Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jan 1998 to 19 Feb 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Jul 1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 96/54/EC, IV.C

Version / remarks:

Jul 1996

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US-EPA FIFRA 81-6

Version / remarks:

Nov 1984

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Currently no LLNA study is available for assessment. The GPMT test has been carried out as an animal test to predict human sensitisation for over a decade and is recommended by international test guidelines such as OECD.

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

EC Number:

276-696-7

EC Name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Cas Number:

72490-01-8

Molecular formula:

C17 H19 N O4

IUPAC Name:

ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Himalayan Spotted

Remarks:

(GOHI)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young healthy adult about 1 - 3 months,
- Weight at study initiation: 353 - 433 g
- Housing: Individually in Macrolon Type 3 cages with soft wood bedding
- Diet: Ad libitum
- Water: Municipal water supply from bottles, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 13 - 14
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: FROM 26 Jan 1998 To: 19 Feb 1998

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

Injection: 0.1 mL
Control: adjuvant/physiological saline mixture, 1:1 (v/v)
Treatment: adjuvant/physiological saline mixture, 1:1 (v/v)

Day(s)/duration:

Day 0: First injection out of the three pairs of intradermal injections

Induction 2

Route:

intradermal

Vehicle:

peanut oil

Concentration / amount:

Injection: 0.1 mL
Control: peanut oil
Treatment: 5.0 % test substance in peanut oil

Day(s)/duration:

Day 0: Second injection out of the three pairs of intradermal injections

Induction 3

Route:

intradermal

Vehicle:

peanut oil

Concentration / amount:

Injection: 0.1 mL
Control: peanut oil, 50 % w/v with 1:1 adjuvant/physiological saline mixture
Treatment: 5.0 % test substance in 1:1 adjuvant/physiological saline mixture

Day(s)/duration:

Day 0: Third injection out of the three pairs of intradermal injections

Induction 4

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

Patch: 2 x 4 cm; approx. 0.4 g per patch
Control: petrolatum
Treatment: 50 % test substance

Day(s)/duration:

Day 8: Occlusive dressing for 48 hours

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

Two treatments were placed on all animals: test substance on the test flank and vehicle on the vehicle flan (0.35mL)
Concentration: 10 % test substance (both control as treatment group)

Day(s)/duration:

Day 21: occlusive dressing for 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test group: 10
Control group: 5

Details on study design:

RANGE FINDING TESTS:
Intradermal Induction Pertest
- Concentrations of test substance and vehicle: 0.5, 1.0, 3.0 and 5.0 % in peanut oil
- Pre-treatment: A 5-cm wide area in the neck-shoulder region of 1 male and 1 female animal was shaved approximately 1 hour before treatment.
- Treatment: Two pair of intradermal injections (0.1 mL) FCA and one pair of injections for each concentration were administered, one of each pair on each side of the spine.
- Observations: The test sites were examined 24 and 48 hours after administration to determine the highest concentration to produce mild to moderate irritation without systemic toxicity.

Epidermal Induction Pertest
- Concentrations of test item and vehicle: 1, 10, 30 and 50% in Vaseline.
- Pre-treatment: Two pairs of intradermal injections (0.1 mL) of a 1:1 mixture of FCA/physiological saline were administered to 1 male and 1 female animal.
- Treatment: Seven days later, the test substance/vehicle mixtures were applied with four Hill Top Chambers, one on each concentration, tow on each flank.
- Observations: The test sites were examined 24 and 48 hours after administration to determine the highest concentration to cause mild to moderate irritation for the induction application and no irritation for the challenge application (highest non-irritant dose).

MAIN STUDY:
Treatment schedule: A set of intradermal induction injections was made on Day 0. An epidermal induction application was made once on Day 8. The epidermal challenge application was made once on Day 21.

A. INDUCTION EXPOSURE
- Intradermal Induction Injections: Concentration of test substance and vehicle: 5.0% in peanut oil.
- Pre-treatment: An area approximately 5 cm x 5 cm on the back of the neck was shaved approximately 1 hour before treatment.

Treatment (Day 0): Three pairs of injections (0.1 mL in volume) were given in the shaved area so that one of each pair was on each side of the middle.

Vehicle control group injections:
- adjuvant/physiological saline mixture, 1:1 (v/v)
- peanut oil
- peanut oil, 50% (w/v) with 1:1 adjuvant/physiological saline mixture

Test substance group injections:
- adjuvant/physiological saline mixture, 1:1 (v/v)
- test substance in peanut oil
- test substance in 1:1 adjuvant/physiological saline mixture

Treatment (Day 8): Epidermal Application Induction
- Concentration of test substance: 50% in Vaseline
- Treatment: A filter paper patch was fully loaded (approximately 0.4 g) with the test substance/vehicle alone (vehicle control group) and held in place with the occlusive dressing for 48 hours.

B. CHALLENGE EXPOSURE
- Epidermal Application Challenge (Day 21)
- Vehicle: Same as for epidermal application induction
- Concentration tested, test substance: 10 % (highest non-irritant dose)
- Pre-treatment: The flanks of all animals were shaved immediately prior to treatment.
- Treatment: One chamber loaded with the test substance/vehicle mixture (approximately 0.35 mL) was placed on one flank (test flank) and one chamber loaded with the vehicle alone was placed on the other flank (vehicle flank) of the animals of both groups. The chambers were held in place with the occlusive dressing for 24 hours.
- Method of sacrifice: CO2 asphyxiation

