4-methylanisole

Endpoint summary

Description of key information

Data from a well performed OECD 429 combined with data from an OET conducted in gunea pigs, as well as human maximization test results demonstrate that 4 -methylanisol is not a skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 13, 2010 to February 16, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Principles of method if other than guideline:

There is no deviation that affects the validty of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
Species/Strain: Mice, CBA/CaOlaHsd
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 19.4 - 22.2 g
- Housing: single, Makrolon Type II, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst / Netherlands)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 20 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

methyl ethyl ketone

Concentration:

10, 25 and 50% (w/v) test item

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was 100% of the undiluted test item. Dilutions were formulated in methyl ethyl ketone.
- Irritation: To determine the highest test item concentration that does not induce local signs of skin irritation or systemic toxicity, a pre-test was performed in two animals. Two mice were treated with test item concentrations of 50% and 100% each on three consecutive days. Prior to the first application of the test item and before sacrifice the ear thickness was determined using a micrometer. Additionally, both animals received ear punches after sacrifice at the apical area using a biopsy punch and were immediately pooled per animal and weighed using an analytical balance. Clinical signs were recorded within 1 hr, 24hrs after each application and on day 6. Ear swelling was observed on day 6 before sacrifice in the animal treated with 100% test item concentration and the ear thiickness measurement on day 6 showed an ear swelling of approximately 28% compared to day 1 prior to treatment. The maximum concentration of test item to be investigated in the LLNA was the dose that could be uniformly applied to the dorsal surface of the ears of the mice and which did not cause excessive skin irritation or systemic toxicity., i.e. 10, 25 and 50% (w/v).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Criteria used to consider a positive response:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
Data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:

Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 10, 25 and 50% (w/v) in methyl ethyl ketone. The application volume, 25 µl, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the vehicle alone (control animals).

Administration of 3H-Methyl Thymidine:
Five days after the first topical application, all mice were administered with 250 µl of 80.9 µCi/ml 3HTdR (corresponds to 20.2 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

For the determination of lymph node weights, after excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Both ears of mice were punched as described in the range finding study and weighed.

Animals were scarificed 5-hours post 3HTdR treatment and determination of incorporation of 3HTdR made as follows. The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group), single cell suspensions were prepared by gentle mechanical disaggregation through stainless steel gauze and the level of 3HTdR incorporation was measured on a scintillation counter.

Data is express as the Stimulation Index (SI) which is expressed as the ratio of 3HTdR incorportaed into the lymph node cells of test lymph nodes relative to controls

Observations were made on: mortality, body weights, ear thickness, ear weights, lymph node weights and clinical signs

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables.The ANOVA (Dunnett-test) was conducted on the ear and lymph node weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, the primary point of consideration is the biological relevance of the results.

Key result

Parameter:

SI

Value:

1.56

Test group / Remarks:

test item at concentration of 10%

Key result

Parameter:

SI

Value:

2.08

Test group / Remarks:

test item at concentration of 25%

Key result

Parameter:

SI

Value:

2.39

Test group / Remarks:

test item at concentration of 50%

Cellular proliferation data / Observations:

The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Pretest:

Ear swelling was observed on day 6 before sacrifice in the animal treated with 100% test item concentration and the ear thickness measurement on day 6 showed an ear swelling of approximately 28% in comparison to the measurement on day 1 prior to

treatment.

Main test:

No deaths, no symptoms of local irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. The body weight of the animals, recorded prior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

A statistically significant increase in lymph node weights or ear weights was not observed in any of the test item treated groups in comparison to the vehicle control group.

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a	number of lymph nodes	DPM per lymph node^b	S.I.
---	BG I	15	---	---	---	---
---	BG II	20	---	---	---	---
0	1	2167	2150	8	268.7
10	2	3380	3363	8	420.3	1.56
25	3	4490	4473	8	559.1	2.08
50	4	5159	5142	8	642.7	2.39

BG = Background ( 1 mL 5% trichloroacetic acid) in duplicate

1 = Control Group

2 -4 = Test Group

S.I. = Stimulation Index

^a)   =  The mean value was taken from the figures BG I and BG II

^b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item p-Cresolmethylether was not a skin sensitiser under the test conditions of this study.

Executive summary:

The purpose of this Local Lymph Node assay was to identify the skin sensitizing potential of p-Cresolmethylether when administered to the dorsum of both ears of mice. 50% test item concentration was chosen as the maximum concentration of test item to be investigated in the LLNA as this concentration could be uniformly applied to the dorsal surface of the ears of the mice and did at the same time not cause excessive skin irritation or systemic toxicity as confirmed by a pre-experiment. Three groups each of four female mice were treated daily with the test item at concentrations of 10, 25, and 50% (w/v) in methyl ethyl ketone by topical application to the dorsum of each ear (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (methyl ethyl ketone) only. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. Signs of local irritation (e.g. reddening of the ear skin, ear swelling) were also not observed during the study period. A statistically significant increase in ear weights was not observed in any of the groups treated with different concentrations of the test item in comparison to the vehicle control group. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3 HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 1.56, 2.08 and 2.39 were determined with the test item at concentrations of 10, 25, and 50% in methyl ethyl ketone, respectively. Under the test conditions, the test material is not a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the key study according to OECD TG 429 and GLP, three groups of four female CBA/CaOlaHsd mice were treated with 10, 25 or 50% 4 -methylanisol in methyl ethyl ketone by topical application at the dorsum of each ear on three consecutive days (BASF58H0506/099149). In a former pre-test, ear swelling was observed on day 6 before sacrifice in the animal treated with undiluted test substance and the ear thickness measurement on day 6 showed an ear swelling of approximately 28% in comparison to the measurement on day 1 prior to treatment. In the main study, the animals did not show any signs of systemic toxicity and no cases of mortality were observed. Signs of local irritation (e.g. reddening of the ear skin, ear swelling) were also not observed during the study period. A statistically significant increase in ear weights was not observed in treated animals compared to control group. Stimulation Indices (S.I.) of 1.56, 2.08 and 2.39 were determined with the test item at concentrations of 10, 25 and 50%, respectively. A statistically significant increase in lymph node weights was not observed in treated groups compared to controls. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3. Overall, 4 -methylanisol did not show a skin sensitizing potential under the test conditions of this study.

 

In support, a review, discussing the open epicutaneous test, presented data for about 300 fragrance raw materials in limited tabular form (Klecak 1985). For 4 -methylanisol, no sensitization reaction were reported using this test protocol.

 

A supportive human maximization test on 25 male volunteers is available as short abstract in a database (Kligman1971). After pretreatment with 5% sodium lauryl sulfate, closed patch applications of 2% 4 -methylanisol in petrolatum for five alternate-day 48-hour periods, followed by occlusive challenge application for 48 hours after a 10-day rest period was performed. No effects on these volunteers were reported. 

Respiratory sensitisation

Endpoint conclusion

Additional information:

No data available.

Justification for classification or non-classification

The present data on dermal sensitization do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.