Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Description of key information

The test item is not a skin sensitiser under the test conditions of the LLNA and guinea pig study.

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 2015-12-11 to 2016-01-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

updated 06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch no.: 13298465

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc.., the Netherlands
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 16.9 - 21.4 g
- Housing: Same experimental group were kept in one cage
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: No, only animals without any visible signs of illness were used for the study.

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

Vehicle:

dimethyl sulphoxide

Concentration:

5, 10, and 20%

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS: 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. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 20% once daily each on three consecutive days.
At the tested concentrations the animals neither showed any signs of local skin irritation nor systemic toxicity.

- Compound solubility: The test item could be dissolved in the vehicle.
- Systemic toxicity: Clinical signs (lsystemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully
- Ear thickness measurements: On day 3 and before sacrifice (day 6). After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Erythema scores: Clinical signs (local irritation at the application site) were recorded at least once daily. Especially the treatment sites were observed carefully

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
• 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.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 20% in DMSO. 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 19.7 μCi of 3H-methyl thymidine (equivalent to 78.6 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal, except for animal 16, for which only one lymph node could be harvested).
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 scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

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, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical program R Script STABW_mitStat.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers (performed with validated R Script).
However, both biological and statistical significance were considered together.

Positive control results:

For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response.
The cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1.
The result is within the historical positive control data.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle Control Group (DMSO)

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

20%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
A statistically significant or biologically relevant increase in lymph node weights or –cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold.

An outlier was identified in the mid group (DPM value determined for animal number 13). However, as exclusion of the outlier did not change the overall test result, the value in question was not excluded from calculation. For animal 16, only one lymph node could be harvested. Therefore, the values obtained from this animal for calculation of mean DPM, mean lymph node weight and –cell count were excluded.

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EC3 CALCULATION
The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. On day 6, at the preparation, scaly ears were observed.
Due to the colour of the test item no assessment of possible erythema was possible in all dose groups on day 3. On day 6, in all dose groups scaly ears were observed.

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 test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Key study

In a GLP and OECD 429 guideline compliant study, the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice.

The following test item concentrations of 5, 10, and 20% (w/w) were used.

The animals showed neither signs of systemic toxicity nor mortality during the course of the study. All animals of the high dose group showed a slight yellow coloration of the ears on day 2. Due to the colour of the test item no assessment of possible erythema was possible in all dose groups on day 3. On day 6, in all dose groups scaly ears were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group.

Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold.

Stimulation Indices (S.I.) of 1.0, 1.1, and 1.2 were determined with the test item at concentrations of 5, 10, and 20% (w/w) in DMSO, respectively.

A statistically significant and biologically relevant increase in radioactive disintegrations per minute (DPM) value and also in lymph node weight and cell count was not observed in any dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group.

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

Supporting study

The test substance was tested in a Guinea Pig Maximization Test (GPMT) according to the OECD TG 406 (Safepharm, 1989). The concentrations used for testing were selected on the basis of the results of a pretest.The induction phase comprises the intradermal and epicutaneous route. For the intradermal Induction, 10% (w/v) in distilled water, 10% (w/v) in Freund's Complete Adjuvant plus distilled water in the ratio 1:1 were applied. For the topical Induction, undiluted test substance were applied. In the challenge phase, 0.1 -0.2 mL of 75% (v/v) in distilled water or vehicle were applied to the test group (20 f) or the control group (10 f). No evidence of erythema was noted at the test substance and vehicle control sites of the test or control animals at the 24 and 48-hour observations. A yellow-coloured staining caused by the test substance was observed at the application site of each animal. Body weight gains of guinea pigs in the test group, between day 0 and day 24, were comparable to those observed in the control group animals over the same period. The test substance produced a 0% (0/20) sensitisation rate in this study and was classified as a non-sensitiser to guinea pig skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the test substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776