2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)

Endpoint summary

Description of key information

Skin irritation

The mean percent viability of the tissue treated with CR SR94 was 81.217% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence, under the conditions of the study, CR SR94 was found to be Non Irritant (OECD TG439).

 

Eye irritation

The percentage viability obtained was 149.334%. Therefore, CR SR94 was classified as No Category for eye irritation or serious eye damage (OECD TG492).

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:

From June 25, 2019 to July 11, 2019

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)

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: recoonstituted human epidermis model

Cell source:

other: Adult human-derived epidermal keratinocytes

Source strain:

other: Adult human

Details on animal used as source of test system:

EPISKIN kits are manufactured according to defined quality assurance procedures (certified ISO 9001).
(a) Biological safety aspects: All biological components of the epidermis and the kit culture medium were tested for the presence of viruses, bacteria and mycoplasma.
(b) Quality control aspects: The quality of the final product was assessed by an MTT cytotoxicity test with Sodium Dodecyl Sulphate (SDS) and by histological examination.

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Standard Model TM is a 0.38 cm^2 reconstituted human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1984). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was tested for its ability to direct formazan reduction. This pre-test was done on the same day of starting the main study.
-6 well of 12 well plate was filled with 2 mL of MTT solution (0.3 mg/L).
-To above 6 wells, 10uL of the test item was added to 3 wells and 10 µL of buffer to other 3 wells and mixed well.
-Incubated the mixture for 3 hours at 37°C protected from light.
Since, no change in the color (blue or purple) of MTT solution was observed, the test item was identified to have no interaction with MTT.

Duration of treatment / exposure:

Pre-Test
The test item was tested for its ability to direct formazan reduction. This pre-test was done on the same day of starting the main study.
- 6 well of 12 well plate was filled with 2 mL of MTT solution (0.3mg/L).
- To above 6 wells, 10uL of the test item was added to 3 wells and 10uL of buffer to other 3 wells and mixed well.
- Incubated the mixture for 3 hours at 37°C protected from light.
Since, no change in the color (blue or purple) of MTT solution was observed, the test item was identified to have no interaction with MTT.

Duration of post-treatment incubation (if applicable):

Post treatment incubation: 42 hours
Incubated the treated and rinsed epidermis at 37°C, 5% CO2, 95% humidified atmosphere for 42 hours.

MTT Test After the 42 hours Incubation Period
Required number of wells of the assay plate were filled with 2 mL per well of 0.3 mg/mL MTT in assay medium.
Treated units were transferred to the MTT filled wells. Plate was covered with lid and incubated for 3 hours at 37°C, 5% CO2, 95% humidified atmosphere.

2.3 Formazan Extraction (Day 3)
(a) 1.5 mL of conic "safelock" type micro tubes were labelled appropriately for test item, negative control, positive control and their replicate Number (1 tube/epidermis).
(b) Tissue units were placed on absorbent paper to dry the tissues.

(c) Using the special biopsy punch a total biopsy of the epidermis was done.
(d) Gently separated the epidermis from the collagen matrix with the aid of forceps, and both parts (epidermis and collagen matrix) were placed into the labelled micro tubes.
(e) When all the tissues were punched and ready in the micro tubes, 500 µL of acidic isopropanol per tube was added.
(f) Each tube was plugged to avoid evaporation and mixed thoroughly using a vortex mixer.
(g) All the tissues were correctly immersed in the solvent and extraction start time for each tissue was noted.
(h) The tubes were stored for 4 hours at room temperature (recommended 18 to 23°C) and protected from light.
(i) Vortex each tube at the middle of the incubation end time was noted.
(j) Tissues were removed from each tube extraction end time was noted.
(k) Extraction solution was homogenized by pipetting 3 times up and down.
(l) From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in using spectrophotometer at 560 nm. Acidified isopropanol solution was used as blank.

Number of replicates:

6

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Main Study
The assay medium supplied with the kits was kept at 2~8°C (refrigerator).
(a) Maintenance medium was pre-warmed to 37°C before using.
(b) EPISKIN kit was kept at room temperature in the Bio safety cabinet until next step.

