Glycine, N-[5-(acetylamino)-4-[2-(2-bromo-4,6-dinitrophenyl)diazenyl]-2-methoxyphenyl]-N-(2-methoxy-2-oxoethyl)-, methyl ester

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 04 December2014 to 26 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

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
- Source: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
- Age at arrival: 7 to 8 weeks old
- Weight at arrival: 19 to 21 grams
- Housing: Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm with nesting material
- Animals per cage: 1/cage during the study; up to 5 during acclimatisation
- Diet (e.g. ad libitum): ad libitum4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C+/-2 °C
- Humidity (%): 55%+/-15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours

IN-LIFE DATES: From: 14 to 26 January 2015

Vehicle:

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

Concentration:

10%, 5% and 2.5% (Test Item); 25% (Positive control)

No. of animals per dose:

4 females/dose

Details on study design:

RANGE FINDING TEST:
- Irritation: Animals treated for three consecutive days (Days 1, 2, 3) with 25 µL/ear/day ofthe vehicle or test item formulations at 1, 2.5, 5, 10, 25%:
The treated sites of all animals were examined daily, ear thickness measured by a suitable micrometer on Day 1 (before dosing),
on Day 3 (before dosing) and on Day 6. After sacrifice, regularly shaped biopsies obtained from both ears and weighed together.

MAIN ASSAY:
- No. of exposures:3
- Test groups: 3 with test item, 1 with positive control
- Control groups: 1
- Site: Ears, 25 µL/ear/day
- Frequency of applications: once daily
- Duration: 3 days
- Concentrations: 2.5%, 5%, 10% (Test Item); 25% (Positive control)
- Day 5: intraperitoneal injection of 0.5 mL/animal of a solution of BrdU at a concentration of 10 mg/mL in physiological saline
- Day 6 : Sacrifice, the auricular lymph nodes were excised, pooled on individual basis and individually collected in a solution of 2 % BSA-PBS [2 % bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation .
BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001, batch no. 10493100).
Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).

The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated. Results for each treatment group were expressed as the mean Stimulation Index (SI). The SI was derived by dividing the mean BrdU labelling index/mouse within each test item
group and the positive control groups by the mean labelling indices for the respective vehicle group.

Positive control substance(s):

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

Statistics:

Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s
test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test
(Cochran and Cox) was applied.

Positive control results:

The Stimulation Index (SI) of the positive control group obtained in this study was 1.52 and not higher than 2.0 as indicated in the protocol. However, the appropriate performance of the assay by responding with adequate sensitivity was demonstrated on the basis of the results obtained with the test item. In addition, the positive control produced an acceptable positive responce in other studies performed in the same period.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

BrdU Labelling index/group (OD, Optical Density) Group 1: 0.241 Group 2: 0.883 Group 3: 1.371 Group 4: 1.461 Group 5 (Positive Control): 0.367 Increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with Stimulation Indices of 6.07, 5.69 and 3.67, respectively.

Preliminary test

Five concentrations (25, 10, 5, 2.5 and 1% w/w) were selected for the preliminary phase. The concentration of 25% was found to be not administrable due to the viscosity of the obtained formulation. No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations. The evaluation of visible reactions showed no erythema at any of the concentrations investigated (10, 5, 2.5 and 1% w/w). A wound, on Days 4 and 5, and a scab, on Day 6, were noted on the right ear of the control animal. The evaluation of ear thickness indicated that no increase was induced by treatment (values of Day 6 compared to Day 1). The evaluation of ear punch weight indicated that an increase, without any dose-relation, was observed in the animals treated at 10, 5 and 1% w/w, ranging from 21 to 71%, when compared to the animal treated with the vehicle. No increase was recorded in the animal treated at 2.5% w/w. In the ear punch weight a range of variations were noted, in some cases >25%, but without dose-relations and not in accordance with the other two parameters measured/observed during the in vivo activities (evaluation of erythema and ear thickness). Based on this rationale, the concentrations selected for the main assay were 10%, 5% and 2.5% w/w.

Main Assay

Neither mortality nor clinical signs were recorded in animals treated at all dose levels (10, 5 and 2.5% w/w). Body weight decreases/reduced body weight gains observed in some animals from all groups (including controls) were considered of low entity and/or incidental and thus not toxicologically relevant.

An increase in cell proliferation was observed in all test item treated groups in which the calculated Stimulation Indices (SI) were 6.07, 5.69 and 3.67, in the high, medium and low dose groups, indicating a dose-response relationship. In addition, Dunnett’s t test showed a statistically significant difference (p <0.01) between treated groups and negative control group.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Conclusions:

A dose-related increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with Stimulation Indices of 6.07, 5.69 and 3.67, respectively. Dunnett’s test showed a statistically significant difference (p < 0.01) between treated and negative control groups, indicating that the test item may elicit a sensitisation response.

Executive summary:

The potential of the test item, Disperse Blue ANT, to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b.

Preliminary phase

Five concentrations [25, 10, 5, 2.5 and 1% w/w in acetone:olive oil 4:1 (v/v)] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. Concentration of 25% w/w was found to be not administrable due to the high viscosity. No signs of toxicity (significant clinical signs or body weight losses) were observed at the administered concentrations. According to the results obtained in the preliminary phase, the concentration of 10% w/w was judged to be not irritant.

Main Assay

In the Main Assay, the test item was topically administered at the concentrations of 10, 5 and 2.5% w/w in acetone:olive oil 4:1 (v/v). No mortality nor clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. A dose-related increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with Stimulation Indices of 6.07, 5.69 and 3.67, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The potential of the test item, Disperse Blue ANT, to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b.

Preliminary phase

Five concentrations [25, 10, 5, 2.5 and 1% w/w in acetone:olive oil 4:1 (v/v)] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. Concentration of 25% w/w was found to be not administrable due to the high viscosity. No signs of toxicity (significant clinical signs or body weight losses) were observed at the administered concentrations. According to the results obtained in the preliminary phase, the concentration of 10% w/w was judged to be not irritant.

Main Assay

In the Main Assay, the test item was topically administered at the concentrations of 10, 5 and 2.5% w/w in acetone:olive oil 4:1 (v/v). No mortality nor clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. A dose-related increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with Stimulation Indices of 6.07, 5.69 and 3.67, respectively.

Migrated from Short description of key information:
The test item caused an increase of the stimulation index and is therefore considered to cause skin sensitisation