N-(2-methoxy-5-methylphenyl)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 OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Constituent 1

Reference substance name:

N-(2-methoxy-5-methylphenyl)acetamide

EC Number:

230-157-2

EC Name:

N-(2-methoxy-5-methylphenyl)acetamide

Cas Number:

6962-44-3

Molecular formula:

C10H13NO2

IUPAC Name:

N-(2-methoxy-5-methylphenyl)acetamide

Details on test material:

- Name of test material (as cited in study report): Acetkresidin TTRS

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 25 g
- Housing: group housing
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45-65
- Artificial light: 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

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 can be technically used was a 25 % (w/v) solution in dimethylformamide. Grinding of the test item and vortexing were necessary to prepare the solution.
To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 10 and 25% each on three consecutive days.

MAIN STUDY:

Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in dimethylformamide. The application volume, 25 µL, was spread over the entire dorsal surface (ø 8 mm) of each ear lobe 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
Five days after the first topical application, all mice were administered with 250 µL of 81.0 µCi/ml 3HTdR (corresponds to 20.2 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.

The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of scintillation liquid and thoroughly mixed.

The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Results and discussion

Positive control results:

EC3: 7.8% (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 5%: 0.85 10%: 1.09 25%: 0.73

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Measurement DPM Control Group: 5166 5%: 4426 10%: 5628 25%: 3778

Any other information on results incl. tables

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	144	---	---	---	---
---	BG II	24	---	---	---	---
---	1	5166	5082	8	635.3
5	2	4426	4342	8	542.8	0.85
10	3	5628	5544	8	693.0	1.09
25	4	3778	3694	8	461.8	0.73

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 all S.I.´s are below 3.

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. After 24 hours after the first application onwards the urine of the treated animals was discoloured, indicating the systemic distribution of the test item.

Body Weights: The body weight of the animals, recordedprior 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 weights (punches) of all animals treated were recorded after sacrifice. A relevant increase in ear weights (punches) was not observed and a statistically significance of the ear weight was not determined.

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 Acetkresidin TTRS was not a skin sensitiser in this assay.

Executive summary:

In the study the test item Acetkresidin TTRS dissolved in dimethylformamide was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. After 24 hours after the first application onwards the urine of the treated animals was discoloured, indicating the systemic distribution of the test item. A relevant increase in ear weights (punches) was not observed and a statistically significance of the ear weight was not determined.

In this study Stimulation Indices (S.I.) of 0.85, 1.09, and 0.73 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide, respectively.

The test item Acetkresidin TTRS was not a skin sensitiser in this assay.