Butyl toluene-4-sulphonate

Endpoint summary

Description of key information

Skin irritation/ corrosion

- in vitro skin corrosion (de Jong, 2019a)

Under the conditions of the study, the test material was not corrosive to skin.

- in vitro skin irritation (de Jong, 2019b)

Under the conditions of the study, the test material was not irritating to skin.

Eye irritation

- in vitro eye irritation (Gijsbrechts, 2019a)

Under the conditions of the study, the test material was not irritating to eyes.

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:

key study

Study period:

12 August 2019 to 16 August 2019

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:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

No correction was made for the purity/composition of the test material.

Test system:

human skin model

Remarks:

EpiDerm Skin Model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Source: MatTek Corporation (Ashland MA, USA)
- Tissue batch number(s): 30939 Kit G and Kit H
- Storage conditions: On the day of receipt, the tissues were kept on agarose and stored in the refrigerator.

TEST FOR DIRECT MTT REDUCTION
To assess the ability of the test material to reduce MTT, 50 μL of the test material or 50 μL Milli-Q water as a negative control were added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time it was checked if a blue/ purple colour change or a blue / purple precipitate was observed.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
To assess the colour interference, 50 μL of the test material or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken, and it was checked if a blue / purple colour change was observed.

MAIN TEST
PRE-INCUBATION
At least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 1 hour at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM just before the test material was applied.

APPLICATION
The liquid test material, positive and negative controls were applied undiluted (50 μL) directly on top of the tissue. To protect the test material from light amber coloured glassware was used or glassware was wrapped in tinfoil.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENTS
The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

ENVIRONMENTAL CONDITIONS USED FOR TEST SYSTEM
All incubations, with the exception of the test material incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 62 - 92 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.5 - 37.2 °C).

NUMBER OF REPLICATE TISSUES: The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to test material and two for a 1-hour exposure. 2 tissues were treated with negative control and 2 tissues were treated with positive control for both the 3-minute and 1-hour time point.

CALCULATION OF CELL VIABILITY
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc / mean ODlt_u+MTT) * 100

DATA EVALUATION
- Acceptability Criteria
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100 % viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30 %.

- Interpretation
A test material is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test material considered non-corrosive (viability ≥ 50 %) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test material is decreased below 15 %.
A test material is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

- Data interpretation and sub-categorisation of test materials
< 50 % after 3-minute exposure = Corrosive
≥ 50 % after 3-minute exposure AND < 15 % after 1-hour exposure = Corrosive
≥ 50 % after 3-minute exposure AND ≥ 15 % after 1-hour exposure = Non-corrosive
For substances/mixtures identified as Corrosive:
< 25 % after 3-minute exposure = Sub-category 1A
≥ 25 % after 3-minute exposure = Sub-categories 1B and 1C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes, 1 hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2 per time point

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute application

Value:

107

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour application

Value:

119

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
No blue / purple precipitate was observed. It was therefore concluded that the test material did not interact with MTT.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
The solutions did not turn blue / purple. It was therefore concluded that the test material did not produce colour interference with the MTT endpoint.

MAIN STUDY
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 107 and 119 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after 3 minutes of treatment and not below 15 % after 1 hour of treatment, the test material is considered to be not corrosive.

- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.
- The mean relative tissue viability following the 3-minute and 1-hour exposures to the positive control were 9.5 and 7.7 %, respectively.
- In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was ≤ 16 %, indicating that the test system functioned properly.

Interpretation of results:

other: not corrosive according to EU criteria

Conclusions:

Under the conditions of the study, the test material was not corrosive.

Executive summary:

The in vitro skin corrosion potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS, under GLP conditions.

During the study, the human three-dimensional epidermal model (EpiDerm (EPI-200)) was used to evaluate the ability of the test material to induce skin corrosion. The possible corrosive potential of the test material was tested through topical application of 50 µL undiluted test material for 3 minutes and 1 hour.

Under the conditions of the study, the positive control had a mean relative tissue viability of 7.7 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was ≤ 16 %, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 107 and 119 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, the test material is considered to be not corrosive.

In conclusion, the test material is not corrosive in the in vitro skin corrosion test under the experimental conditions described.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2019 to 28 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

18 July 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

No correction was made for the purity/composition of the test material.

Test system:

human skin model

Remarks:

EPISKIN Small Model™

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
- Source: SkinEthic Laboratories, Lyon, France.
- Tissue batch number(s): EPISKIN-SM™, 0.38 cm², Batch no.: 19 EKIN 043)

TEST FOR INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
The test material was checked for possible direct MTT reduction and colour interference in an in vitro skin corrosion test using EpiDerm as a skin model. As solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test material did not interfere with the MTT endpoint.

MAIN TEST
PRE-INCUBATION
On the day of receipt, tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 23 hours at 37 °C.

APPLICATION
The liquid test material, positive and negative controls were applied undiluted (25 μL) directly on top of the tissue. The positive control was re-spread after 7 minutes of contact time.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline (PBS) to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

CELL VIABILITY MEASUREMENTS
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for approximately 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test material incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 73 - 94 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.8 – 37.2 °C).

NUMBER OF REPLICATE TISSUES: The test was performed on a total of 3 tissues per test material together with 3 tissues per negative and 3 tissues per positive controls.

CALCULATION OF CELL VIABILITY
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc / mean ODlt_u+MTT) * 100

DATA EVALUATION
- Acceptability criteria
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40 % relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

- Interpretation
A test material is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤ 50 % of the mean viability of the negative controls.
A test material is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is > 50 % of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL

NEGATIVE CONTROL
- Amount(s) applied: 25 µL

POSITIVE CONTROL
- Amount(s) applied: 25 µL
- Concentration (if solution): 5 % solution in phosphate buffered saline

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours followed by 3 hours with MTT

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

TEST FOR INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
As no colour changes were observed it was concluded that the test material did not interact with the MTT endpoint.

