3-tert-butyladipic acid

Endpoint summary

Description of key information

Skin

In an in vitro skin irritation study (OECD 439) a mean tissue viability of 72 % was determined.

In an in vitro skin corrosion study (OECD 431) a mean tissue viability of 101 % after 4 h exposure to the test substance was determined.

Eye

In an in vitro eye irritation study (OECD 438) the test substance overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, it has been categorized as “No prediction can be made”.

In an in vitro Reconstructed Human Cornea like Epithelium (RhCE) eye irritation study (OECD 492) the tissue viability was ≤ 60% and, thus, no prediction can be made for the test item.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-07 to 2017-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU method B.40 Bis: In Vitro Skin Corrosion: Human Skin Model Test

Version / remarks:

28 April 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test”

Version / remarks:

February 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20170601
- Expiration date of the batch: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Justification for test system used:

This method is approved by international regulatory agencies as a replacement for the identification of corrosives in the in vivo Rabbit skin assay (OECD 404).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM (Supplier: SKINETHIC Laboratories)
- Tissue batch number: 17-EKIN-049

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL PBS 1 x solution
- 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 hours
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0.2136 – 3.1752

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposure OR
the mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure
- The test substance is considered to be non-corrosive to skin if the mean tissue viability is ≥ 35 % after 4 hours exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 mg

NEGATIVE CONTROL
- Amount applied: 50 µL
- Concentration (if solution): 9 g/L

POSITIVE CONTROL
- Amount applied: 50 µL (undiluted)

Duration of treatment / exposure:

4 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

103

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

99

Vehicle controls validity:

not examined

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:
No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

- Colour interference with MTT:
The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissues and lead to false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD value of the two negative control tissues was 0.713.
- Acceptance criteria met for positive control: Yes, the positive control result showed 3 % viability.
- Acceptance criteria met for variability between replicate measurements: Yes, the difference of viability between the two tissue replicates was between 2 % to 14 %.

Interpretation of results:

other: non-corrosive

Conclusions:

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.

Executive summary:

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro in accordance with OECD guideline 431 (TOXI-COOP, 2018).

Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1 °C in 5±1 % CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal to 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 101 %.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-07 to 2018-03-09

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 April 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

February 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20170601
- Expiration date of the batch: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

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 (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin (TM) SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model
- Tissue batch number(s): 17-EKIN-035
- Expiry date: 04 September 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume washing steps: 25 mL PBS 1 x solution
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

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 corrosive to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq. solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42±1 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

66

Vehicle controls validity:

not examined

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

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

71

Vehicle controls validity:

not examined

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: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is white and therefore considered to be not able to significantly stain the tissues and lead to a false estimate of viability. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD value of the three negative control tissues was 0.911.
- Acceptance criteria met for positive control: Yes, the mean OD value obtained for the positive control was 0.062 and this result corresponds to 7 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: Each calculated standard deviation value (SD) for the % viability was below 18.

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions.

Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro in accordance with OECD guideline 439 (TOXI-COOP, 2018).

Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) 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±1°C for 42 hours (± 1h) in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

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 chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item 3-Tert Butyladipic Acid did not show significantly reduced cell viability in comparison to the negative control (mean viability: 72 %). 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.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

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:

2018-03-12 to 2018-03-15

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

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals, for use with MatTek Corporation's Reconstructed Human EpiOcular™ Model

Version / remarks:

14. July 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20170601
- Expiration date of the batch: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

Species:

other: reconstructed human cornea-like epithelium

Details on test animals or tissues and environmental conditions:

Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Batch No.: 27027
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 ± 5 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used

Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.

Exposition and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, the controls and a defined amount of the test item were applied in duplicate in 1-min- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

- RhCE tissue construct used, including batch number

Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27027
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: 50 mg (test substance), 50 µL (controls)
- Duration and temperature of exposure; post-exposure incubation periods: 6 hours at 37 ± 1 °C; 25 min at room temperature + 18 hours at 37 ± 1 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary. Furthermore, no colour development was visible. Therefore, the main test was performed without colourant controls.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 3 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a Microtiter plate photometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is: < 50 % of negative control
3. Acceptable variability between tissue replicates: < 20 %

Irritation parameter:

other: % viability

Run / experiment:

Tissue 1

Value:

1.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: % viability

Run / experiment:

Tissue 2

Value:

1.9

Vehicle controls validity:

not examined

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: No
- Colour interfrence with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD = 1.6
- Acceptance criteria met for positive control: Yes, % mean relative viability of positive control was 38.5 %
- Variation within replicates:
2.9 % (negative control)
4.9 % (positive control)
0.0 % (test item)

Interpretation of results:

other: No prediction can be made.

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was ≤ 60%. Thus, no prediction can be made.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model according to OECD guideline 492 (LAUS, 2018).

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours. 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 25 minutes and further 18 hours with fresh medium.

