Anisole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study is performed according to OECD 406 and in compliance with GLP . However, the purity is not identified.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An appropriate guinea pig maximisation test is available which would not justify conducting an additional LLNA due to animal welfare. In addition, this study predates the adoption of the Local Lymph Node Assay (LLNA; TG 429).

Test material

Test material information

Constituent 1

Reference substance name:

Anisole

EC Number:

202-876-1

EC Name:

Anisole

Cas Number:

100-66-3

Molecular formula:

C7H8O

IUPAC Name:

anisole

Test material form:

other: liquid stored at room temperature

Details on test material:

- Name of test material (as cited in study report): Anisole
- Physical state: colourless liquid
- Stability under test conditions: assumed to be stable by the sponsor
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Centre d'Elevage Lebeau, 78950 Gambais, France
- Age at study initiation: no data
- Weight at study initiation: the mean body weight was 339 +/- 16 g
- Housing: individually
- Diet (e.g. ad libitum): "Guinea-pigs sustenance reference 106 diet" (U.A.R., 91360 Villemoisson-sur-Orge, France)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): about 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: no data

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Remarks:

(batch no. 5830)

Induction 2

Concentration / amount:

- Intradermal Induction: Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCL solution, Anisole at a concentration of 10% (w/w) in the vehicle, a mixture 50/50 (w/v) of Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCL solution, and, the test substance at a concentration of 10% (w/w) in the vehicle
- Topical Induction: 0.5 mL of a non-irritant concentration of the test substance (100%)
- Topical Challenge: 0.5 mL of the maximum non-irritant concentration of the test substance (100%)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Remarks:

(batch no. 5830)

Challenge 2

Concentration / amount:

- Intradermal Induction: Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCL solution, Anisole at a concentration of 10% (w/w) in the vehicle, a mixture 50/50 (w/v) of Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCL solution, and, the test substance at a concentration of 10% (w/w) in the vehicle
- Topical Induction: 0.5 mL of a non-irritant concentration of the test substance (100%)
- Topical Challenge: 0.5 mL of the maximum non-irritant concentration of the test substance (100%)

No. of animals per dose:

- Test group: 10
- Control group: 5

Details on study design:

RANGE FINDING TESTS: two females were exposed by intradermal injection to the test item at different concentrations (from 1 to 25%) and the signs of skin irritancy were observed 24 hours after the exposure. The same animals were exposed by topical application to the test item at two concentrations (50 and 100%) and the signs of skin irritancy were analysed 24 and 48 hours following the exposure. Based on the results of this pre-test, the test item concentration of 10% (w/w) was selected for intradermal induction in the main study. For the induction by epidermal application, the concentration of 100% test material was chosen. For the topical challenge, the test material concentration of 100% was also chosen. (see the results of the range findings study in Tables 7.4.1/1 and 7.4.1/2).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: D0 and D7
- Test groups: FCA + NaCL, test substance at 10% (w/w) in paraffin oil, FCA + NaCL + test substance in paraffin oil. Before the topical application, sodium Lauryl sulphate was applied on the skin to promote an irritant reaction.
- Control groups: FCA + NaCl, paraffin oil alone, FCA + NaCL + paraffin oil
- Site: dorsal skin of the scapular region
- Frequency of applications: 2 applications
- Duration: 48 hours (epidermal application)
- Concentrations: 10% (w/w) (intradermal), 100 % (v/v) (epidermal)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 hrs
- Test groups: topical application of the test item in its original form (M.N.I.C)
- Control group: vehicle alone
- Site: right flank (test item) and left flank (vehicle)
- Concentrations: 100%
- Evaluation (hr after challenge): 24 and 48 hours after challenge

Challenge controls:

Negative control animals were treated by vehicle topical application (paraffin oil)

Positive control substance(s):

yes

Remarks:

Dinitro 2.4 chlorobenzene

Results and discussion

Positive control results:

