2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Endpoint summary

Description of key information

In a LLNA-study all treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. A statistically significant or biologically relevant increase in ear weight was not observed 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 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 0.96, 0.98 and 0.58 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil (4+1 v/v), respectively. A dose response was not observed.

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

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 2

Endpoint:

skin sensitisation: in chemico

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 APR 2012 to 05 JUN 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

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)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test: 10 - 11 weeks (beginning of treatment) Main study: 8 – 9 weeks (beginning of treatment)
- Weight at study initiation: 18.2 - 21.7 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Vehicle:

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

Concentration:

2.5, 5, and 10% (w/w)

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 10 % suspension in acetone:olive oil (4+1 v/v). Sonicating of the test item was necessary 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. vortexing, warming to 37°C).
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 5 and 10% 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 skin irritation were documented and a score was used to grade a possible erythema 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 an erythema 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 (for detailed results see Annex 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.
Thus, the test item in the main study was assayed at 2.5, 5 and 10% (w/w).


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 2.5, 5 and 10% (w/w) in acetone:olive oil (4+1 v/v). 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 futher 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): 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 for the body weight.

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.

Positive control results:

Experiment performed in April 2012 (Harlan study number 1486301) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 0.88, 1.51, and 3.73, respectively.
The EC3 value calculated was 20.1 % (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.

Parameter:

SI

Remarks on result:

other: Dose group /SI 0% / 1 2.5% / 0.96 5% / 0.98 10% / 0.58

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Dose group / DPM per lymph node 0% / 491.6 2.5% / 473.6 5% / 482.2 10% / 286.4

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 or biologically relevant increase in ear weights was not observed.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

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 2.5, 5, and 10% (w/w) in acetone:olive oil (4+1 v/v) by topical application to the dorsum of each ear for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil (4+1 v/v)) 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 ß-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. A statistically significant or biologically relevant increase in ear weight was not observed 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 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 0.96, 0.98 and 0.58 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil (4+1 v/v), respectively. A dose response was not observed.

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)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

In an in.vivo LLNA Study no skin sensitizing effect was observed.