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Description of key information

No irritating effects observed

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2015 to 29 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guidelines, coherence between data, results and conclusion.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Remarks:
reconstructed human epidermis
Source species:
human
Details on animal used as source of test system:
Test system: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon – France).
Batch numbers: Alive tissues: 15-EKIN-020 (Main Assay I), 15-EKIN-025 (Main Assay II).
Killed tissues: 15-EKIN-005 and 15-EKIN-006 (Main Assay I), 15-EKIN-020 (Main Assay II).
Vehicle:
unchanged (no vehicle)
Details on test system:
The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 +/- 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.


Positive control item: Sodium Dodecyl Sulphate (SDS) (SIGMA, batch no. 041M8713V), diluted at the final concentration of 5% (v/v) in sterile water for injection (Baxter, batch nos.14C2402 and 14C2403).

Negative control item: D-PBS (GIBCO, batch no. 1605086).

Non Specific Colour (NSC) control: test item treated tissues without MTT.

Non Specific MTT Reduction (NSMTT): killed tissues treated with the test item.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
20 mg/epidermis unit, each measuring 0.38 cm² (treatment level: 53 mg/cm²)
Duration of treatment / exposure:
An exposure time of 15 +/- 0.5 minutes was allowed in a ventilated cabinet at room temperature. In Main Assay I, treatment was carried out staggering samples of approximately 1 minute, the exposure time for test item treated samples was approximately 30 minutes, due to the adhesion of the substance to the test system. In Main Assay II, test item treatment was carried out staggering samples of approximately 2 minutes while control tissues were staggered of 1 +/- 0.5 minutes.
Observation time was 42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean cell viability
Value:
86.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks on result:
other:
Other effects / acceptance of results:
In Main Assay I, the baseline value of the negative control was slightly lower than the acceptable one (mean OD value = 0.53313), hence a
second experiment (Main Assay II) was performed. However, result obtained for the non specific colour (NSC=3.0%) was considered reliable from Main Assay I for demonstrating the absence of colour interaction; this additional control was not repeated in Main Assay II.

In Main Assay II, the non specific MTT reduction (NSMTT) induced by the test item was 2.7%, thus only the OD-blank background subtraction was performed for the evaluation of irritant properties of Acid Red EAY 9656.

The test item did not induce cell death in any replicate with a mean cell viability of 86.6%, when compared to the negative control.
Acceptable intra-replicate variability was obtained (SD of % variability lower than 18).

Preliminary test

A reddish suspension was observed, with plentiful precipitate, which indicates a colouring capacity of the test item. For this reason, additional controls were added in the Main Assay for the evaluation of non specific colouring potential (NSC).

Main Assay I

The Non Specific Colour (NSC) was 3.0%, while the non specific MTT reduction (NSMTT) induced by the test item was 6.0%. Based on this result, appropriate background subtraction was carried out for the evaluation of test item mean cell viability. Using alive tissues, a mean cell viability of 89.0%, when compared to the negative control, was obtained after treatment with the test item.

Positive control results indicated appropriate relative cell viability (5.6 % of the negative control value) and variability between replicates (SD of % viability = 1.7). However, the baseline value of the negative control was slightly lower than the acceptable one (mean OD value = 0.53313), hence a second experiment was performed.

Since low values were obtained for the Non Specific Colour (NSC), results were considered reliable for demonstrating the absence of colour interaction and this additional control was not repeated in the second experiment. Relevant variability between replicates was noted in NSMTT determination, hence this additional control was repeated in Main Assay II.

 

Main Assay II

Using alive tissues, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications. According to the method, the mean negative control value is considered the baseline value of the experiment and thus represents 100% of cell viability.

Positive control results indicated an appropriate cell death with an acceptable relative cell viability (2.8 % of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 0.1). Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.

The Non Specific Colour (NSC), relative to the negative control and obtained in Main Assay I, was 3.0%, while the non specific MTT reduction (NSMTT) induced by the test item was 2.7%. Based on these results, only the OD-blank background subtraction was performed for the evaluation of test item mean cell viability.