OTHER:
- Period of observation: Through 48 hours after completion of the challenge application.
- Skin irritation check: Application sites were examined 1 hour after removal of the epidermal induction dressing on Day 10 and rated for positive or negative skin irritation reactions.
- Scoring intervals: 24 and 48 hours after removal of the dressing for the challenge application.
- Grading of skin reactions: Dermal reactions were graded according to the Draize scale
- Clinical observations: Checked daily; any remarkable findings, with indication of severity, were recorded.
- Mortality: Checked daily
- Body weight: Measured and recorded immediately before treatment and at study termination.
- Grading of skin reactions: Please, see 'Any other information on materials and methods incl. tables' section

Challenge controls:

Vehicle without test substance

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole

Results and discussion

Positive control results:

Positive reactions were observed in 4 males and 4 females of the test group animals on the test anks at the 24-hour examination and in 5 males and 4 at the 48-hour examination; the sensitization rate for Benzocaine was therefore 45 %. There were no positive responses among the control group animals nor on the vehicle flank.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % w/v in peanut oil

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 % w/v in peanut oil

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % w/v in peanut oil

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50% w/v in peanut oil

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

30% w/v in vaseline

No. with + reactions:

8

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

30% w/v in vaseline

No. with + reactions:

9

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

PRE-TESTS, INTRADERMAL INDUCTION

Injections of 0.5, 1.0, 3.0, and 5.0 % test substance in peanut oil produced irritation at the injection site for all concentrations. The 5.0 %concentration was selected for the induction injection for the definitive test, in that it was suitable for injection, systematically well tolerated, and produced mild to moderate skin irritation.

 

PRE-TEST, EPIDERMAL APPLICATION

The maximum obtainable concentration of test substance in Vaseline was 50 %. Epidermal application of 1, 10, 30, and 50 % concentrations produced skin irritation at 30 % and 50 %. (both sexes). The 50 % concentration was selected for epidermal induction as the highest concentration to produce mild to moderate irritation. The 10 % concentration was the highest concentration to produce no irritation (highest non-irritant dose), and was therefore selected for the epidermal challenge application.

 

SKIN IRRITATION CHECK

To document irritation by the test article during epidermal induction, application sites were examined 1 hour after removal of the dressing for the epidermal induction. Positive skin irritation reactions were noted for all animals in the test substance. group. There were no positive reactions among the vehicle control group animals.

 

SKIN REACTIONS FOLLOWING CHALLENGE APPLICATION

Positive reactions were observed in 3 males and 1 female of the test group animals on the test flanks at the 24 hour examination and in 3 males at the 48-hour examination; the sensitisation rate for test substance. was therefore 20 %. There were no positive skin responses on the vehicle flanks and among the vehicle control group.

 

Mortality: There was no mortality in the test

Clinical Observations: There were no remarkable clinical observations.

Body weights: Body weights were not affected by treatment.

Table 3 Intraderrnal Induction Pretest, Test Article in peanut oil

Draize cores

Sex/Animal	After 24 hrs				After 48 hrs
	M/1	F/2	M/1	F/2	M/1	F/2	M/1	F/2
Concentration	Erythema		Oedema		Erythema		Oedema
0.5 %	1	1	0	0	1	1	0	0
1.0 %	1	1	0	0	1	1	0	0
3.0 %	1	1	1	0	1	1	0	0
5.0 %	1	1	1	1	1	1	1	1

Note: A different pair of animals(one male, one female) was used for each of the two pretests.

Table 4 Epidermal Application Pretest, Test Article in vaseline

Draize cores

Sex/Animal	After 24 hrs				After 48 hrs
	M/1	F/2	M/1	F/2	M/X1	F/2	M/1	F/2
Concentration	Erythema		Oedema		Erythema		Oedema
1 %	0	0	0	0	0	0	0	0
10 %	0	0	0	0	0	0	0	0
30 %	1	1	0	0	1	1	0	0
50 %	1	1	0	0	1	1	0	0

Note: A different pair of animals (one male, one female) was used for each of the two pretests.

Table 5 Summary of Positive Skin Reactions after Challenge Application

Number of Animals with Positive Responses/Number of Animals in Group

	Vehicle flank		Test flank
	24 Hours	48 Hours	24 Hours	48 Hours
Control group	0/10	0/10	0/10	0/10
Test article group	0/20	0/20	4/20	3/20

Table 6 Positive Skin Reactions after Challenge Application

Number of Animals with Positive Responses/Number of Animals in Group

	Vehicle flank		Test flank
	24 Hours	48 Hours	24 Hours	48 Hours
Control group	0/10	0/10	0/10	0/10
2-Mercapto benzothiazole	0/20	0/20	8/20	9/20

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance has been found to be not skin sensitising.

Executive summary:

A dermal sensitisation test was conducted with the test substance using the Maximization procedure in accordance with OECD TG 406 following GLP principles. Two groups of male and female Himalayan Spotted (GOHI) guinea pigs (10 vehicle control, 20 test) were included. On Day 0, the test group animals were given a set of intradermal injections (including 5.0% test substance in peanut oil (vehicle) for the test group). The epidermal induction was made on Day 8; the test animals with 50 % test substance in vaseline and the vehicle control animals were treated with the vehicle alone. The epidermal challenge application on Day 21 consisted of a paired application of the vehicle alone and 10 % test substance in vaseline in both groups. Skin reactions on both the vehicle and test flanks were scored 24 and 48 hours after completion of the challenge application, according to the Draize scale.

Very slight to severe erythema in 4/20 test animals and was observed 24 hours after topical challenge. Very slight to well-defined erythema in 3/20 test animals and very slight oedema in 1/20 test animals was observed 48 hours after topical challenge. The sensitisation rate for test substance was therefore 20 %. There were no positive skin responses on the vehicle flanks and among the vehicle control group. There was no mortality, and there were no remarkable clinical observations in either group. Body weights were not affected by treatment.

According to the test results, the test substance is not required to be classified for skin sensitisation since the sensitisation rate is below 30%.