Reaction products of Hydrogen amino-4-hydroxynaphthalene-2-sulfonate, Potassium substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate and Sodium substituted-({4-[(2-chloroethyl)sulfonyl]butanoyl}amino)benzenesulfonate

Endpoint summary

Description of key information

FAT 40875/A was found to be non-irritant for skin but may cause serious damage to eyes.

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:

2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: 40875/A TE
Physical Appearance: Dark red powder
Purity as per Certificate of Analysis (Content): 86.4% all organic components
Batch No.: BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured Date: 20 December 2017
Expiry Date: 11 December, 2022
Recommended Storage Condition : Refrigeration (+2 to +8 °C), Protect from light
pH: 4.3 (aq. soln.(2% (w/w) at room-temperature)

Test system:

human skin model

Source species:

human

Cell type:

other: normal human keratinocytes

Justification for test system used:

The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals. One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 20 - 28 °C
- Temperature of post-treatment incubation: 37 ± 1 °C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 42h
- Spectrophotometer: TECAN Infinite® M200 Microplate Reader
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES: Triplicate

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg/tissue of test substance was applied topically to the EpiSkin three dimensional RhE model.

Negative control : 10 microliter of Phosphated Buffered Saline (PBS).
Positive control : 10 microliter of 5% Sodium Dodecyl Sulfate (SDS).

Duration of treatment / exposure:

Incubation for 15 minutes at room temperature (20 - 28 °C)

Duration of post-treatment incubation (if applicable):

Tissues were incubated with MTT solution for 4 hours and then extracted with acidified isopropanol to quantify spectrophotometrically at wavelength of 570 nm.

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

True tissue viability

Value:

131.23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 0.753% mean cell viability.
- Acceptance criteria met for positive control: 6.89% mean cell viability.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation was within the range.

Individual tissue viability of epidermis units (relative)

	% Individual Viability
	R1	R2	R3	Mean	SD
Positive control	6.63	7.40	6.64	6.89	0.44
Test item	88.07	88.30	91.89	89.42	2.14
% NSC living tissue	4.13	4.30	-	4.22	0.12
% NSMTT killed tissue	4.86	6.61	-	5.74	0.24
% NSC killed tissue color	51.21	52.32	-	51.77	0.78

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative tissue viability for the test item, FAT 40875/A TE was 131.23 % and hence, the test item is predicted to be non-irritant.

Executive summary:

The test item, FAT 40875/A TE was tested for its skin irritation potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. This test was conducted in accordance with OECD test guideline 439 in a GLP certified laboratory.

 

10 mg/tissue of test item, 10µL of negative control (PBS) & 10µL of positive control (5% of SDS) respectively was exposed for 15 minutes.

In the preliminary tests, FAT 40875 was identified as producing direct MTT reduction and color interference and thus adapted controls evaluation was performed.

After approximately 42-hour post incubation period, irritation potential was evaluated by assessing the cytotoxic (irritancy) effect. The cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured on formazan production from MTT and the end of treatment.

Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 mins of treatment with the test item is compared with negative control tissues.

The absolute mean Optical density (OD) at 570nm of negative control, positive control and test item was 0.7530, 0.0519 and 0.6733 respectively. The positive control had a mean cell viability of 6.89% after 15 mins exposure, indicating the test system functioned properly.

The true tissue viability of the test item was found to be 131.23%.

The study indicated that the test item FAT 40875/A TE is predicted to be non-irritating in this In-vitro skin irritation test using Reconstructed Human Epidermis under the condition of testing employed.

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: FAT 40875/A TE
Physical Appearance: Dark red powder
Purity as per Certificate of Analysis (Content): 86.4% all organic components
Batch No.: BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured Date: December, 20, 2017
Expiry Date: December 11, 2022
Recommended Storage Condition : Refrigeration (+2 to +8°C)
pH: 4.3 of 2% (w/w) aq. Solution.
Solubility: 499.9 g/l

Species:

other: Bovine eye

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES

- Source: Local abattoir (Slaughter house), Near Frazer town, Bengaluru.
- Storage, temperature and transport conditions of ocular tissue: The bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillinstreptomycin (100 IU & 100 μg/mL) in an ice box.
- indication of any existing defects or lesions in ocular tissue samples: none

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

Amount / concentration applied:

100 mg of test item per cornea

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

10 min

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Pretest examination:
Immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches.

