Methyl myristate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Apr - 29 May 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions, the analytical purity of the test substance was not specified.

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:

adopted in 1992

Deviations:

yes

Remarks:

analytical purity of test substance not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

adopted in 1996

Deviations:

yes

Remarks:

analytical purity of test substance not specified

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Data for skin sensitisation already existed with a non-LLNA method

Test material

Test material information

Constituent 1

Reference substance name:

Methyl laurate

EC Number:

203-911-3

EC Name:

Methyl laurate

Cas Number:

111-82-0

Molecular formula:

C13H26O2

IUPAC Name:

methyl laurate

Details on test material:

- Name of test material (as cited in study report): methyl laurate
- Physical state: colourless liquid
- Analytical purity: not indicated by the sponsor; nevertheless, the test item was considered as 100% pure
- Storage: 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. 5 weeks
- Weight at study initiation: 381 g (mean value)
- Housing: groups of 5 animals in metal cages with wire mesh floors
- Diet: free access to standard guinea pig diet (LC 23-B, pellet diameter 4 mm; Hope farms, Woerden, The Netherlands), including ascorbic acid (1600 mg/kg); in addition, hay was provided once a week (B.M.I., Helmond, The Netherlands)
- Water: tap water, diluted with decalcified water, ad libitum (by automatic drinking system: ITL, Bergen, The Netherlands)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Induction 2

Concentration / amount:

Induction: 50% intradermally, 100% epicutaneously
Challenge: 20% epicutaneously

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

Induction: 50% intradermally, 100% epicutaneously
Challenge: 20% epicutaneously

No. of animals per dose:

10 females for the experiment group; 5 for control

Details on study design:

RANGE FINDING TESTS: Prior to the start of the main study the intradermal and epidermal irritancy of the test substance were investigated for selection of suitable test substance concentrations for induction and challenge of the main study. The selection was based on the absence of toxicity. For the Induction phase the highest possible concentration that produced moderate irritation should be determined (including slight necrosis with diameter <3 mm for intradermal application. The challenge concentration should be the maximum non-irritant concentration. Selection of this concentration depended on a number of factors and exact criteria did not always apply.
The test substance concentrations used were from the series: Undiluted, 50%, 20%, 10%, 5%, 2%, 1% and, if needed, further lower concentrations using the same steps.
The test system, procedures and techniques were identical to those used in the main study; Animals were between 5 and 9 weeks old; therefore, body weights could exceed 500 g. Body weights were determined prior to treatment.

Intradermal injections: A series of four test substance concentrations were used; the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.

Epidermal application: A series of four test substance concentrations was used; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on medical tape, 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 24 hours the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 hours after exposure

Based on the results of the preliminary study a 50% dilution of the test substance in corn oil was used for intradermal induction, and the undiluted substance was used for the epidermal induction exposure. A 20% test substance concentration was selected for the challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Intradermal induction on Day 1 and epidermal induction at Day 8 (for 48 h)
- Test groups: Intradermal: FCA with water (1:1 w/w), 50% TS in vehicle, undiluted TS with FCA (1:1 w/w); Epidermal: 0.5 mL undiluted test substance
- Control group: FCA with water (1:1 w/w), vehicle (corn oil), vehicle with FCA (1:1 w/w)
- Site: Three pairs of intradermal injections into the scapular region of both flanks; one of each pair injected on each side of the midline and from cranial to caudal)
- Frequency of applications: 7 days
- Duration: 10 days
- Concentrations: Intradermal: 50%; Epidermal: undiluted


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 h
- Test groups: TS
- Control group: TS
- Site: On one of the flanks (previously clipped)
- Concentrations: 20% in vehicle
- Evaluation (hr after challenge): 48 and 72 h (24 and 48 h after removal of the dressing)

Challenge controls:

none

Positive control substance(s):

yes

Remarks:

alpha-hexyl cinnamic aldehyde, tech. 85%

Results and discussion

Positive control results:

The skin reactions in the experimental animals observed in response to the 10% and 5% test substance concentration in the challenge phase of an independent control experiment were considered indicative of sensitisation, based on the absence of any response in the respective control animals. The test substance exposure lead to a sensitisation rate of 100% to both the 10% and the 5% concentrations. 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 sensitisation studies in a Maximisation type of test.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20% . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 2

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

20%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 3

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 20% . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

20%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Any other information on results incl. tables

Induction readings:

Animal number	Intradermal injection (reading at day 3)			Epidermal exposure (reading at day 10)
	A	B	C	100%
Control				Erythema	Edema
31	E2	NA	E3	0	0
32	N2	NA	E4	0	0
33	E4	NA	E4	0	0
34	E2	NA	E3	0	0
35	E3	E1	E2	0	0
Experimental
36	E2	E1	E2	3	2
37	E3	NA	E3	3	2
38	E3	E2	E4a	4	3
39	E2	E1	E3a	3	1
40	E3	E1	E4a	4n	1
41	E3	E2	E3	4	1
42	E4	E3	E4	4	3
43	E4	E4	E4	4	3
44	E4	E3	E4a	4	3
45	E3	E2	E3a	4	1

A: 1:1 mixture of Freuds´complete adjuvant with water

B: Test substance (50%) in corn oil

C: 1:1 mixture of the undiluted test substance with Freuds´complete adjuvant

a: Moderate erythema visible approx. 2 cm caudally of the injection sites

n: Signs of necrosis

 

Skin effects intradermal injections:

NA: No abnormalities

E(.): Erythema (grade)

N(.): Signs of necrosis (mm in diameter)

Challenge readings

Animal No.	Day 24		Day 25			Comments
	20%	Vehicle	20%		Vehicle
Control
31	0	0	0	0
32	0	0	0	0
33	0	0	0	0
34	0	0	0	0
35	0	0	0	0
Experimental
36	0	0	0	0		Not sensitized
37	0	0	0	0		Not sensitized
38	0	0	0	0		Not sensitized
39	0	0	0	0		Not sensitized
40	0	0	0	0		Not sensitized
41	0a	0a	0	0		Not sensitized
42	0	0	0	0		Not sensitized
43	0	0	0	0		Not sensitized
44	0	0a	0	0		Not sensitized
45	0	0	0	0		Not sensitized

 a: Skin reactions grade 1 noted at the edges of the application area are considered non-specific, possibly provoked by the edges of the patches.

No deaths occurred. No significant differences in the gain of body weight were observed between treatment and control group.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified