3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

Endpoint summary

Description of key information

The following in vitro skin and eye irritation studies showed negative results:

Skin:

assessment of the skin irritancy of the test item Macrolex Fluoreszenzrot G with reconstructed human epidermis (RhE).

Eye:

ocular irritation properties of the test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM.

Evaluation of corrosive/severe irritant properties by using the Bovine Corneal Opacity and Permeability (BCOP) Test Method

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:

Batch number: CHA91249
Purity: 97.6 %

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

other: reconstructed human epidermis

Cell source:

other: reconstructed human epidermis

Source strain:

other: reconstructed human epidermis

Justification for test system used:

Human full thickness skin models and reconstituted epidermal equivalents are in vitro engineered tissue cultures that provide a three dimensional architecture which is biochemically, morphologically and functionally comparable to human epidermal tissue/skin in vivo. In contrast, organotypic skin explant systems are based on ex vivo skin removed from human or mouse and cultured in toto, afterwards. According to the literature all the models are useful in screening for topically applied irritant, corrosive or photocytotoxic substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

The model used for this study has a functional stratum corneum with an underlying layer of living cells as recommended by the test guidelines. The barrier function of the stratum corneum is adequate, as has been shown by the supplier.

The viability of the living cells in the model must be sufficiently high to discriminate well between the positive and negative control substances. Cell viability is measured by the amount of MTT reduction, i.e. an OD value, following exposure to the negative control substance or the test item.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

Three replicates of the epiCS inserts were exposed to the test item (30 mg), negative or positive control (each 30 µl). For the solid test item 30 µl of 0.9% NaCl were used to moisten and ensure good contact with the epidermis surface. A piece of nylon mesh was used as a spreading aid.

Duration of treatment / exposure:

The incubation of the treated epiCS inserts was carried out at RT for 20 min.

Duration of post-treatment incubation (if applicable):

After a post treatment incubation period of 42 h of the rinsed tissue in the incubator a MTT assay was performed

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 replicates

Value:

98.81

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item Macrolex Fluoreszenzrot G with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.

The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.

The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.

Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%.

The results of the concurrent negative control (NC, 0.9 % NaCl) and positive control (PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

The following value of cell viability was recorded for the test item: 99 % (rounded).

In conclusion the results of the assay used show no skin irritant properties of the test item Bayscript Magenta BB and thus, the test item requires no classification according to UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

Version / remarks:

Adopted 09 Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: CHA91249
Purity: 97.6 %

Species:

human

Strain:

other: three-dimensional human cornea model tissue model

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
In principle the EpiOcular™ eye irritation test (EIT) measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). This differentiation would need to be addressed by another tier of a test strategy. As a BCOP has already been conducted and Category 1 can be excluded based on the result, this test can be used to determine, if the substance needs to be classified (Category 2) or not.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium (Part#: OCL-200, OCL -212; Lot No.: 23755; Testing date: 06 Dec 2016).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µg

Duration of treatment / exposure:

6h

Duration of post- treatment incubation (in vitro):

18h

Number of animals or in vitro replicates:

each 2 tissues for the test item, positive and negative controls

Details on study design:

- RhCE tissue construct used, including batch number: The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Doses of test chemical and control substances used: 50 µg of the neat test item, 50 µL negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 37 ± 2°C; 6h exposure, 25 min. post-soak immersion, 18h post-treatment incubation

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Killed control and colour control not required.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): each 2 tissues for the test item, positive and negative controls

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 µL).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Positive and negative control means and acceptance ranges based on historical data: The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Irritation parameter:

other: mean percent tissue viability

Value:

95.5

Vehicle controls validity:

not examined

Remarks:

no vehicle used

Negative controls validity:

valid

Remarks:

100 % final cell viability

Positive controls validity:

valid

Remarks:

24.1 % final cell viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

GHS criteria not met

Remarks:

not irritant

Conclusions:

The study was conducted under GLP according to OECD guideline 439 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritation potential of the test item to the eye in vitro.
As the final test item-treated tissue viability was > 60% (76.2%) relative to negative control, the test item can be characterized as NOT having eye irritating properties.

Executive summary:

The model used is standardized and commercially available.

The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492.

The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period.

Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model.

The final mean percent tissue viability recorded for the test item is 96 % (rounded).

According to the results of this study the test item was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Reference 2

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:

Batch number: CHA91249
Purity: 97.6 %

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test system: isolated cornea from eyes of slaughtered cattle

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

750 µl of the test material formulations were applied to the corneas.

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

3

Details on study design:

In this test method, eye damaging properties of a test item is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a visible light spectrophotometer, respectively. Both measurements are used to calculate an IVIS, which is used to assign an in vitro irritancy hazard classification
category for prediction of the in vivo ocular irritation potential of a test item.

The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber of the corneal holder. Test items are applied to the epithelial surface ofthe cornea by addition to the anterior chamber of the corneal holder.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

13.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean

Value:

-0.002

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: IVIS (in vitro irritation score)

Run / experiment:

Mean

Value:

13.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye

Tabular In vitro irritancy score

	Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group; mean (SD)
Negative control isotonic saline solution	1 2 3	4.0 1.1 1.1	0.008 0.010 0.011	4.1 1.3 1.3 0.5	2.2 (1.6)
Positive Control 20% Imidazole	4 5 6	85.7 55.1 79.6	0.667 0.624 0.448	95.7 64.4 86.3	82.1 (16.0)
Test item 20 % Bayscript Magenta BB	10 11 12	30.5 1.9 8.2	-0.001 -0.002 -0.002	30.5 1.9 8.2	13.5 (15.0)

IVIS < 55: classification as Category 1 is not warranted

Interpretation of results:

GHS criteria not met

Executive summary:

Based on the experimental conditions reported in an OECD TG 437 study the test item is classified as not seriously damaging the eye (no Category 1).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification is warranted because the following in vitro skin and eye irritation studies showed negative results:

Skin:

assessment of the skin irritancy of the test item Macrolex Fluoreszenzrot G with reconstructed human epidermis (RhE).

Eye:

ocular irritation properties of the test item was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcularTM.

Evaluation of corrosive/severe irritant properties by using the Bovine Corneal Opacity and Permeability (BCOP) Test Method