The test item did not induced any relevant reduction of MTT staining in any replicate. The mean cell viability was 86.6%, when compared to the negative control. Acceptable intrareplicate variability was obtained (SD of % viability = 5.2 lower than 18).

Interpretation of results:
not irritating
Conclusions:
The test item Acid Red EAY 9656 is classified as not irritant to the skin.
Executive summary:

The potential of the test item Acid Red EAY 9656 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

Before the Main Assays, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se; a red coloured suspension, with dark red precipitate, was observed at the end of the incubation period, indicating that the test item could interact directly with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a reddish suspension, with plentiful precipitate, was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were added.

Two experiments were performed. In both experiments, the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2). Positive and negative controls [a 5% (v/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSC) and the non specific MTT reduction (NSMTT) were also evaluated.

In Main Assay I, the negative control did not give the acceptable baseline value, thus a second experiment was performed. However, result obtained for the non specific colour (NSC=3.0%) was considered reliable for demonstrating the absence of colour interaction and this additional control was not repeated in the second experiment.

In Main Assay II, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (2.8% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.1). Based on the stated criteria (mean viability 40% and SD of % viability 18 ), the assay was regarded as valid. The non specific MTT reduction (NSMTT) induced by the test item was 2.7%, thus only the OD-blank background subtraction was performed for the evaluation of irritant properties of Acid Red EAY 9656.

The test item did not induce cell death in any replicate with a mean cell viability of 86.6%, when compared to the negative control. Based on the results obtained, the test item Acid Red EAY 9656 is classified as not irritant to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February 2015 to 10 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Compliant to GLP and testing guidelines; coherence between data, results and conclusion.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD series on testing and assesment no. 160
Qualifier:
according to guideline
Guideline:
other: Use of an alternate testing framework for classification of eye irritation potential of EPA pesticide products - Office of Pesticide Programs, U.S. Environmental Protection Agency (31 May 2013).
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Remarks:
bovine cornea
Details on test animals or tissues and environmental conditions:
Test system: isolated corneas from the eyes of freshly slaughtered bovine.
Age of animals: 6-12 months

Transport solution: Hanks balanced salt solution (HBSS) Modified supplemented with Penicillin sulphate and Streptomycin sulphate.
Transport conditions: in transport solution at approximately 4°C.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
The test item was spread on each epithelial surface of three idoneous bovine corneas as supplied (150 mg/cornea); 0.75 mL of physiological saline was then added on each cornea in order to better distribute the substance on the cornea surface (final concentration on cornea = 200 mg/mL, approximately 20% w/v)
Number of replicates: 3
Duration of treatment / exposure:
Corneas were exposed in horizontal position for 4 hours ± 10 minutes, incubated in a liquid bath at 32 ± 1°C.
There was no post-exposure period.
Details on study design:
Positive control item: corneas treated 20% (w/v) Imidazole (Sigma, batch no. 0001422794) in physiological saline (0.9% NaCl) (Baxter, batch no. 13D0406).
Negative control item: corneas treated with physiological saline (0.9% NaCl) (Baxter, batch no. 13D0406).
Volume of treatment: 0.75 mL
Number of replicates: 3
Irritation parameter:
cornea opacity score
Remarks:
mean score of three corneae
Value:
22.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Remarks:
mean permeability OD490 score
Value:
0.186
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
IVIS = mean opacity score + (15 x mean permeability OD490 score)
Value:
25.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean opacity detected with an opacitometer at the end of the test item exposure period was 22.7. This was confirmed at the macroscopic observation in which slight opacity of the treated corneas was noted. Plentiful residual test item, adhering on the surface of all treated corneas, was observed. Slight increases in cornea permeability were observed after treatment with the test item, with a mean permeability OD490 value of 0.1863.

The following results were obtained in the negative control:
– mean opacity value of the negative control = 0.00
– mean permeability value of the negative control = 0.0077

The following results were obtained in the positive control:
– IVISpositive control = 72.4

Based on the evaluation criteria the experiment was accepted as valid.

