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

Endpoint summary

Description of key information

The contact sensitisation potential of the test material has been address using two key studies. Both studies were conducted under GLP conditions and in line with standardised guidelines, which utilised different traditional sensitisation test methods. Both studies are in agreement that the test material is not sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

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.

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.

Route:

intradermal and epicutaneous

Vehicle:

propylene glycol

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

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

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)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 1997 - 5 June 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-6 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF Testing Guideline

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

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

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 4 to 8 weeks of age.
- Weight at study initiation: Weighing from 367 to 585 g.
- Housing: During acclimatisation animals were individually housed in suspended, screen-bottom stainless steel cages.
- Diet: Certified guinea pig diet ad libitum
- Water: Water was provided ad libitum
- Acclimation period: at least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C (irritation screening phase Days 1 and 2) or 18 to 26 °C (irritation screening phase Day 3 and the entire definitive study).
- Humidity (%):50 % ± 20 %
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 23 April 1997 To: 5 June 1997

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

Irritation screen: 10 %, 25 %, and 50 % w/v
Induction and challenge tests: 100 % (moistened in 0.5 mL of mineral oil to ensure good skin contact)

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

Irritation screen: 10 %, 25 %, and 50 % w/v
Induction and challenge tests: 100 % (moistened in 0.5 mL of mineral oil to ensure good skin contact)

No. of animals per dose:

Irritation screening group: 4 animals
Test group: 20 animals
Naive control group: 10 animals

Details on study design:

RANGE FINDING TESTS:
> Irritation Screening Study:
An irritation screening study using four animals was conducted to determine the irritation threshold of the test material. The test material was administered as a 0.4 g dose (moistened with approximately 0.5 mL of mineral oil) and at concentrations of 10 %, 25 %, and 50 % w/v in mineral oil with each animal receiving all four concentrations of the test material. The appropriate test material concentrations, in the amount of 0.4 g or 0.4 mL, were applied to adhesive patches. The patches were then placed on four shaved sites (two on the right side and two on the left side) on each animal, covered with an overlapping strip of dental dam, and overwrapped with tape. The patches remained in place for 6 hours after which they were removed. Any residual material was then removed from the application sites with water and disposable paper towels. The application sites were observed for dermal reactions at 24 and 48 hours after test material application. All test material mixtures used in the irritation screening study were stored at room temperature until administered.

MAIN STUDY
> Induction Exposure:
- Test site preparation: On each day of test material application, the hair was removed from the back of each animal in the test group with electric clippers.
- Application: The test material was applied to each animal in the test group by placing 0.4 g (moistened with approximately 0.5 mL of mineral oil) on an adhesive patch and placing the patch on the induction site along the dorsal anterior left quadrant. The patch was covered with dental dam and overwrapped with tape. The dressing remained in place for a period of 6 hours after which it was removed. Any residual material was then removed from the application sites with water and disposable paper towels. The animals in the test group received one application per week for 3 weeks for a total of three applications. The naive control animals were not treated during this phase of the study.

> Challenge Exposure:
Two weeks following the administration of the third induction dose, a challenge dose of 0.4 g of the test material (moistened with approximately 0.5 mL of mineral oil) was administered along the dorsal anterior right quadrant of the test group animals in the same manner as during the induction phase of the study.

> Observations:
- Dermal irritation: On the day of the 24-hour examination following the irritation screening and challenge applications, the application sites of the respective animals were depilated by applying a depilatory. After approximately 10 to 20 minutes, the depilatory was washed from the application sites.
The respective application sites were examined and scored for dermal reactions according to the Buehler (Buehler and Ritz, 1980) scoring scale at 24 and 48 hours following the irritation screening, induction, and challenge applications.
- Clinical observations: Observations were conducted daily throughout the study.
- Body weights: The body weights on the irritation screening animals were determined only on the day of treatment. Body weights on the definitive study animals were determined before test material administration and at termination of the in-life phase.
- Termination: At termination of the respective in-life phase for each group, all animals were euthanized and discarded. Gross necropsy was not performed.

Challenge controls:

10 naive (previously untreated) control animals were also treated with a challenge application of the test material.

Positive control substance(s):

yes

Remarks:

2,4-dinitrochlorobenzene

Positive control results:

The positive control was demonstrated to be a sensitiser.