2, 4-Dinitrochlorobenzene (DNCB) at a concentration of 0.5% induced positive skin sensitization reactions in 100% of the guinea-pigs. This is considered a satisfactory response rate for DNCB under the conditions of the test.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: - . No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: -. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.5%

No. with + reactions:

5

Total no. in group:

5

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.5%

No. with + reactions:

5

Total no. in group:

5

Any other information on results incl. tables

Table 7.4.1/1: results of the preliminary test/intradermal route

Animal number	Test item concentration % (w/w)	Scoring after treatment 24 hours
01	1	Irritation
02	1	Irritation
01	10	Irritation
02	10	Irritation
01	25	Necrosis
02	25	Necrosis

Concentration used in the main study is 10% (w/w) of the test substance

Table 7.4.1/2: results of the preliminary test/cutaneous route

Animal number	Test item concentration % (w/w)	Scoring after removal of the dressing (1)
		24 hours		48 hours
		E	O	E	O
01	50	0	0	0	0
02	50	0	0	0	0
01	100	0	0	0	0
02	100	0	0	0	0

 

M.N.I.C: maximum non- irritant concentration = 100%

M.I.C: minimum irritant concentration. Not determined

E: Erythema

O: oedema

(1): no residual was observed

Table 7.4.1/3: Reaction at challenge at 24 h and 48 h in negative control group and test group

Animal number	Reactionat challenge
	24 h	48 h
Control group
61	0	0
62	0	0
63	0	0
64	0	0
65	0	0
Test group (100% test material)
66	0	0
67	0	0
68	0	0
69	0	0
70	0	0
71	0	0
72	0	0
73	0	0
74	0	0
75	0	0

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions of this study, the test item Anisole is not classified as skin sensitiser according to the criteria of the EU Regulation 1272/2008 (CLP) and of the Annex VI of the Directive 67/548/EEC.

Executive summary:

In a guinea pig maximization test performed according to the OECD No. 406 guideline and in compliance with GLP, Anisole was tested for its skin sensitizing potential in female Dunkin-Hartley guinea pigs.

A preliminary test on two animals allowed determining the appropriate concentrations of the test item for the induction and the challenge phases of the main study. Indeed, 24 hours after an intradermal injection strong irritant skin effects were observed with signs of necrosis. Those effects were observed from concentrations of 25%. However at 1 and 10%, only irritation was observed. The concentration of intradermal induction of 10% was therefore chosen for the main test. 24 and 48 hours after a topical application, the test item applied on the skin at 50 and 100% induced no sign of irritation. Therefore the concentration of 100% was chosen for the topical induction and for the challenge phases.

For the main test, 5 animals were treated only with the vehicle (paraffin oil) (control group) and 10 animals were treated with the test substance.

For the induction phase, animals received an intradermal injection of the test substance at 10% in paraffin oil or in Freund’s Complete adjuvant (FCA) emulsion. One week later, a second induction was performed by a topical application of the test item at 100% in paraffin oil. Before this topical induction, the skin was treated with Sodium Lauryl sulfphate to promote an irritant reaction.

Two weeks after the topical induction phase, challenge was performed by applying the test substance at 100% on the skin of guinea pigs. The observation of skin irritation 24 and 48 h after the challenge treatment informs on the skin sensitising potential of the test item.

No clinical signs and no deaths related to treatment were noted during the study. After 24 and 48 hours following removal of the dressing of the cutaneous challenge application of the test substance, no cutaneous reactions were recorded.

2, 4-Dinitrochlorobenzene (DNCB) at a concentration of 0.5% induced positive skin sensitization reactions in 100% of the guinea-pigs. This is considered a satisfactory response rate for DNCB under the conditions of the test.

Therefore, under the test conditions, Anisole is not considered as a skin sensitiser according to the criteria of Regulation (EC) 1272/2008 (CLP) and Directive 67/548/EEC.

This skin sensitisation study is classified as acceptable. It satifies the guideline requirement for a skin sensitisation in the guinea pig.