The following results were obtained:

– mean opacity value of the negative control = 0.00

– mean permeability value of the negative control = 0.0077

– IVISpositive control = 72.4

Based on the stated criteria (§ 4.6.2) the experiment was accepted as valid.

The calculated in vitro irritancy score for the test item was:

– IVIStest item = 25.5

indicating that the test item cannot be clearly classified.

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
The test item Acid Red EAY 9656 cannot be clearly classified.
Executive summary:

The potential of the test item, Acid Red EAY 9656, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on 26 July 2013), the Guidance Document OECD series on testing and assessment no. 160 and the method described in the document issued by U.S. EPA/OPP (31 May 2013). The test item was spread on each epithelial surface of three idoneous bovine corneas as supplied (150 mg/cornea); 0.75 mL of physiological saline was then added on each cornea in order to better distribute the substance on the cornea surface (final concentration on cornea = 200 mg/mL, approximately 20% w/v); an exposure period of 4 hours ± 10 minutes was used. The mean opacity detected with an opacitometer at the end of the test item exposure period was 22.7. This was confirmed at the macroscopic observation in which slight opacity of the treated corneas was noted. Plentiful residual test item, adhering on the surface of all treated corneas, was observed. After the determination of opacity, the epithelial surface was treated with a 0.5% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD490 value of the corneas treated with the test item was 0.1863. The calculated in vitro irritancy score (IVIS) for the test item was 25.5. Positive and negative controls [a 20 % (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar conditions and gave the expected results. According to the OECD Guideline no. 437, the test item can not be clearly classified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The potential of the test item Acid Red EAY 9656 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42±1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

Before the Main Assays, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se; a red coloured suspension, with dark red precipitate, was observed at the end of the incubation period, indicating that the test item could interact directly with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a reddish suspension, with plentiful precipitate, was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were added.

Two experiments were performed. In both experiments, the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2). Positive and negative controls [a 5% (v/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSC) and the non specific MTT reduction (NSMTT) were also evaluated.

In Main Assay I, the negative control did not give the acceptable baseline value, thus a second experiment was performed. However, result obtained for the non specific colour (NSC=3.0%) was considered reliable for demonstrating the absence of colour interaction and this additional control was not repeated in the second experiment.

In Main Assay II, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (2.8% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.1). Based on the stated criteria (mean viability40% and SD of % viability18 ), the assay was regarded as valid. The non specific MTT reduction (NSMTT) induced by the test item was 2.7%, thus only the OD-blank background subtraction was performed for the evaluation of irritant properties of Acid Red EAY 9656.

The test item did not induce cell death in any replicate with a mean cell viability of 86.6%, when compared to the negative control. Based on the results obtained, the test item Acid Red EAY 9656 is classified as not irritant to the skin.

The potential of the test item, Acid Red EAY 9656, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on 26 July 2013), the Guidance Document OECD series on testing and assessment no. 160 and the method described in the document issued by U.S. EPA/OPP (31 May 2013). The test item was spread on each epithelial surface of three idoneous bovine corneas as supplied (150 mg/cornea); 0.75 mL of physiological saline was then added on each cornea in order to better distribute the substance on the cornea surface (final concentration on cornea = 200 mg/mL, approximately 20% w/v); an exposure period of 4 hours ± 10 minutes was used. The mean opacity detected with an opacitometer at the end of the test item exposure period was 22.7. This was confirmed at the macroscopic observation in which slight opacity of the treated corneas was noted. Plentiful residual test item, adhering on the surface of all treated corneas, was observed. After the determination of opacity, the epithelial surface was treated with a 0.5% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD490 value of the corneas treated with the test item was 0.1863. The calculated in vitro irritancy score (IVIS) for the test item was 25.5. Positive and negative controls [a 20 % (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar conditions and gave the expected results. According to the OECD Guideline no. 437, the test item cannot be clearly classified.

Justification for classification or non-classification

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