Disodium 2-[4-[[2-cyano-3-[4-[methyl(2-sulphonatoethyl)amino]phenyl]-1-oxoallyl]amino]phenyl]-6-methylbenzothiazole-7-sulphonate

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:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: Envigo RMS B.V., Inc. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test and main study: 9 - 10 weeks
- Weight at study initiation: mean 20.3 +/- 1.4 g (at the start of the experiment)
- Housing: Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: ad libitum, tap water
- 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
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: 1% aqueous Pluronic

Concentration:

5, 10, 25 %

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 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 25% suspension in 1% aqueous Pluronic® (v/v). At a concentration of 10% and below, the test item could be dissolved in the vehicle. Grinding of the test item in a mortar was used 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, sonicating, warming to 37°C)
- Irritation: 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 25% 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 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. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (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. At the tested concentrations the animals neither showed any signs of local skin irritation nor systemic toxicity. Substance residuals were observed at the ears of the animals at every single time of assessment but not on days 5 and 6. Thus, the test item in the main study was assayed at 5, 10, and 25%. 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. Thus, the test item in the main study was assayed at 5, 10, and 25%. 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.

MAIN STUDY
TREATMENT
- Criteria used to consider a positive response:
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 HTdR 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.
A test item is regarded as a sensitiser 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 HTdR 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.

Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated 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 and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1 . However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item preparation
The test item was placed into a mortar on a tared balance and 1% aqueous Pluronic® (v/v) was gradually added whilst constantly grinding. The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied

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 5, 10, and 25% in 1% aqueous Pluronic® (v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface 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 78.3 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated HTdR
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). 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 ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY® DT, Schärfe System). The values obtained were taken down manually.

Determination of Ear Weights
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.

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 DecisionTree_2.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 program R Script Outlier.Rnw). Two outliers (DPM values for animal 1 and 19, respectively) were detected in both outlier tests but were not excluded from calculations since exclusion of the outlier values would not change the overall test result.
However, both biological and statistical significance were considered together

Parameter:

SI

Remarks on result:

other: In this study Stimulation Indices (S.I.) of 0.82, 1.11, and 1.03 were determined with the test item at concentrations of 5, 10 and 25 % (w/w) in PG, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM values per animal were 822.5, 675.3, 913.3, 846.7 % for control, 5, 10 and 25 % test item concentration.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity were observed during the study period. Possible redness of the ear skin could not be detected due to the colour of the test item.

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.

Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. 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.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase in ear weights was observed in all dose groups in comparison to the vehicle control group (p<0.05). 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A weight of evidence approach was performed to fulfill the data requirements of this endpoint. A combination of several in vitro methods and one in vivo study is described in the following section.

The objective was to assess the skin sensitizing potential of test substance. A combination of several in vitro methods addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

- protein reactivity (DPRA),

- activation of keratinocytes (LuSens), and

- activation of dendritic cells (h-CLAT).

DPRA: The reactivity of test substance towards synthetic cysteine (C)- or lysine (K)- containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in de-ionized water. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was assessed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. The following results were obtained in the DPRA: The test substance was solved in de-ionized water at a concentration of 100 mM. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides. The mean K-peptide depletion, caused by the test substance was determined to be 100.00%. The C-peptide depletion could not be determined due to interference using the standard analytical procedure. However, due to the unambiguous result of the assay, resulting already in the most stringent reactivity category considering the K-peptide reaction, only, no further analyses with the C-peptide were performed. As no C-peptide depletion could be determined, exact mean peptide depletion of C- and K peptide cannot be given. However, the mean peptide depletion must be ≥ 50%. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that test substance shows a high chemical reactivity in the DPRA under the test conditions chosen.

LuSens: The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by ATP assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test luciferase activity was measured after 48 hour exposure. In parallel an ATP assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments (4th and 5th) were performed. The 1st until 3rd experiment was performed using the MTT assay for cytotoxicity determination. The experiments were not evaluable due to an artificial effect noticed in the MTT assay, which is attributed to the test substance. Hence, these experiments are not included in this report but available with the raw data. The following results were observed: At concentrations used in the main experiment the test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations). Solubility in final concentrations and precipitates after 48 hours could not be assessed as opaque-walled 96-well plates had to be used for both test plates. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable. In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

h-CLAT: The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test after 24 hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid experiments were performed. The following results were observed: At concentrations used in the main experiment the test substance was soluble in culture medium (2 x stock preparations) and in 0.2% DMSO in culture medium (final concentrations).No precipitates were noticed in any concentration after 24 hours. The EC150 (the concentration resulting in a RFI of 150) for CD86 was calculated to be 16.7 μg/mL (experiment 2). Calculation of an EC 200 for CD54 (the concentration resulting in a RFI of 200) was not applicable as fold inductions above 200 were obtained in all tested concentrations. In summary, after 24 hours of exposure to test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance induces dendritic cell activation. Based on the results the evaluation criteria described in the test item is peptide reactive, does not activate keratinocytes and activates dendritic cells. Applying the evaluation criteria the test item is predicted to be a skin sensitizer based only on the in vitro tests.

LLNA

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item formulations at different concentrations were prepared in the vehicle 1% aqueous Pluronic® (v/v). The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could be technically achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

The animals neither showed any signs of systemic toxicity nor mortality during the course of the study. Possible redness of the ear skin could not be detected due to the colour of the test item. A statistically significant increase in ear weights was observed in all dose groups in comparison to the vehicle control group (p<0.05). 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, thus the statistically significant increased ear weights were considered not to be biologically relevant.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results 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 test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 0.82, 1.11, and 1.03 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in 1% aqueous Pluronic® (v/v), respectively. A dose response was not observed.

An outlier was identified in the vehicle control group and in the high dose group (DPM values determined for animal number 1 or 19, respectively). However, as exclusion of the outliers did not change the overall test result, the values in question were not excluded from calculation.

A statistically significant or biologically relevant increase in DPM values as well as for lymph node weights and –cell counts were not observed in any treated 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.

Conclusion: As shown in the summary of the in-vitro test battery 2 of 3 results were positive. The Direct Peptide Reactivity Assay (DPRA) and the Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT) were positive. The Keratinocyte Activation Assay (LuSens) was negative. The results determined in the in vitro test battery are not consistent with the fact that no structural alert for the substance for sensitizing potential was evident. Furthermore, taking into account the physicochemical properties of the substance (molecular weight > 500 Da (656.7 Da), water solubility > 10000 mg/L (250 g/L), log Pow < -1 (-4.02; estimated)) dermal uptake of the substance as a prerequisite for skin sensitisation is will be low. To clear this inconsistency a further in vivo study (LLNA) was performed. This in vivo test was negative. A statistically significant or biologically relevant increase in DPM values as well as for lymph node weights and –cell counts were not observed in any treated group in comparison to the vehicle control group. The in vivo animal study has a higher relevance than the in vitro studies. A lot of parameters play an important role in the process of sensitization especially in humans. These effects can be determined in a more realistic and adequately way in one in vivo study, which is closer to biological processes in humans than in vitro studies. Therefore the test substance is classified as non-skin sensitizer.

Migrated from Short description of key information:
A weight of evidence approach was performed with three in vitro skin sensitization methods (DPRA, LuSens and h-CLAT assay) and one in-vivo (LLNA) study. The DPRA and the h-CLAT assays showed positive effects. The LuSens assay showed negative effects. A further in-vivo study showed also no signs of sensitizing effects. The in-vivo study is closer to humans and the result is more reliable. Therefore the substance was classified as non skin sensitizer.

Justification for selection of skin sensitisation endpoint:
GLP and guideline study

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 (EC) No 1272/2008. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008.