MAIN TEST RESULTS
The relative mean tissue viability obtained after 15 ± 0.5 minutes of treatment with the test material compared to the negative control tissues was 74 %. Since the mean relative tissue viability for the test material was above 50 % the test material is considered to be non-irritant.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 6.6 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 5 %, indicating that the test system functioned properly.

Interpretation of results:

other: not classified as a skin irritant according to EU criteria

Conclusions:

Under the conditions of the study, the test material was not irritating.

Executive summary:

The in vitro skin irritation potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions, using a human three-dimensional epidermal model (EPISKIN Small model (EPISKIN-SM™)).

During the study, 0.25 µL test material was applied (unchanged), directly on top of the skin tissue, for 15 minutes.

The test material was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes of treatment with the test material compared to the negative control tissues was 74 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.

The positive control had a mean cell viability of 6.6 % after 15 ± 0.5 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 5 %, indicating that the test system functioned properly.

In conclusion, the test material is non-irritant in the in vitro skin irritation test under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2019

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)

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from a slaughterhouse (Vitelco, 's-Hertogenbosch, The Netherlands). The eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

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): 750 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

Three corneas were allocated to the test material, three corneas were allocated to the negative control item, and three corneas were allocated to the positive control item.

Details on study design:

PREPARATION AND SELECTION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1 % (v/v) L-glutamine and 1 % (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for a minimum of 1 hour at 32 ± 1 °C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test material was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or test material over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed with MEM (Earle’s Minimum Essential Medium) with phenol red and thereafter with cMEM. Possible pH effects of the test material on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.

POST-EXPOSURE INCUBATION
Corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS
After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
- Opacity measurement: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to the following equation:
Opacity = [(I0/I) - 0.9894] / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Application of Sodium Fluorescein: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.
- Permeability Determinations: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

The test material was classified according to the following prediction model:
IVIS ≤ 3: No Category (UN GHS)
IVIS >3; ≤ 55: No prediction can be made (UN GHS)
IVIS > 55: Category 1 (UN GHS)

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test material (mean)

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative control (mean)

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control (mean)

Value:

55

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The corneas treated with the test material showed opacity values ranging from -0.2 to 0.5 and permeability values ranging from 0.006 to 0.018. The corneas were clear after the 10 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.1 to 0.7 after 10 minutes of treatment with the test material. The test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.
The individual in vitro irritancy scores for the negative controls ranged from 0.5 to 2.6. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. The corneas treated with the negative control item were clear after the 10 minutes of treatment.
The individual positive control in vitro irritancy scores ranged from 54 to 61. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

ACCEPTANCE OF RESULTS:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 55 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Summary of results

Treatment	Final opacity	Final OD490	In vitro Irritancy Score
Negative control	2.7	-0.005	2.6
	0.5	-0.003	0.5
	5.2*	0.029*	5.7*
Mean	1.6	-0.004	1.6
Positive control	21	2.218	54
	22	2.592	61
	30	1.370	50
Mean	24	2.060	55
Test material	0.5	0.013	0.7
	-0.2	0.006	-0.1
	0.2	0.018	0.5
Mean	0.2	0.012	0.4

Positive control and test material are corrected for the negative control.

*Excluded from analysis

Interpretation of results:

other: not classified according to EU criteria

Conclusions:

Under the conditions of the study, the test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.
In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye irritation potential of the test material was evaluated, as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was conducted in accordance with the standardised guideline OECD 437 and under GLP conditions.

During the study, the test material was applied as supplied (750 μL) directly on top of the corneas. The eye damage of the test material was tested through topical application for 10 minutes. After exposure the cornea was thoroughly rinsed to remove the test material and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 55 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of the study, the test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.

In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/ corrosion

- in vitro skin corrosion (de Jong, 2019a)

The in vitro skin corrosion potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the human three-dimensional epidermal model (EpiDerm (EPI-200)) was used to evaluate the ability of the test material to induce skin corrosion. The possible corrosive potential of the test material was tested through topical application of 50 µL undiluted test material for 3 minutes and 1 hour.

Under the conditions of the study, the positive control had a mean relative tissue viability of 7.7 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was ≤ 16 %, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 107 and 119 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, the test material is considered to be not corrosive.

In conclusion, the test material is not corrosive in the in vitro skin corrosion test under the experimental conditions described.

- in vitro skin irritation (de Jong, 2019b)

The in vitro skin irritation potential of the test material was assessed in a study which was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions, using a human three-dimensional epidermal model (EPISKIN Small model (EPISKIN-SM™)). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, 0.25 µL test material was applied (unchanged), directly on top of the skin tissue, for 15 minutes.

The test material was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes of treatment with the test material compared to the negative control tissues was 74 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.

The positive control had a mean cell viability of 6.6 % after 15 ± 0.5 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 5 %, indicating that the test system functioned properly.

In conclusion, the test material is non-irritant in the in vitro skin irritation test under the experimental conditions described.

Eye irritation

- in vitro eye irritation (Gijsbrechts, 2019a)

The eye irritation potential of the test material was evaluated, as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study was conducted in accordance with the standardised guideline OECD 437 and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the test material was applied as supplied (750 μL) directly on top of the corneas. The eye damage of the test material was tested through topical application for 10 minutes. After exposure the cornea was thoroughly rinsed to remove the test material and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 55 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of the study, the test material did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.4 after 10 minutes of treatment.

In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin or eye irritation.