100 % test material (0.3 g moistened with 5 drops of propylene glycol)
2) Challenge control (test site No. 2): 100 % test material (0.3 g moistened with 5 drops of propylene glycol)
The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage.
Approximately 24 hours after dosing, the elastic wrap, tape and chamber were removed. The test sites were wiped with gauze moistened with deionized water followed by dry gauze to remove test material residue. The animals were then returned to their cages.

> Rechallenge Exposure:
- Test site preparation: On the day prior to rechallenge dose administration, the hair was removed from the left side of the test and rechallenge control animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- Application: On the following day (day 28), the appropriate concentration of the test material was prepared and applied to the animals as indicated below:
1) Test (test site No. 1): 75 % test material (0.3 mL)
2) Rechallenge control (test site No. 1): 75 % test material (0.3 mL)
The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage.
Approximately 24 hours after dosing, the elastic wrap, tape and chamber were removed. The test sites were wiped with gauze moistened with deionized water followed by dry gauze to remove test material residue. The animals were then returned to their cages.

> Second Rechallenge Exposure:
- Test site preparation: On the day prior to second rechallenge dose administration, the hair was removed from the right side of the test and challenge control animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- Application: On the following day (day 36), the appropriate concentration of the test material was prepared and applied to the animals as indicated below:
1) Test (test site No. 4): 25 % test material (0.3 mL)
2) Rechallenge control (test site No. 4): 25 % test material (0.3 mL)
The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage.
Approximately 24 hours after dosing, the elastic wrap, tape and chamber were removed. The test sites were wiped with gauze moistened with deionized water followed by dry gauze to remove test material residue. The animals were then returned to their cages.

> Observations:
- Dermal observations: The test sites at challenge, rechallenge and second rechallenge were graded for dermal irritation at approximately 24 and 48 hours following chamber removal. Erythema and edema formation was scored using the Draize scale. All other observations were recorded.
- Clinical observations: Any unusual observations and mortality were recorded. The animals were observed for general health/mortality twice daily, once in the morning and once in the afternoon.
- Body weight: Individual body weights were obtained on the day prior to intradermal induction (day -1), for the test and challenge control animals on the day prior to challenge and second rechallenge dosing and for the test and rechallenge control animals on the day prior to rechallenge dosing. All animals were also weighed prior to scheduled euthanasia.
- Gross necropsy: All animals were euthanized by carbon dioxide inhalation following each animal’s final scoring interval. Gross necropsy examinations were not required for these animals.

Challenge controls:

Challenge controls were include for all challenge and re-challenge exposures.

Positive control substance(s):

yes

Remarks:

1-Chloro-2,4-dinitrobenzene (DNCB) and Hexylcinnamaldehyde (HCA)

Results and discussion

Positive control results:

> Historical Control

The test facility has completed two studies during the past six months which provided historical control data for contact sensitization to this agent utilizing the test system described herein (Maximization Design).
DNCB: Following intradermal induction at 0.1 % w/v DNCB in acetone/propylene glycol, topical induction at 0.1 % w/v DNCB in acetone/propylene glycol and challenge at 0.05 % and 0.1 % w/v DNCB in acetone/propylene glycol, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent.
HCA: Following intradermal induction at 5 % w/v HCA in propylene glycol, topical induction at 5 % w/v HCA in propylene glycol and challenge at 0.5 % and 1 % w/v HCA in propylene glycol, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 % w/v

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: challenge. . Hours after challenge: 24.0. Group: test group. Dose level: 100 % w/v. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: none.

Results 2

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100 % w/v

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: other: challenge. . Hours after challenge: 48.0. Group: test group. Dose level: 100 % w/v. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100 % w/v

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: challenge. . Hours after challenge: 24.0. Group: other: challenge control. Dose level: 100 % w/v. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Key result

Reading:

other: challenge

Hours after challenge:

48

Group:

negative control

Dose level:

100 % w/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: challenge. . Hours after challenge: 48.0. Group: other: challenge control. Dose level: 100 % w/v. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 5

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

75 % w/v

No. with + reactions:

6

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 75 % w/v. No with. + reactions: 6.0. Total no. in groups: 20.0. Clinical observations: none.

Results 6

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

75 % w/v

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 75 % w/v. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Results 7

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

75 % w/v

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: other: rechallenge control. Dose level: 75 % w/v. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: none.

Results 8

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

75 % w/v

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: other: rechallenge control. Dose level: 75 % w/v. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: none.

Results 9

Key result

Reading:

other: second rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % w/v

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: other: second rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 25 % w/v. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.

Results 10

Key result

Reading:

other: second rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % w/v

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: Reading: other: second rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 25 % w/v. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.