Preparation and Pre-incubation (Day 0)
This step was conducted in a Biosafety cabinet
(a) 3 wells of a 12 well assay plates were filled with pre-warmed maintenance medium (2mL per well).
(b) EPISKIN kit was opened and epidermis units were transferred into maintenance medium filled wells, using sterile forceps avoiding air bubbles.
Labelled plate lids by replicate treatment order as follows:
Negative control Positive control Test item
Rep 1 (NCR1) Rep 1 (PCR1) Rep 1 (9243 R1)
Rep 2 (NCR2) Rep 2 (PCR2) Rep 2 (9243 R2)
Rep 3 (NCR3) Rep 3 (PCR3) Rep 3 (9243 R3)
Key: Rep: Replicate; NC: Negative control; PC: Positive control; 9243 = Study number for test item CR SR94

(c) Incubated at 37°C, 5% CO2, in a >95% humidified atmosphere for 24 hours.

Duration of treatment / exposure:

Application of test item and rinsing (Day 1)
(a) Pre-warmed maintenance medium (2 mL per well) was filled by taking into account of the 3 wells per replicate. Plate lids were labeled with test item (3 wells per test item), or negative control (3 wells) or positive control (3 wells).

Topical applications: 15 minutes treatment
(b) 5 µL of Distilled water was applied to the epidermal surface in order to improve further contact between the test item and epidermis.
(c) 10 µL of negative control, 10 mg of test item and 10 µL of positive control were applied on the top of the epidermis.
(d) Followed the defined application order (Rep 1, Rep 2 and Rep 3).
(e) After application, plate containing the treated epidermis was closed with lid and placed in the same biosafety cabinet for 15 minutes at room temperature.
Only for positive control, re-spread after 7 minutes contact time. And each tissue applications were done at 1 minute interval.

End of the treatment and removal of the test chemical: After 15 minutes exposure:
(f) Treated epidermal units were removed using forceps, and rinsed thoroughly with 25 mL sterile DPBS by filling and emptying the tissue inserts to remove residual material from the epidermal surface.
(g) Epidermal units were placed on an absorbent paper and remaining DPBS were removed from the epidermal surface by gently taping, and swept the epidermal surface with a cotton-bud without damaging the epidermis.
(h) Blotted tissue units were transferred to the new maintenance medium pre-filled wells.

Details on study design:

The assay medium supplied with the kits was kept at 2-8°C (refrigerator).
(a) Maintenance medium was pre-warmed to 37°C before using.
(b) EPISKIN kit was kept at room temperature in the Bio safety cabinet until next step.

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean

Value:

81.217

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

85.306

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

79.26

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

79.084

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results, the mean percent viability of the tissue treated with test item CR SR94 was 81.217% and it was greater than 50% of the negative control. Hence, under the conditions of the study the test item was found to be 'Non Irritant' to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemicals.

Executive summary:

Test item, CR SR94 was applied topically to the EPISKIM TM epidermal model (three epidermis units are used per each test item, positive and negative controls) for 15 minutes. Exposure to the test item was terminated by rinsing with Dulbecco's phosphate buffered saline (DPBS). Epidermis was then incubated at 37℃for 42 additional hours. The viability was assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/mL in assay medium; 2 mL per well). The precipitated formazan was then extracted using acidified isopropanol (0.5 mL) for 4 hours at room temperature and quantified spectrophotometrically at 560nm using 96 well plates (200µL/well). SDS5% and DPBS treated epidermis were used as positive (PC) and negative controls (NC) respectively. Each treated tissue % viability was obtained by comparing with the mean of the negative control tissues.

The negative controls mean Optical Density (OD) was found to be 0.852, which is well within the acceptability range. The mean percent viability of the tissue treated with positive control was 18.763% and it was less than 50% of the negative control, which reflects the irritant nature of positive control and the sensitivity of the tissues used in the study.

The mean percent viability of the tissue treated with test item was 81.217% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence, under the conditions of the studym, the test item was found to be Non Irritant.

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:

From August 16, 2018 to April 30, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Species:

other: stratified human keratino cytes

Details on test animals or tissues and environmental conditions:

Description of the test system
The EpiOcular TM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

Justification for selection of the test system
The EpiOcular TM Eye Irritation Test (EIT), using the MatTek EpiOcular TM tissue model OCL-200, was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular TM EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose, as an alternative to Dralze Rabbit Eye Test with excellent correlation - in vivo to in vitro test results.