Dissection:
Post pretest examination, selected eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container with fresh HBSS (Hank’s Balanced Salt Solution).

Mounting:
The isolated corneas were mounted on the cornea holder. During mounting, it was ensured that the epithelium of the cornea projected into the anterior chamber. The EMEM without phenol red was added in to both the chambers and kept in an incubator (32°C) for 1 hour.
The blank value I0 (lux) was obtained with each of 30 available cornea holders containing medium but without cornea. The mean of the blank value obtained was 502 lux (I0). This value was considered while calculating the initial opacity and final opacity readings.

After pre-test incubation, the EMEM from both the chambers was removed and refilled with the fresh EMEM. The pre-treatment cornea reading (lux) was measured and initial opacity was calculated for all corneas.
The initial opacity was calculated. The corneas which exhibited initial opacity < 7 were considered for further experiment.

NUMBER OF REPLICATES : Three for each test group.

NEGATIVE CONTROL : Distilled water

POSITIVE CONTROL : Imidazole

Irritation parameter:

in vitro irritation score

Value:

680.328

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Negative Control: Distilled water
The corneas treated with distilled water exhibited IVIS score of 1.234 hence classified as “No Category”.

Positive Control: Imidazole
The corneas treated with Imidazole exhibited IVIS score of 78.2905 hence classified under “Category 1”.

Test Item: FAT 40875/A TE
The corneas treated with FAT 40875/A TE exhibited IVIS score of 680.3285 hence classified under “Category 1”.

Calculation of In Vitro Irritation Score (IVIS*)

Treatment group	Mean opacity value	Mean Permeability value	IVIS	Classification
Negative control (Distilled water)	1	0.0156	1.234	No category
Imidazole	64.33	0.9307	78.2905	Category 1
FAT 40875/A TE	680	0.0219	680.3285	Category 1

*IVIS = Mean Opacity value + (15 × Mean Permeability value)

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item FAT 41047/A TE exhibited an IVIS score of 680.3285. The test item FAT 40875/A TE is predicted to cause serious eye damage.

Executive summary:

The evaluation of the occular irritancy of “FAT 40875/A TE” was carried out using the Bovine Corneal Opacity and Permeability assay (BCOP) in accordance with OECD TG 437 in a GLP certified laboratory.

100 mg of test substance per cornea, 0.75 mL negative control per cornea and positive control (enough quantity covering the entire corneal surface) respectively were applied undiluted on the corneal surface by means of open chamber method. The control and treated corneas were then subjected to the opacity and permeability measurements.

The negative control (distilled water) was classified as UN GHS "No Category" (IVIS score 1.234) and the positive control (Imidazole) was classified under Category 1 (IVIS score 78.2905).

The test item FAT 40875/A TE exhibited an IVIS score of 680.3285 and was classified under ‘Category 1’ as per UN GHS.

From the results, it can be concluded that the test item FAT 40875/A TE can be predicted to cause serious eye damage to bovine corneal eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item, FAT 40875/A TE was tested for its skin irritation potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. This test was conducted in accordance with OECD test guideline 439 in a GLP certified laboratory.

The study indicated that the test item FAT 40875/A TE is predicted to be non-irritating in this In-vitro skin irritation test using Reconstructed Human Epidermis under the condition of testing employed.

 

The evaluation of the ocular irritancy of “FAT 40875/A TE” was carried out using the Bovine Corneal Opacity and Permeability assay (BCOP) in accordance with OECD TG 437 in a GLP certified laboratory.

The negative control (distilled water) was classified as UN GHS "No Category" (IVIS score 1.234) and the positive control (Imidazole) was classified under Category 1 (IVIS score 78.2905). The test item FAT 40875/A TE exhibited an IVIS score of 680.3285.

From the results, it can be concluded that the test item FAT 40875/A TE can be predicted to cause serious eye damage to bovine corneal eye.

Justification for classification or non-classification

Skin: The mean relative tissue viability for the test item, FAT 40875/A TE was 131.23 % and hence, the test item is was classified as non-irritant to the skin.

Eye: The test item FAT 40875/A TE exhibited an IVIS score of 680.3285 and was classified under ‘Category 1’ as per UN GHS.