Reaction products of tetrazotized 5-amino-2-[(E)-2-(4-amino-2-sulfophenyl)ethenyl]benzenesulfonic acid with 6-amino-4-hydroxynaphthalene-2-sulfonic acid and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid in 2-[bis(2-hydroxyethyl)amino]ethanol

Endpoint summary

Description of key information

skin irritation in vitro (human epidermis model test): not irritating

eye irritation in vitro (human cornea epithelium test): not irritating

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:

Nov 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2015)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test system epiCS is commercially available and the laboratory has been validated with this test system in a multicenter validation study.

Vehicle:

unchanged (no vehicle)

Details on test system:

DETAILS OF THE TEST PROCEDURE USED
The irritant potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item. All tests were performed in triplets for each time point. The test item was applied unchanged, i.e. 30 mg per insert for 20 min. (room temperature), plus 30 µL saline to moisten and ensure good contact to the tissue surface. After the exposure a post-treatment incubation period of 42 h of the rinsed tissue in the incubator followed. Then, cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density compared to negative control).

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): 37 ± 2° C (incubator temperature)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Incubation conditions: 37 ± 2° C, 5 % CO2, maximum humidity
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 for test substance, positive and negative control (for determination of cell viability the absorption of the isopropanol-extracts were measured in duplicates = 6 OD values for each)

DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- Classification according to UN GHS: The test substance is considered to be at least irritant to skin if the viability after 20 minutes exposure is less than or equal to 50% (Category 2 or 1; depending on outcome of corosivity test). The test substance is considered to be non-irritant to skin if the viability after 20 minutes exposure is greater than 50% .

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 mg per insert

Duration of treatment / exposure:

20 min. (room temperature)

Duration of post-treatment incubation (if applicable):

42 hours (37 °C)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

91.75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- DEMONSTRATION OF TECHNICAL PROFICIENCY:
The reliability of the test conduction was previously confirmed in an interlaboratory validation study.

Summary of results

Sample No.	Test item	OD mean*	StdDev	% Viability
1 -3	Negative control (NaCl 0.9 %)	2.33	0.04	100.00
4 -6	Positive control (SDS 5 %)	0.03	0.00	1.09
10 -12	Test item, undiluted	2.14	0.11	91.75

*: 6 values

Conclusions:

No irritant potential to the skin conlcuded when tested in an in vitro test according to OECD TG 439.

Executive summary:

An in vitro study was performed for the assessment of skin irritation on a reconstructed human epidermis (RhE; epiCS®). The experiment was carried out in accordance to OECD TG 439 with the neat substance. 30 mg of the test item was applied topically on the RhE, plus 30 µL saline to moisten and ensure good contact with the tissue surface. After an exposure period of 20 minutes (room temperature) and a post-exposure incubation of 42 hours (37 °C, 5 % CO2, maximum humidity), the cell viability was 91.8 %, as measured by a MTT conversion assay. The test item was thus not considered to have an irritant potential 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:

weight of evidence

Study period:

Jan 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Test system HCE: Assessment of the ocular irritation potential by determination of the cytotoxic effect of a test item on the human corneal epithelium model (exposure 60 min./rt followed by 16 hours incubation at 37 °C, subsequently MTT).

The HCE model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for severe eye damage/eye irritancy (e.g. Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537), and is routinely used by cosmetic and pharmaceutical companies. It has been prevalidated (van Goethem et. al., Tox in Vitro 20, 2006, 1-17; Alépée et al., Tox in Vitro 27, 2013, 1476-1488) and has entered formal ECVAM validation in 2010. Although a high reproducibility was attested within the validation process, a further need for optimization was identified. A recently conducted multi-laboratory validation study again demonstrated that the assay in general is a promising tool that may be used in combination with others for a solid eye irritation risk assessment (Alépée et al., Toxicol in Vitro 31, 2016, 43–53).

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per insert

Duration of treatment / exposure:

60 min. (room temperature)

Duration of post- treatment incubation (in vitro):

16 hours (37°C, 5% CO2, maximum humidity)

Number of animals or in vitro replicates:

3 inserts

Details on study design:

DETAILS OF THE TEST PROCEDURE USED
The irritation potential of the test item is assessed by determination of its cytotoxic effect on a reconstructed human ocular epithelium. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
The test substance was applied unchanged, i.e. 30 mg per insert (plus 30 µL PBS to moisten and ensure good contact with the tissue surface) for 60 min at room temperature. After the exposure period the inserts were washed carefully with PBS. MTT reduction was performed after a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity). Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item.

NUMBER OF TISSUE REPLICATES
Tests were performed in triplets for test substance, positive and negative control.

DECISION CRITERIA:
A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Irritation parameter:

other: cell viability (%)

Run / experiment:

decision criteria: ocular irritant if cell viability

Value:

93.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Summary of results:

Sample No.	Test item	%Viability (mean)
1 -3	Negative control (PBS)	100.00
4 -6	Positive control (SDS 0.3%)	27.66
13 -15	test substance	93.41

Conclusions:

No indication for an eye irritant potential was seen in an in vitro test (human coreneal epithelial model; cp. Cotovio et al., Toxicol. in Vitro 24, 523-537, 2010).

Executive summary:

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial cell model (HCE; Episkin, France). This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol. in Vitro 24, 523 -537, 2010), and is routinely used by cosmetic and pharmaceutical companies.

In this study 30 mg of this test item was applied topically to the reconstructed HCE tissue (plus 30 µL PBS to moisten and ensure good contact with the skin). After an exposure period of 60 minutes (room temperature), followed by a 16 hours post-treatment incubation period (37 °C, 5 % CO2, maximum humidity), the cell viability was 93.4 %, as measured by a MTT conversion assay. Based on this assay no potential for eye irritation was concluded.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro study was performed for the assessment of skin irritation on a reconstructed human epidermis (RhE; epiCS®). The experiment was carried out in accordance to OECD TG 439 with the neat substance. 30 mg of the test item was applied topically on the RhE, plus 30 µL saline to moisten and ensure good contact with the tissue surface. After an exposure period of 20 minutes (room temperature) and a post-exposure incubation of 42 hours (37 °C, 5 % CO2, maximum humidity), the cell viability was 91.8 %, as measured by a MTT conversion assay. The test item was thus not considered to have an irritant potential to the skin.

Further available is an in vitro study for assessing ocular irritation. This study was conducted in a human corneal epithelial cell model (HCE; Episkin, France). The model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol. in Vitro, 24, 2010, 523 -537), and is routinely used by cosmetic and pharmaceutical companies.

In this study 30 mg of this test item was applied topically to the reconstructed HCE tissue (plus 30 µL PBS to moisten and ensure good contact with the skin). After an exposure period of 60 minutes (room temperature), followed by a 16 hours post-treatment incubation period (37 °C, 5 % CO2, maximum humidity), the cell viability was 93.4 %, as measured by a MTT conversion assay. Based on this assay no potential for eye irritation was concluded.

Justification for classification or non-classification

No classification concluded for Skin Irritation/Corrosion or Damage to the Eye according to Regulation (EC) No 1272/2008, Annex I.