N-(2,5-dimethoxyphenyl)acetamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (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

Constituent 1

Reference substance name:

N-(2,5-dimethoxyphenyl)acetamide

EC Number:

222-423-1

EC Name:

N-(2,5-dimethoxyphenyl)acetamide

Cas Number:

3467-59-2

Molecular formula:

C10H13NO3

IUPAC Name:

N-(2,5-dimethoxyphenyl)acetamide

Details on test material:

- Name of test material (as cited in study report): Acetchinonbase (2,5-Dimethoxyacetanilid)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS:
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 18.0 - 21.0 g
- Housing: group
- Cage Type: Makrolon Type II / III, with wire mesh top
- Diet: pelleted standard diet (Harlan Laboratories), ad libidum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 5, 10 and 25%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25 % (w/v) solution in acetone:olive oil (4+1 v/v). Vortexing and sonicating were used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. warming to 37°C or sonicating).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance.
Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation.
At the tested concentrations the animals did not show any signs of systemic toxicity. Only on day 5 of the pre-test, an erythema of the ear skin (Score 1 = very slight erythema) and flaky ear skin was observed in both animals.

MAIN STUDY:

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 5, 10 and 25% (w/w) in acetone/olive oil (4:1). The application volume, 25 µL was spread over the entire dorsal surface (ø~8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-First, that 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.
-Second, that the 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.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability At least once daily from experimental start to necropsy.
Body weights In the pre-test prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test and main test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.

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 weights to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

Experiment performed in May 2011 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.30, 2.82, and 9.03, respectively.
The EC3 value calculated was 10.4 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: S.I. at 0 % (w/v): 1.00 S.I. at 5 % (w/v): 1.37 S.I. at 10 % (w/v): 1.25 S.I. at 25 % (w/v): 1.60

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 0 % (w/v): 392.1 DPM per lymph node 5 % (w/v): 537.7 DPM per lymph node 10 % (w/v): 488.2 DPM per lymph node 25 % (w/v): 625.8 DPM per lymph node

Any other information on results incl. tables

EC3 VALUE The EC3 valuecould not be calculated, since all S.I.´s are below the threshold value of 3.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symtoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

EAR WEIGHTS

The measured ear weight of all animals treated was recorded on day 6 after necropsy. A statistically significant increase in ear weight was seen in the high dose group in comparison to the vehicle control group (p < 0.05).

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The test item Acetchinonbase (2,5-Dimethoxyacetanilid) was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item Acetchinonbase (2,5-Dimethoxyacetanilid) dissolved in acetone:olive oil (4+1 v/v) was assessed for its possible contact allergenic potential according to OECD guideline 429.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10 and 25%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.37, 1.25, and 1.60 were determined with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4+1 v/v), respectively.

The test item Acetchinonbase (2,5-Dimethoxyacetanilid) was not a skin sensitiser under the test conditions of this study.