Results 11

Key result

Reading:

other: second rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

25 % w/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: second rechallenge. . Hours after challenge: 24.0. Group: other: second rechallenge control. Dose level: 25 % w/v. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 12

Key result

Reading:

other: second rechallenge

Hours after challenge:

18

Group:

negative control

Dose level:

25 % w/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: second rechallenge. . Hours after challenge: 18.0. Group: other: second rechallenge control. Dose level: 25 % w/v. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 13

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.05 %

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

sensitization response was observed

Any other information on results incl. tables

Topical Range-Finding Study

The results of the range-finding study indicated that a test concentration of 100 % was appropriate for topical induction as the 100 % concentration did not cause irritation that would hinder the interpretation of the study results.

Intradermal Range-Finding Study

The results of the range-finding study indicated that a test concentration of 5.0 % w/v in propylene glycol was appropriate for intradermal induction as there was no difference in the response for all concentrations tested.

 

Definitive Sensitization Study

Following challenge with a test concentration of 100 %, dermal scores of 1 (all with slight edema) were noted in 7/20 test animals and in 6/10 challenge control animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 1/20 test animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 or ±. Group mean dermal scores were noted to be similar in the test animals as compared to the challenge control animals.

A rechallenge was conducted in order to substantiate and clarify the equivocal challenge results since the primary irritation was too high in the control group at challenge. Following rechallenge with a test concentration of 75 % w/v, dermal scores of 1 were noted in 6/20 test animals and in 3/10 rechallenge control animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 2/10 rechallenge control animals. Dermal reactions in the remaining test and rechallenge control animals were limited to scores of 0 or ±. Group mean dermal scores were noted to be similar in the test animals as compared to the rechallenge control animals.

A second rechallenge was conducted in order to substantiate and clarify the rechallenge results since the primary irritation was too high in the control group at rechallenge. Following second rechallenge with a test concentration of 25 % w/v, dermal scores of 1 were noted in 1/20 test at the 24 and 48-hour scoring intervals. Dermal reactions in the remaining test and all challenge control animals were limited to scores of 0 or ±. Group mean dermal scores were noted to be similar in the test animals as compared to the challenge control animals.

Body Weight Data

The sensitization study animals gained weight during the test period. The animals appeared in good health, since there were no positive clinical observations noted.

Conclusion

Based on the results of this study, the test material is not considered to be a contact sensitizer in guinea pigs. Results at both challenge and rechallenge were considered equivocal (uncertain) due to the primary irritation observed in the controls. The single response observed in the final second rechallenge may have been within the potential variance of primary dermal irritation. In addition, the single response (1/20) in the second rechallenge is well below the OECD 30 % requirement of response in an adjuvant test for a mild to moderate sensitizer. The results of the DNCB and HCA historical control studies demonstrated that the study design utilized by the test facility would detect potential contact sensitizers.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, the test material was determined to be not sensitising.

Executive summary:

The dermal sensitization potential of the test material was evaluated in Hartley-derived albino guinea pigs in a maximisation test. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 406, EPA OPPTS 870.2600, EU Method B.6 and Japan MAFF Dermal Sensitization Study, 1985.

Ten male and ten female guinea pigs received intradermal injections of the test material at 5.0 % w/v prepared in propylene glycol along with injections of FCA and 5.0 % w/v (test material) in FCA. One week later, the test animals received a topical application of 100 % (test material). Challenge and rechallenge control animals received similar intradermal and topical treatments except propylene glycol was used in place of the test material. Following a two-week rest period, a challenge was performed whereby the twenty test and ten challenge control guinea pigs were topically treated with the test material at 100 %. Challenge responses in the test animals were compared with those of the challenge control animals. Following a seven-day rest period, a rechallenge was performed whereby the twenty test and ten rechallenge control guinea pigs were topically treated with the test material at 75 % w/v. Rechallenge responses in the test animals were compared with those of the re- challenge control animals. Following an eight-day rest period, a second rechallenge was performed whereby the twenty test and ten challenge control guinea pigs were topically treated with the test material at 25 % w/v. Second rechallenge responses in the test animals were compared with those of the challenge control animals. 1-Chloro-2,4-dinitrobenzene (DNCB) and Hexylcinnamaldehyde (HCA) were used as positive controls.

Results at both challenge and rechallenge were considered equivocal (uncertain) due to the primary irritation observed in the controls. The single response observed in the final second rechallenge may have been within the potential variance of primary dermal irritation. In addition, the single response (1/20) in the second rechallenge is well below the OECD 30 % requirement of response in an adjuvant test for a mild to moderate sensitizer.

Under the conditions of the test, the test material was determined to be not sensitising.