1,2-Benzisothiazole, 3-methyl-, 1,1-dioxide

Endpoint summary

Description of key information

The test substance was determined to be not irritating in the in vitro human skin model test with EPISKIN model.

In an Isolated Chicken Eye Test the test substance was determined to be neither corrosive nor non-irritating to the eye.

The test substance was determined to be neither corrosive nor non-irritating to the eye.

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:

2016-09-14 to 2016-09-16

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Lot: 216-106
- Expiry date: 2018-06-16
- Storage conditions: Keep at room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number: 16-EKIN-037
- Expiry date: 2016-09-19
- Date of initiation of testing: 2016-09-14

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL, 1 step
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control mean: 13 %, min.: 2 %, max.: 39 %

NUMBER OF REPLICATE TISSUES:
3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls and 2 replicates color controls were used.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item showed no direct interaction with MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 μL
- Concentration: 1x

POSITIVE CONTROL
- Amount applied: 10 μL
- Concentration: 5 %

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls and 2 replicates color controls were used.

Irritation / corrosion parameter:

% tissue viability

Value:

143

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

16

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: Non Specific Colour % (NSC %): 2.1

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method the test laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes, each calculated standard deviation value (SD) for the % viability was below 18.

Substance	Replicate	Optical Density (OD)	Viability (%)
Negative Control: 1x PBS	1	1.018	117
	2	0.823	95
	3	0.765	88
	mean	0.869	100
	standard deviation (SD)	15.22
Positive Control: SDS (5 % aq.)	1	0.167	19
	2	0.168	19
	3	0.072	8
	mean	0.136	16
	standard deviation (SD)	6.34
Test Item	1	1.355	156
	2	1.102	127
	3	1.281	147
	mean	1.246	143
	standard deviation (SD)	14.97

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be not irritating in the in vitro human skin model test with EPISKIN model.

Executive summary:

The reconstituted human epidermis in the EPISKIN model study according to OECD 439 was performed to assess the irritation potential of the test substance. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. As the test item has an intrinsic colour (tan), two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test substance did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 143 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin and required no classification.

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:

weight of evidence

Study period:

2017-01-18 to 2017-01-19

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

29 June 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Lot: 216-106
- Expiry date: 2018-06-16
- Storage conditions: Keep at room temperature

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) 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™ 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.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcular™ (OCL-200-EIT), Lot: 23759, Expiry date: 19 January 2017, Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma, resulting in "not detected". The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3 % (v/v) Triton X-100).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amountapplied: 50 mg

Duration of treatment / exposure:

6 hours (± 15 min) at 37 ± 1 °C

Duration of post- treatment incubation (in vitro):

Post-Soak: 25 ± 2 minute at room temperature
Post-treatment Incubation: 18 hours ± 15 minutes at 37 ± 1 °C

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT), Lot: 23759, Expiry date: 19 January 2017, Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Doses of test chemical and control substances used: see above
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: see above
- Description of any modifications to the test procedure: None
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The test item showed no direct interaction with MTT. Using of additional control was not necessary.
- Number of tissue replicates used per test chemical and controls: see above
- Wavelength used for quantifying MTT formazan, and measuring device: 96-well plate spectrophotometer at the wavelength of 570 nm
- Description of the method used to quantify MTT formazan: Absorbance / Optical Density of the samples
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. The test chemical is identified as requiring classification and labelling 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 % of the negative control. The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.
- Positive and negative control means and acceptance ranges based on historical data: The mean historical OD value of the negative control tissue was between 1.138 and 2.378. The mean historical OD value of the positive control tissue was between 0.064 and 0.5 with a viability of 3 to 22 %.
- Acceptable variability between tissue replicates for positive and negative controls: The mean OD value of the two negative control tissues should be between 0.8 and 2.5. The acceptable percentage viability for positive control (mean of two tissues) is for 30 minute exposure below 50 % of control viability and for 6 hours exposure below 50 % of control viability.
- Acceptable variability between tissue replicates for the test chemical: The difference of viability between the two relating tissues of a single chemical is < 20 % in the same run (for positive and negative control tissues and tissues of single chemicals).

Irritation parameter:

other: tissue viability [%]

Run / experiment:

mean

Value:

68

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

15 %

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values: The mean historical OD value of the negative control tissue was between 1.138 and 2.378. The mean historical OD value of the positive control tissue was between 0.064 and 0.5 with a viability of 3 to 22 %.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be not irritating to the eyes in the in vitro Reconstructed human Cornea-like Epithelium.

Executive summary:

The in vitro eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 was performed to assess the irritation potential of the test substance. Independent duplicate tissues of EpiOcular were exposed to either the test item, the negative control (deionised water, 50 μL) or the positive control (Methyl acetate, 50 μL) for 6 hours ± 15 minutes. 50 mg of the test item were dispensed directly onto duplicate EpiOcular tissue surface. The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 68 %). Positive (15 %) and negative (100 %) controls showed the expected cell viability values within acceptable limits. All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to eye.