Following treatment with the test item, the tissue viability was ≤ 60% and, thus, according to OECD 492 no prediction can be made for the test item identified. The test substance either needs to be classified in cat 1 or cat 2 with respect to eye irritation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-12-12

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:

09 October 2017

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:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20170601
- Expiration date of the batch: June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse, TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129., Hungary
- Characteristics of donor animals (e.g. age, sex, weight):
- Storage, temperature and transport conditions of ocular tissue: Heads were provided within 2 hours from collection at ambient temperature (19.4-20.3 ºC). After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline. Then they were placed in a plastic box that can be closed (4-5 heads/box).
- indication of any existing defects or lesions in ocular tissue samples: No.
- Indication of any antibiotics used: No.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g

Duration of treatment / exposure:

10 sec

Number of animals or in vitro replicates:

3

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 carefully 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 eyeball was carefully removed from the orbit by holding the nictitating membrane with 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
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 2%) were observed in the eyes. However, this finding is considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

OBSERVATION PERIOD
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.
The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole and test item treated eyes after 30, 75, 120 and 180 minutes of observation. The cornea surface of Imidazole and test item treated eyes were not totally cleared at 240 minutes after the post-treatment rinse.

METHODS FOR MEASUREMENT:
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

SCORING SYSTEM: Please refer to "Any other information on materials and methods incl. tables"

DECISION CRITERIA: The decision criteria as indicated in the TG was used. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an Isolated Chicken Eye (ICE) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

Irritation parameter:

percent corneal swelling

Remarks:

Mean

Run / experiment:

at up to 75 min

Value:

7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Remarks:

Mean

Run / experiment:

at up to 240 min

Value:

7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Remarks:

Mean

Value:

2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Remarks:

Mean

Value:

3

Vehicle controls validity:

not examined

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: None.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
Positive and negative control values were within the corresponding historical control data ranges.

Table 1 test substance results and interpretation

Observation	Value		ICE Class
Mean maximum corneal swelling at up to 75 min	7 %		II
Mean maximum corneal swelling at up to 240 min	7 %		II
Mean maximum corneal opacity	2.0		III
Mean fluorescein retention	3.0		IV
Other Observations		None
Overall ICE Class		1xII, 1xIII, 1xIV

Interpretation of results:

other: No prediction can be made.

Conclusions:

The overall in vitro classification of the test substance is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, test item has been categorized as “No prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes according to OECD guideline 438 (TOXI-COOP, 2017). The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. 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. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item and Imidazole (positive control) was ground before use in the study. Both were 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 Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole and test item treated eyes after 30, 75, 120 and 180 minutes of observation. The cornea surface of Imidazole and test item treated eyes were not totally cleared at 240 minutes after the post-treatment rinse.

In this ICET, the test substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xII, 1xIII, 1xIV. Positive and negative controls showed the expected results. The experiments were considered to be valid. According to the guideline OECD 438, the test substance overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two in vitro test batteries were performed in a weight of evidence approach in order to evaluate if the test substance is irritating or corrosive to either skin or eye.

Skin

in vitro skin corrosion

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro in accordance with OECD guideline 431 (TOXI-COOP, 2018).

Disks of EPISKIN (two units) were treated with the test item and incubated for 4 hours at room temperature. Exposure of the test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1 °C in 5±1 % CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal to 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 101 %.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

in vitro skin irritation

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro in accordance with OECD guideline 439 (TOXI-COOP, 2018).

Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) 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±1°C for 42 hours (± 1h) in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1°C in 5±1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. 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 chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 72 %). 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. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Conclusions

Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance does not show a skin irritation potential in the two EpiDerm™ in vitro skin irritation and corrosion tests under the test conditions chosen.

Eye

in vitro eye corrosion

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes according to OECD guideline 438 (TOXI-COOP, 2017). The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. 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. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

The test item and Imidazole (positive control) was ground before use in the study. Both were 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 Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole and test item treated eyes after 30, 75, 120 and 180 minutes of observation. The cornea surface of Imidazole and test item treated eyes were not totally cleared at 240 minutes after the post-treatment rinse.

In this ICET, the test substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xII, 1xIII, 1xIV. Positive and negative controls showed the expected results. The experiments were considered to be valid. According to the guideline OECD 438, the test substance overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

in vitro eye irritation

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model according to OECD guideline 492 (LAUS, 2018).

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours. 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 25 minutes and further 18 hours with fresh medium.

Following treatment with the test item, the tissue viability was ≤ 60% and, thus, according to OECD 492 no prediction can be made for the test item. The test substance either needs to be classified in in UN GHS Classification Category I or II with respect to eye irritation.

Conclusions

Based on the results for ICET and EpiOcular Test and considering the evaluation criteria, the test substance needs to be regarded as eye irritating in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

Based on the study results of the in vitro test battery and taking into account the provisions laid down in Regulation (EC) No 1272/2008, the test substance does not need to be classified as irritating or corrosive to skin. With regard to eye irritation the test substance needs to be classified as eye irritating cat. 2 (H319).