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

0.4 g 2,4-dinitrochlorobenzene in 0.5 ml mineral oil

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

positive indication of sensitisation observed

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.4 g 2,4-dinitrochlorobenzene in 0.5 ml mineral oil

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

positive indication of sensitisation observed

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.4 g 2,4-dinitrochlorobenzene in 0.5 ml mineral oil

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

positive indication of sensitisation observed

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

0.5 ml mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.5 ml mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.5 ml mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

0.4 g test material in 0.5 ml mineral oil

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Non-formed faeces were noted in three test animals

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.4 g test material in 0.5 ml mineral oil

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Non-formed faeces were noted in three test animals

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.4 g test material in 0.5 ml mineral oil

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Non-formed faeces were noted in three test animals

Remarks on result:

no indication of skin sensitisation

Irritation Screening Study

No dermal irritation was observed with any test material concentration. All animals appeared normal during this phase of the study.

 

Definitive Study

- Clinical Observations and Body Weights: Non-formed faeces were noted in three test animals and one naive control animal at various times during the study. A thin appearance, few feces, and hypothermia were also noted for one naive control animal. These findings are not considered to be test material-related. The remaining animals appeared normal throughout the study. All animals exhibited body weight gain during the definitive study.

- Dermal Reactions: No dermal reactions were observed in the animals in the test group when administered the test material during the induction or challenge phases of the study. None of the naive control animals reacted to the challenge application of the test material.

Positive Control

Information from a positive control run within 6 months of this study was used.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material did not cause delayed contact hypersensitivity in guinea pigs and was not considered to be a dermal sensitizer.

Executive summary:

The delayed contact hypersensitivity potential of the test material was evaluated in albino guinea pigs using the Buehler method. The study was conducted under GFLP conditions and in accordance with the standardised guidelines OECD 406, EPA OPP 81-6, EU Method B.6 and Japanese MAFF Testing Guideline.

Twenty male Hartley albino guinea pigs received one application of 0.4 g of the test material moistened with approximately 0.5 mL of mineral oil per week for three weeks during the induction period. The condition of the test sites was assessed approximately 24 and 48 hours after the application. Two weeks following the administration of the third induction dose, a challenge dose of 0.4 g of the test material moistened with mineral oil was administered to the test group animals in the same manner as during the induction phase of the study. At this time, 10 naïve control animals were also treated in the same manner with a challenge application of the test material. The condition of the test sites was assessed approximately 24 and 48 hours after the challenge application. The test material moistened with mineral oil did not elicit any dermal reactions during the induction or challenge phases of the study. All animals were in apparent good health and all animals gained weight throughout the study.

Under the conditions of this study, the test material did not cause delayed contact hypersensitivity in guinea pigs and was not considered to be a dermal sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The contact sensitisation potential of the test material has been address using two key studies. Both studies were conducted under GLP conditions and in line with standardised guidelines, which utilised different traditional sensitisation test methods. Both studies are in agreement that the test material is not sensitising.

In the first study (Glaza, 1997) the delayed contact hypersensitivity potential of the test material was evaluated in albino guinea pigs using the Buehler method. The study was conducted under GFLP conditions and in accordance with the standardised guidelines OECD 406, EPA OPP 81-6, EU Method B.6 and Japanese MAFF Testing Guideline.

Twenty male Hartley albino guinea pigs received one application of 0.4 g of the test material moistened with approximately 0.5 mL of mineral oil per week for three weeks during the induction period. The condition of the test sites was assessed approximately 24 and 48 hours after the application. Two weeks following the administration of the third induction dose, a challenge dose of 0.4 g of the test material moistened with mineral oil was administered to the test group animals in the same manner as during the induction phase of the study. At this time, 10 naïve control animals were also treated in the same manner with a challenge application of the test material. The condition of the test sites was assessed approximately 24 and 48 hours after the challenge application. The test material moistened with mineral oil did not elicit any dermal reactions during the induction or challenge phases of the study. All animals were in apparent good health and all animals gained weight throughout the study.

Under the conditions of this study, the test material did not cause delayed contact hypersensitivity in guinea pigs and was not considered to be a dermal sensitizer.

In the second study (Bonnette 1999) 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.

Migrated from Short description of key information:

Not sensitising, Hartley guninea pigs, Buehler test, OECD 406, EPA OPPTS 81-6, EU Method B.6, Japanese MAFF Testing Guideline, Glaza 1997.

Not sensitising, Hartley guninea pigs, Maximization test, OECD 406, EPA OPPTS 870.2600, EU Method B.6, Japan MAFF Dermal Sensitization Study, 1985, Bonnette 1999.

Justification for selection of skin sensitisation endpoint:

Two key studies were available to address this endpoint. Both studies were conducted under GLP conditions, in accordance with standardised guidelines and are fully reported. The studies were assigned reliability scores of 1, reliable without restrictions, in line with the principles of Klimisch (1997).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No study available.

Justification for selection of respiratory sensitisation endpoint:

No study available.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I (Classification and Labelling Requirements for Hazardous Substances and Mixtures), Regulation (EC) No. 1272/2008 (CLP), the substance does not require classification with respect to skin sensitisation.