N-(2-ethoxyphenyl)-N'-(2-ethylphenyl)oxamide

Endpoint summary

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:

from 5th March to 1st April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to international guideline. No relevant deviations have been reported so that the study is considered reliable.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age: Pre-test: 8 - 9 weeks (beginning of treatment) Main study: 9 - 10 weeks (beginning of treatment)
Body weight: 21 - 22g

Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.

Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C relative humidity approx. 45-65% (except for several hours, see deviations) artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

dimethylformamide

Concentration:

The test item in the main study was assayed at 1, 2.5, and 5% (w/w). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

No. of animals per dose:

Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1

Details on study design:

Topical application:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 1, 2.5, and 5% (w/w) in DMF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) 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 (day 6) 250 μL of phosphate-buffered saline containing 19.6 μCi of 3H-methyl thymidine (equivalent to 78.6 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

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, see attachment in the field "attached background materials"

Positive control results:

In this study Stimulation Indices of 1, 1.8, 3.2, 5.8 were determined with the test item at concentrations of 0, 5, 10, 25 % (w/w) in acetone/olive oil (4+1 v/v).
The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value that was 9.3% (w/v)

Parameter:

SI

Value:

1

Test group / Remarks:

Test group 1 (control)

Parameter:

SI

Value:

0.86

Test group / Remarks:

Test group 2 (test item 1% w/w)

Remarks on result:

other: see Remark

Remarks:

Stimulation Indices of 0.86, 0.94, and 0.94 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Parameter:

SI

Value:

0.94

Test group / Remarks:

Test group 3 (test item 2.5% w/w)

Remarks on result:

other: see remarks

Remarks:

Stimulation Indices of 0.86, 0.94, and 0.94 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Parameter:

SI

Value:

0.94

Test group / Remarks:

Test group 4 (test item 5% w/w)

Remarks on result:

other: see remarks

Remarks:

Stimulation Indices of 0.86, 0.94, and 0.94 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 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. DPM per limph node: 653.3, 562.3, 615.1 and 615.9 at 0, 1, 2.5 and 5 % (w/w) in DMF

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

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

Executive summary:

In order to study a possible skin sensitising potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 1, 2.5, and 5% (w/w) in DMF by topical application to the dorsum of each ear for three consecutive days. 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. A control group of four mice was treated with the vehicle (DMF) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. On day 3 and 4, the animals treated with test item concentrations of 2.5 and 5% showed an erythema of the ear skin (Score 1). On day 5 and 6 the animals treated with 5% continued to show an erythema of the ear skin (Score 1). Animals treated with 1% test item concentration did not show any signs of local skin irritation.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR 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 0.86, 0.94, and 0.94 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In order to study a possible skin sensitising potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 1, 2.5, and 5% (w/w) in DMF by topical application to the dorsum of each ear for three consecutive days. 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. A control group of four mice was treated with the vehicle (DMF) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. On day 3 and 4, the animals treated with test item concentrations of 2.5 and 5% showed an erythema of the ear skin (Score 1). On day 5 and 6 the animals treated with 5% continued to show an erythema of the ear skin (Score 1). Animals treated with 1% test item concentration did not show any signs of local skin irritation.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR 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 0.86, 0.94, and 0.94 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

A supporting study of Klimish 4 reliability is available which anyway confirms that the substance does not show sensitizing effects on the on rats skin.

Migrated from Short description of key information:
Skin sensitization (LLNA, OECD 429): not sensitizing

Justification for selection of skin sensitisation endpoint:
GLP study conducted according to international guideline. No relevant deviations have been reported so that the study is considered reliable.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The substance is not considered as a skin sensitization toxicant and there is no data indicating that the substance has respiratory sensitization potential.

Justification for classification or non-classification

Skin sensitization: the substance is not expected to cause a skin sensitization (allergic) reaction on the basis of the LLNA study enclosed. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.4, as amended by Regulation (EC) No 286/2011.

Respiratory sensitization: The substance is not considered as a skin sensitization toxicant and there is no data indicating that the substance has respiratory sensitization potential. As a result, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.4, as amended by Regulation (EC) No 286/2011.