Methyl (R)-amino(4-hydroxyphenyl)acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

end on 18-AUG-2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to OECD guideline and GLP. In the end nineties Deretil has noticed that in particular esters resulted in positive M&K tests, whereas the practice did not show any effect. Deeper analysis by an expert and of existing studies revealed that the use of corn oil could be the cause of that. Therefore we decided to redo the test for HPGM with 1% aqueous carboxymethylcellulose.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (R)-amino(4-hydroxyphenyl)acetate

EC Number:

253-657-2

EC Name:

Methyl (R)-amino(4-hydroxyphenyl)acetate

Cas Number:

37763-23-8

Molecular formula:

C9H11NO3

IUPAC Name:

methyl (2R)-2-amino-2-(4-hydroxyphenyl)acetate

Details on test material:

- Name of test material (as cited in study report): HPGM
- Substance type: monoconstituent substance
- Physical state: powder
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: < 500 grams
- Housing: group housing of 5 animals per labelled metal cage with wire-mesh floors
- Diet: Free access to standard guinea pig diet, including ascorbic acid (1000 mg/kg)
- Water: Free access to tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature 21 °C
- Humidity: 70%
- Air changes: approximately 15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light and 12 hours dark per day
(deviations from these optima conditions were noted, but were considered not to have affected study integrity)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Induction 2

Concentration / amount:

induction (intradermal): 2%
induction (epidermal): 50%
challenge (epidermal): 25% (1st challenge) and 25%, 10%, 5% (2nd challenge)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Challenge 2

Concentration / amount:

induction (intradermal): 2%
induction (epidermal): 50%
challenge (epidermal): 25% (1st challenge) and 25%, 10%, 5% (2nd challenge)

No. of animals per dose:

Experimental group: 10 females.
Control group: 5 females.

Details on study design:

RANGE FINDING TESTS:
Prior to the start of the Main study, the intradermal and epidermal irritancy of FGHM was investigated to select test substance concentrations suitable for the induction and challenge phase o f the Main Study.
Intradermal injections:
A series of four test substance concentration was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1mL/site) in the clipped scapular region. The injected site were assessed for irritation 24 and 48h after treatment.
Epidermal application:
A series of four test substance concentration was used the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5mL each) per animal to the clipped flank, using Metalline Patches mounted on medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage.
The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations.
After 24h, the dressing was removed and the skin cleaned of residual test substance using water. The treated skin areas were assessed for irritation 24 and 48h after exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
INDUCTION - Experimental animals:
Day 1 The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made in this area as follows:
A) A 1 :1 w/w mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 2 % concentration.
C) A 1 :1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 8 The scapular area between the injection sites was treated with 0.5ml of a 50% test substance concentration using a Metalline patch (2x3cm) mounted on Metalline tape and secured with Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance with water and the dermal reactions caused by the epidermal exposure were assessed for irritation.
INDUCTION - Control animals:
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

B. CHALLENGE EXPOSURE (all animals)
Day 21 All animals were treated epidermally with 0.15 ml at test substance concentration of 50 % on a clipped flank , using Patch test Plasters, attached to Micropore tape and secured with Coban elastic bandage.
The dressing was removed after 24h exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48h after removal of the dressing.
Day 28 A re-challenge was conducted approximately one week after the first challenge. The contralateral flank of all animals was similarly treated, except that multiple test substance concentrations and the vehicle were applied. All animals were treated epidermally with 0.15mL of each of the following concentrations, 25%, 10% and 5% and the vehicle.
The dressing was removed after 24h exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48h after removal of the dressing.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMICALDEHYDE

Results and discussion

Positive control results:

The skin reactions in the experimental animals observed in response to the 10% test substance concentrations in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals.
These results lead to a sensitisation rate of 80 per cent for the 10% concentration.
From these results, it was concluded that the female guinea pig of the albino Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance in a Maximisation type of test.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effect

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effect.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effect

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effect.

Results 3

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

25%, 10%, 5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effect

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 25%, 10%, 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effect.

Results 4

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

25%, 10%, 5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effect

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 25%, 10%, 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effect.

Results 5

Reading:

other: 1st and 2nd challenge

Hours after challenge:

24

Group:

negative control

Dose level:

25%, 10%, 5%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no effect

Remarks on result:

other: Reading: other: 1st and 2nd challenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 25%, 10%, 5%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effect.

Any other information on results incl. tables

Challenge phase:

First challenge: No skin reactions were evident after the challenge exposure to the 25% test substance concentration in the experimental and control animals.

Second challenge: No skin reactions were evident after the challenge exposure to the 25%, 10% and 5% test substance concentrations in the experimental and control animals.

 

Toxicity Symptoms / Mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

 

Body Weights:

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

Table 1: results of first challenge reading

Animal number	Day 23		Day 24		Comments
	25%	Vehicle	25%	Vehicle
Control
136	0	0	0	0
137	0	0	0	0
138	0	0	0	0
139	0	0	0	0
140	0	0	0	0
Experimental
141	0	0	0	0	Not sensitised
142	0	0	0	0	Not sensitised
143	0	0	0	0	Not sensitised
144	0	0	0	0	Not sensitised
145	0	0	0	0	Not sensitised
146	0	0	0	0	Not sensitised
147	0	0	0	0	Not sensitised
148	0	0	0	0	Not sensitised
149	0	0	0	0	Not sensitised
150	0	0	0	0	Not sensitised

 

Table 2: results of second challenge reading

Animal number	Day 30				Day 31				Comments
	25%	10%	5%	Vehicle	25%	10%	5%	Vehicle
Control
136	0	0	0	0	0	0	0	0
137	0	0	0	0	0	0	0	0
138	0	0	0	0	0	0	0	0
139	0	0	0	0	0	0	0	0
140	0	0	0	0	0	0	0	0
Experimental
141	0	0	0	0	0	0	0	0	Not sensitised
142	0	0	0	0	0	0	0	0	Not sensitised
143	0	0	0	0	0	0	0	0	Not sensitised
144	0	0	0	0	0	0	0	0	Not sensitised
145	0	0	0	0	0	0	0	0	Not sensitised
146	0	0	0	0	0	0	0	0	Not sensitised
147	0	0	0	0	0	0	0	0	Not sensitised
148	0	0	0	0	0	0	0	0	Not sensitised
149	0	0	0	0	0	0	0	0	Not sensitised
150	0	0	0	0	0	0	0	0	Not sensitised

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

not sensitising to the skin

Executive summary:

In a dermal sensitization study with HPGM in 1% aqueous carboxymethylcellulose, young adult Dunkin Hartley guinea pigs (10+5 females) were tested using the method of Maximisation (OECD 406, GLP).

Test substance concentrations selected for the Main study were based on the results of a preliminary study.

 

In the Main study, ten experimental animals were intradermally injected with a 2% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with vehicle only. Two weeks after the epidermal application all animals were challenged with a 25% test substance concentration and the vehicle. A second challenge was performed one week later at 25%, 10%, 5% and vehicle.

 

Challenge phase:

First challenge: No skin reactions were evident after the challenge exposure to the 25% test substance concentration in the experimental and control animals.

Second challenge: No skin reactions were evident after the challenge exposure to the 25%, 10% and 5% test substance concentrations in the experimental and control animals.

 

Toxicity Symptoms / Mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

 

Body Weights:

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

There was no evidence that HPGM had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in the first and second challenge phase to any of the test substance concentrations tested.

The result indicates a sensitisation rate of 0%.

 

Based on these results, HPGM does not have to be classified as sensitising to the skin according to EU criteria (DSD and CLP regulation).