Methyl (R)-2-(4-hydroxyphenoxy)propionate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 January 1992 to 07 February 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1989

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted prior to the LLNA becoming the first choice of method in the EU.

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

EC Number:

411-950-4

EC Name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

Cas Number:

96562-58-2

Molecular formula:

C10H12O4

IUPAC Name:

methyl (2R)-2-(4-hydroxyphenoxy)propanoate

Test material form:

solid

Details on test material:

- Appearance: Brown solid
- Storage conditions of test material: Refrigerator

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

Pirbright White, Dunkin Hartley HOE DHPK [SPF-LAC] BÖ

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: Young adult
- Weight at study initiation: 308 to 343 g
- Housing: 5 animals in cages with bedding
- Diet: ad libitum
- Water: ad libitum access to tap water; about 2 g of ascorbic acid per 10 L of water was added to the drinking water twice a week
- Acclimation period: 7 days
- Indication of any skin lesions: None reported

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 30 to 70 % (relative)
- Air changes: Not specified; the animals were housed ln fully air-conditioned rooms
- Photoperiod: 12 hours of light / 12 hours of darkness (lights on 06:00 to 18:00)

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

olive oil

Remarks:

DAB 9

Concentration / amount:

Intradermal induction: 5 % in olive oil DAB 9 resp. in Freund’s adjuvant/0.9 % aqueous NaCl solution (1:1) resp. olive oil DAB 9
Percutaneous induction: 25 % in olive oil DAB 9 resp. olive oil DAB 9

Day(s)/duration:

For the intradermal induction 6 injections were carried out in groups of two per animal. Percutaneous induction was carried out one week after intradermal induction.

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, open

Vehicle:

olive oil

Remarks:

DAB 9

Concentration / amount:

10 % in olive oil DAB 9 resp. olive oil DAB 9

Day(s)/duration:

21 days after intradermal induction

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Ten animals per dose in the test group; 5 animals in the control group

Details on study design:

RANGE FINDING TESTS: For detecting a possible influence on irritating effects of previous intradermal treatment with Freund’s adjuvant, animals were pre-treated with Freund’s adjuvant/0 .9 % aqueous NaCl-solution (1:1) each, in the same manner as intradermal induction in the main test about 4 weeks prior to the application of the test material.
In the preliminary test after two 24-hour percutaneous occlusive applications within 96 hours the minimum irritant concentration was found to be a 25 % test material preparation in olive oil DAB 9 and the maximum non-irritant concentration was a 10 % test material preparation in olive oil DAB 9 (48 hours after the beginning of application).
It was possible to inject a 5 % test material preparation in olive oil DAB 9 resp. in Freund’s adjuvant/0.9 % aqueous NaCl-solution (1:1) with a syringe.
In the pre-test, 2 x 2 cm filter paper strips were applied to the skin of the flanks under an occlusive dressing. The test filter paper strip was soaked in the test material formulation; thus, the animals were exposed to about 0.15 g of the test material formulation. The test material was applied 2 times for 24 hours within a period of 96 hours in order to detect non-specific phenomena that are not caused by a sensitisation reaction but could possibly be attributed to a shift in the irritation threshold.
The site of application was the flank, respective on the same area. Four animals were treated per test concentration and readings taken about 24 and 48 hours after the beginning of application. Skin finding were assessed according to Draize.

MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal induction
6 intradermal injections in groups of two were administered per animal. Injections for the test group were as follows:
A) Front row: 2 injections each of 0.1 mL Freund’s adjuvant without test material emulsified with 0.9 % aqueous NaCl-solution in a ratio of 1:1.
B) Middle row: 2 injections each of 0.1 mL of the test material formulation
C) Back row: 2 injections each of 0.1 mL Freund’s adjuvant/ 0.9 % aqueous NaCl-solution 1:1 with test material.
Control animals were administered the same injections (A, B and C) but without the test material. The site of application was the shoulder and readings were carried out 24 hours after the beginning of application in accordance with the Draize scale.

- Percutaneous induction
Percutaneous induction was carried out one week after intradermal induction. 2 x 4 cm filter paper strips were applied to the skin of the shoulder under an occlusive dressing.The filter paper strip was soaked in the test material formulation. Thus, the animals were exposed to about 0.3 g of the test material formulation.
Control animals were treated analogously to the test group with the vehicle only. The site of application was the shoulder in the same area as the intradermal induction and readings were carried out 48 hours after the beginning of application in accordance with the Draize scale.
Olive oil was used as vehicle because of poor solubility in water. The test material formulations were prepared immediately before test material application with Ultraturrax or with a magnetic stirrer. Formulations were prepared gravimetrically; all concentrations were determined in weight/weight.
Assessment of skin findings (according to Draize)
- Erythema and eschar formation
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well-defined erythema
3 = Moderate to severe erythema
4 = Severe erythema (beet redness) to slight eschar formation (injuries in depth)

- Oedema formation
0 = No oedema
1 = Very slight oedema (barely perceptible)
2 = Slight oedema (edges of area well defined by definite raising)
3 = Moderate oedema (raised approximately 1 mm)
4= Severe oedema (raised more than 1 mm and extending beyond the area of exposure)

Weight check of the individual animals was performed before induction and before the end of the study. Clipping of the test animals was performed about 3 to 6 hours before test material application. Animals were examined each work day to check for any that were sick and for those showing a deteriorated state.