 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-08-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Lot: 216-106
- Expiry date: 2018-06-16
- Storage conditions: Keep at room temperature

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: multiple
- Characteristics of donor animals: slaughter house chicken, no further characteristics were determined
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after sedation of the chickens. The heads were transported within 2 hours from collection. The ambient temperature was optimal (20.5 ºC) during the transport. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed. After removal eyes were inspected for cornea damage with fluorescein. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity.
- Time interval prior to initiating testing: used directly after arrival and preparation
- indication of any existing defects or lesions in ocular tissue samples: Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g

Positive control: 0.03 g

Saline control: 30 μL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

3 test item treated eyes
3 positive control eyes
1 negative control eye

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye.

EQUILIBRATION AND BASELINE RECORDINGS
Acclimatisation was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods. At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness, opacity, and fluorescein retention to serve as a baseline (t=0) for each individual eye.

NUMBER OF REPLICATES
3 test item treated eyes
3 positive control eyes
1 negative control eye

NEGATIVE CONTROL USED
saline solution (9 g/L saline)

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
The test substance and positive control substance were used undiluted and 0.03 g were applied. The negative control was done with 30 μL of saline solution (9 g/L). All substances were left on the respective eyes for 10 sec.

OBSERVATION PERIOD
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the eyes had been rinsed.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline, was repeated after each observation if substance (test item or positive control) was still present on cornea
- Indicate any deviation from test procedure in the Guideline: No.

METHODS FOR MEASURED ENDPOINTS:
A slit lamp microscope (Haag-Streit BQ 900) was used for evaluation of the eyes. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: The conclusion on eye irritancy was based on the OECD guideline on quantitative assessments for ICE and classified according to UN GHS.

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 75 min

Value:

8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2 %

Positive controls validity:

valid

Remarks:

31 %

Remarks on result:

not determinable

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 240 min

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2 %

Positive controls validity:

valid

Remarks:

38 %

Remarks on result:

not determinable

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

4.0 %

Remarks on result:

not determinable

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

3.0 %

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Test substance: None
- Positive control: Cornea opacity score 4 was observed in two eyes at 30 minutes after the post-treatment rinse.
- Negative control: None

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

Interpretation of results:

other:

Remarks:

not Category 1

Conclusions:

The test substance was determined to be not corrosive to the eye.

Executive summary:

An Isolated Chicken Eye Test (ICE) according to OECD 438 was conducted to evaluate the corrosion and irritation potential of the test substance to the eye. The undiluted test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. The test item and Imidazole (positive control) was ground before use in the study. The test item and positive control was applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. The test substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xII. For the test substance a cornea opacity score of 1.0, a fluorescein retention score of 1.0 and a corneal swelling of max. 10 % were detected. The values for the positive control were 4.0, 3.0 and 38 % respectively, while for negative control 0.0, 0.0 and 2 % were established. Due to these observations it has to be concluded according to the guideline OECD 438, that the test item is to be categorized as “No prediction can be made”. The overall in vitro classification is neither UN GHS Classification Category 1 nor No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The reconstituted human epidermis in the EPISKIN model study according to OECD 439 was performed to assess the irritation potential of the test substance. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. As the test item has an intrinsic colour (tan), two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test substance did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 143 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control.Therefore the test item was considered to be non-irritant to skin and required no classification.

 

Eye irritation

To determine the corrosion or irritation potential of the test substance for the eye a stepwise weight of evidence approach was done. An Isolated Chicken Eye Test according to OECD 438 was conducted resulting in no classification in Category 1. Therefore an eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 was conducted in accordance with the top down approach described in the draft guideline on IATA for eye hazard (version 2016 -12 -22).

 

An Isolated Chicken Eye Test (ICE) according to OECD 438 was conducted to evaluate the corrosion and irritation potential of the test substance to the eye. The undiluted test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. The test item and Imidazole (positive control) was ground before use in the study. The test item and positive control was applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. The test substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 3xII. For the test substance a cornea opacity score of 1.0, a fluorescein retention score of 1.0 and a corneal swelling of max. 10 % were detected. The values for the positive control were 4.0, 3.0 and 38 % respectively, while for negative control 0.0, 0.0 and 2 % were established. Due to these observations it has to be concluded according to the guideline OECD 438, that the test item is to be categorized as “No prediction can be made”. The overall in vitro classification is neither UN GHS Classification Category 1 nor No Category.

The in vitro eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 was performed to assess the irritation potential of the test substance. Independent duplicate tissues of EpiOcular were exposed to either the test item, the negative control (deionised water, 50 μL) or the positive control (Methyl acetate, 50 μL) for 6 hours ± 15 minutes. 50 mg of the test item were dispensed directly onto duplicate EpiOcular tissue surface. The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 68 %). Positive (15 %) and negative (100 %) controls showed the expected cell viability values within acceptable limits. All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to eye.

 

Conclusion

Based on the result from the Isolated Chicken Eye Test according to OECD 438 a categorization into Category 1 was excluded. The eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 determined the test item to be non-irritant to eye. Using the top down approach described in the draft guideline on IATA for eye hazard (version 2016 -12 -22) an overall conclusion for the test item can be reached. The weight of evidence with the two conducted studies determined that the test substance is not irritating to the eyes.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on available data on skin irritation/corrosion, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.

Based on available data on eye irritation/corrosion, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.