Characterisation of the test system
MatTek’s EpiOcular TM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure with progressively stratified, but not cornified cells which closely parallel the corneal epithelium. QC results for the specific lot of models received were checked in-house for MatTek acceptance range with the following outcome:
Morphology – PASS
Tissue viability – PASS
Skin barrier function (ET50 value for 0.3% Triton X-100) where ET50 is the time taken for 0.3% Triton X-100 to reduce the viability of the skin model to 50% relative to the negative control) – PASS
Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Duration of treatment / exposure:

After pre-wetting tissues with 20uL PBS (sterile Dulbecco's Phosphate Buffered Saline) for 30±2 minutes, a single, topical application of approximately 50mg of neat test item using a spatula, or 50uL of reference items was applied to the surface of the Epiocular TM model for 6 hours ± 15 minutes, followed by a 25±2 minutes post-treatment immersion, and 18 hour ± 15 minutes post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n=3).

Details on study design:

Preliminary test:
CR SR94 was first checked for its potential for MTT interference and solvent interference (water and isopropanol).

Main test overview:
Day 0: On the day of receipt, EpiOcular TM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH.
Day 1: Exposure to, and removal of approximately 50 mg of CR SR94 (addition of test item was by spatula) or 50uL of reference item for 6 hours ±15 minutes, followed by a 25±2 minutes post-treatment immersion, and 18 hours ±15 minutes post-treatment incubation.
Day 2: End of MTT viability test, optical density readings at 570 nm without reference filter.

Irritation parameter:

other: %viability

Value:

149.334

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: Corrected %vialbility

Value:

149.331

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Prior to the study, the required preliminary test confirmed that the test item interfered with the solvent and so colourant controls were performed in parallel.
CR SR94 was classified as No category i.e. not requiring a warning label in the European chemical classification systems for eye irritation or serious eye damage as the viability was above 60%.

Table 1. Viability measurements after 6 hours (±15 minutes) of application and 18 hours (±15 minutes) post-incubation of test and reference items.

Condition	Tissue #	Raw Data		Blank Corrected Data		Mean OD	%Viability
		Aliquot 1	Aliquot 2	Aliquot 1	Aliquot 2
NC	Tissue 1	2.224	2.253	2.068	2.097	2.083	100.349
	Tissue 2	0.169	0.179	0.013	0.023	0.018	1.295
	Tissue 3	2.209	2.239	2.053	2.083	2.068	99.651
PC	Tissue 1	0.876	0.832	0.720	0.676	0.698	50.234
	Tissue 2	0.623	0.624	0.467	0.468	0.468	33.645
	Tissue 3	0.75	0.78	0.594	0.624	0.609	43.829
TA1	Tissue 1	1.335	1.343	1.179	1.187	1.183	85.139
	Tissue 2	2.143	2.118	1.987	1.962	1.975	142.101
	Tissue 3	2.31	2.353	2.154	2.197	2.176	156.567

Table 2. Mean and SD of viability measurements and of viability percentages after 6 hours (±15 minutes) of application and 18 hours (±15 minutes) post-incubation.

Name	Code	Mean of OD	SD of OD	Mean if viability (%)	Corrected Mean of viability(%)	SD of viability (%)	CV%	Classification
Sterile water	NC	2.075	0.01	100	N/A	0.494	0.494	No-Category
Methyl Acetate	PC	0.592	0.116	42.569	N/A	8.366	19.652	No Prediction
CR SR94	TA1	2.075	0.142	149.334	149.331	10.229	6.85	No-Category

Interpretation of results:

GHS criteria not met

Conclusions:

A test item is considered as " No Category", i.e. not requiring a warning label in the European chemical classification systems for eye irritation or serious eye damage (UN GHS No Category), if the eye model viability after exposure and post-treatment incubation is >60%. The percentage of viability obtained with CR SR94 was 149.331%, therefore it is classified as No Category.

Executive summary:

In this study, the eye irritation potential of CR SR94 was assessed in vitro according to OECD Test Guideline 492 (Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage).

 

After 6 hours ±15 minutes exposure on the surface of EpiOcular TM reconstructed ocular epithelium and 18 hours ±15 mins post-incubation time, the viability of the tissues was assessed and compared to a negative control.

 

The percentage viability obtained was 149.334% and therefore:

CR SR94 was classified as No Category i.e. not requiring a warning label in the European chemical classification systems for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available