B. CHALLENGE EXPOSURE
2 x 2 cm filter paper strips were applied to the skin of the flank under an occlusive dressing. The test filter paper strip was soaked in the test material formulation. Thus the animals were exposed to about 0.15 g of the test material formulation.
Treatment of the test group and of control group 1 was with the test material formulation. Additionally olive oil DAB 9 was applied as a vehicle. Control group 2 only received olive oil DAB 9.
The duration of exposure was for 24 hours on the intact skin of the clipped flank. Readings were made 24 and 48 h after the removal of the test patch and assessed according to the Draize scale.

Challenge controls:

Control group 1 was treated with the test material and vehicle 21 days after the induction period.
Two control groups were designated to be used as part of the challenge. Control group 2 that had been intended for a potential 2nd challenge was not treated with the test material since a 2nd challenge was not necessary on the basis of the unambiguous results of the 1st challenge.

Positive control substance(s):

no

Remarks:

A positive control with a known sensitiser was not included in this study; however a separate study was performed twice a year in the testing facility. The most recent positive control was with 1-chlor-2,4-dinitro-benzol.

Results and discussion

Positive control results:

Following induction in the positive control test, at the challenge application positive skin reactions were seen in all surviving animals (two animals died of pneumonia). The substance was therefore shown to exhibit a positive skin sensitising effect and the positive control showed that the test system was able to detect sensitising compounds under the laboratory conditions chosen.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 % in olive oil DAB 9

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10 % in olive oil DAB 9

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Remarks:

control group 1

Results 3

Key result

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Remarks:

Conducted as a separate study (performed twice a year in the testing facility)

Any other information on results incl. tables

After intradermal induction well-defined erythema and slight oedema were observed at the injection sites of the control group animals and test group animals at which only Freund's adjuvant/0.9 % aqueous NaCl-solution (1:1) was applied. Injection of test material preparation in olive oil DAB 9 caused in the half of the injection sites well-defined erythema and slight oedema and in the other half necrotic skin changes and slight oedema. After application of test material preparation in Freund’s adjuvant/0.9 % aqueous NaCl-solution (1:1) the test group animals showed necrotic skin changes and slight oedema.

After percutaneous induction (25 % in olive oil DAB 9) necrotic skin changes and slight oedema could be observed in the test group animals (caused by the intradermal induction). The control groups which were applied with olive oil DAB 9 (vehicle) exhibited incrustation, partially open (caused by the intradermal induction) in addition to well defined erythema and slight oedema.

After the percutaneous challenge with the test material preparation no skin reactions were observed in any animal. Olive oil DAB 9 applied as vehicle did not cause any reaction.

 

Table 1: Summary of Reactions at Challenge

	1^stChallenge
	Test Material (10 % in Olive oil DAB 9)	Olive oil DAB 9
Control Group 1	0/5	0/5
Control Group 2*	N/A	0/5
Test Group	0/10	0/10

The results are presented as the number of animals showing a positive reaction per number of animals treated.

*Control group 2 was not administered the test material due to being unnecessary after the unambiguous results.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was determined not to be a skin sensitiser.

Executive summary:

The potential of the test material to act as a sensitiser was investigated in accordance with the standardised guideline EU Method B.5 under GLP conditions in a guinea pig maximisation test. 

Following a pre-test to select doses, 10 female Dunkin Hartley guinea pigs were exposed to the test material in olive oil DAB 9 in both the induction and challenge phases of the study. At intradermal induction, the test material concentration was 5 % in in the vehicle and at percutaneous induction the test material concentration was 25 % in the vehicle. Twenty one days after the intradermal induction, the test and control animals (5 females) were exposed to the test material in olive oil DAB 9 at a concentration of 10 % in the challenge.

After intradermal induction well-defined erythema and slight oedema were observed at the injection sites of the control group animals and test group animals at which only Freund's adjuvant/0.9 % aqueous NaCl-solution (1:1) was applied. Injection of test material preparation in olive oil DAB 9 caused in the half of the injection sites well-defined erythema and slight oedema and in the other half necrotic skin changes and slight oedema. After application of test material preparation in Freund’s adjuvant/0.9 % aqueous NaCl-solution (1:1) the test group animals showed necrotic skin changes and slight oedema.

After percutaneous induction (25 % in olive oil DAB 9) necrotic skin changes and slight oedema could be observed in the test group animals (caused by the intradermal induction). The control groups which were applied with olive oil DAB 9 (vehicle) exhibited incrustation, partially open (caused by the intradermal induction) in addition to well defined erythema and slight oedema.

After the percutaneous challenge with the test material preparation no skin reactions were observed in any animal. Olive oil DAB 9 applied as vehicle did not cause any reaction.

Under the conditions of this study, the test material was determined not to be a skin sensitiser.