Methyl o-toluate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 2004-09-15 and 2004-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Skin Sensitisation: Local Lymph Node Assay method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Methyl o-toluate

EC Number:

201-932-2

EC Name:

Methyl o-toluate

Cas Number:

89-71-4

Molecular formula:

C9H10O2

IUPAC Name:

methyl o-toluate

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Age Range: 11 weeks at start of dosing; records of dates of birth for animals used in this study are retained in the Calvert archives.
- Body Weight Range: 20 - 26 grams at the outset (Day 1) of the study.
- Animal Source: Jackson Laboratories, Bar Harbor, ME 04609
- Experimental History: Purpose-bred and experimentally naive at the onset of the study.
- Identification: Tail marked with an indelible marker and cage card.
- Housing: Animals were group housed (5 per cage) upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". The room in which the animals were kept was documented in the study records. No other species were kept in the same room
- Lighting: 12 hours light/12 hours dark
- Room Temperature: 21.1 to 25.6°C
- Relative Humidity: 30 - 54%
- Food: Animals had access to Certified Rodent Chow 7012C ad libitum.
- Water: Tap water was available ad libitum.
- Acclimation: Study animals were acclimated to their housing for six days prior to their first day of dosing.

All animals used in this study were assessed as to their general health by a member of the veterinary staff or other authorized personnel. During the acclimation period, each animal was observed at least once daily for any abnormalities or for the development of infectious disease. Only animals that were determined by the veterinary staff and/or Study Director to be suitable for use were assigned to this study.

Animals were assigned to study groups by a computerized randomization program (LABCAT Randomization module version 2.48, developed by Innovative Program Associates, Inc. 303 Wall Street, Princeton, NJ 08540-1515) designed to achieve similar group mean body weights.

Treatment of animals was in accordance with the study protocol and also in accordance with Calvert SOP's which adhere to the regulations outlined in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press). The Calvert IACUC approved the study protocol prior to dose administration, outlined in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press). The Calvert IACUC approved the study protocol prior to dose administration.

Study design: in vivo (LLNA)

Vehicle:

other: Diethyl phthalate/ethanol 3:1

Concentration:

Concentrations of 0, 7.5, 15, and, 30% v/v in vehicle 3:1 were tested.

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Test Item Administration:
- Route: Topically on the dorsal surface of both ears
- Frequency: Once daily for 3 consecutive days (Days 1 - 3). The timing of dose administration remained consistent(± 2 hours) during the dosing phase.
- Procedure: A volume of 25 µL/ear was applied using a micropipette.

Observations and Measurements:
- Mortality/Morbidity: Daily on Days 1 to 6
- Clinical Observations: Prior to dose administration and once post-dose on Days 1 to 3. Clinical observations were performed once daily on Days 4 to 6. Particular attention was given to the application sites. Any significant alterations (e.g., erythema and edema) to the application sites, and the general appearance of the pinnae, including build up of test article, was recorded.
- Body Weight: Animals were weighed at the time of randomization/selection, and on Days 1 and 6.

Method of Performance:
Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and, 3. Approximately 24 ± 2 hours between applications of test article was maintained. On Day 6 the mice were injected i.v. with 20 µCi of 3H­ thymidine in 250 µL of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation. Ear thickness measurements of each mouse were recorded and the draining auricular lymph nodes removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2 - 8°C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H­ thymidine was measured in a B-scintillation counter.

Positive control substance(s):

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

Statistics:

The mean DPM for each group was evaluated using SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control. Body weight data and ear thickness measurements were also evaluated. Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM, body weight and ear thickness data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett's test was used to determine the degree of significance from the control means.

Results and discussion

Positive control results:

The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 5.03. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.001) when compared to the vehicle control group.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Remarks on result:

other: see Remark

Remarks:

The following SI values were derived at 7.5, 15, and 30% (w/v): 1.13, 1.59, and 1.22, respectively. A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index 2 3.0 is regarded as a positive response. However, treatment with the test substance at 7.5, 15 and 30% (w/v) did not result in a stimulation index of 3 or greater.

Results 2

Key result

Parameter:

SI

Remarks on result:

other: at 7.5, 15 and 30%: 1.13, 1.59 and 1.22

Any other information on results incl. tables

Interpretation of Results: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non‑sensitiser". For chemicals, which the lowest concentration tested resulted in a stimulation index of greater than 3, an EC3 value was extrapolated from the two lowest doses utilized. The extrapolated EC3 value was calculated by log‑linear interpolation between the two points on a plane where the x-axis represents the dose level and the y‑axis represents the stimulation index. The point with the higher stimulation index was denoted (a, b) and the point with the lower stimulation index was denoted (c, d). The formula for the extrapolated EC3 value was as follows: EC3= 2^ {log2(c) + (3-d)/(b-d)] x [log2(a)-log2(c)]} a= mid concentration giving a stimulation index >3 b = actual stimulation index caused by ‘a’ c = lowest concentration giving a stimulation index of >3 d = actual stimulation index caused by ‘c’ The radioactive disintegrations per minute per animal and the stimulation index were recorded. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are provided.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Remarks:

according to the CLP Regulation EC 1272/2008 and its updates

Conclusions:

The test item was considered not to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay method. At 7.5, 15, and 30% the substance showed SI values of 1.13, 1.59, and 1.22, respectively. No statistically significant differences in the ear measurements were observed in any of the treatment groups when compared to the vehicle group. The test item was considered not to be a sensitiser under the conditions of the test.