5,5'-diisopropyl-2,2'-dimethylbiphenyl-4,4'-diyl dihypoiodite

Endpoint summary

Description of key information

Skin Corrosion (in vitro)

The test material was not corrosive in the in vitro skin corrosion test.

Skin Irritation (in vitro)

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

Eye Irritation (in vitro)

Under the conditions of the study, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.  

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:

26 March 2018 to 30 March 2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Tissue lot number: 28307

TEST FOR DIRECT MTT REDUCTION
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, at least 25 mg 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
The test material was checked for possible colour interference before the study was started. Some non-coloured test mateials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, at least 25 mg 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
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM just before the test material was applied.

APPLICATION OF TEST MATERIAL AND CONTROLS
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. The skin was moistened with 25 µL Milli-Q water to ensure close contact of the test material to the tissue and 25.5 to 30.2 mg of the solid test material was added into the 6-well plates on top of the skin tissues.
For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

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 MEASUREMENT
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.

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 item 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 %.

ANALYSIS
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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.5 to 30.2 mg (with 25 µL Milli-Q water to increase tissue surface contact)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes or 60 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION AND ASSESSMENT OF COLOUR INTERFERENCE
The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test material did not interfere with the MTT endpoint.

TEST MATERIAL AND CONTROLS
Mean OD570 values and viabilities for the negative control, positive control and test material are given in Table 1.
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with test material compared to the negative control tissues was 97 and 104 % respectively. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1 hour treatment the test material is considered to be not corrosive.

QUALITY CRITERIA
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 6.3 %. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was ≤ 16 %, indicating that the test system functioned properly.

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissue	Exposure period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative control	3 minutes	1.913	1.896	± 0.024	1.8	100
		1.880
	60 minutes	2.082	2.074	± 0.011	0.7
		2.066
Positive control	3 minutes	0.208	0.220	± 0.017	10	12
		0.231
	60 minutes	0.142	0.131	± 0.016	16	6.3
		0.120
Test material	3 minutes	1.683	1.839	± 0.221	16	97
		1.996
	60 minutes	2.071	2.165	± 0.132	8.3	104
		2.258

OD = optical density

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

Under the conditions of the study, the test material was not corrosive in the in vitro skin corrosion test.

Executive summary:

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.bis under GLP conditions using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.

Under the conditions of the study, the positive control had a mean relative tissue viability of 6.3 % 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 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 97 and 104 %, respectively. Because the mean relative tissue viability for 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 was not corrosive in the in vitro skin corrosion test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 2018 to 04 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

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

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™ (EPISKIN-SM™, 0.38 cm²)
- Tissue batch number(s): Lot no.: 18-EKIN-022
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Source: SkinEthic Laboratories, Lyon, France

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

NUMBER OF REPLICATE TISSUES: 3

CELL CULTURE
- Tissues: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 21 hours at 37 °C. Maintenance medium and Assay medium were supplied by SkinEthic Laboratories.
- MTT medium: MTT concentrate (3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL).
- 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 to 100 % (actual range 59 - 84 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.5 - 37.2 °C).

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 the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20140962). Because solutions did not turn blue / purple and/or a blue / purple precipitate was observed it was concluded that the test material did interfere with the MTT endpoint.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The test was performed on a total of 3 tissues per test material together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water to ensure close contact of the test material to the tissue and the solid test material (14.0 to 17.2 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5 % SDS (positive control), respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline 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 MEASUREMENT
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 68 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

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 were calculated by subtracting the value of 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) x 100

INTERPRETATION
- Acceptability of the assay
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.

- 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.

≤ 50 % of the mean viability of the negative controls = Category 1 or Category 2 (additional information on corrosion needed)
> 50% of the mean viability of the negative controls = No category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 14.0 to 17.2 mg

NEGATIVE CONTROL
- Amount(s) applied: 25 μL

POSITIVE CONTROL
- Amount(s) applied: 25 μL
- Concentration: 5 %

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours and then incubated for 3 hours with MTT

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

117

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

No colour changes were observed so it was concluded that the test material did not interact with the MTT endpoint.

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 treatment with the test material compared to the negative control tissues was 117 %. 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 4.6 %. 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 < 4 %, indicating that the test system functioned properly.

Table 1: Mean Absorption in the In Vitro Skin Irritation Test

Treatment	A (OD570)	B (OD570)	C (OD570)	Mean (OD570) ± SD
Negative Control	0.998	0.972	1.039	1.003 ± 0.034
Test Material	1.133	1.176	1.198	1.169 ± 0.033
Positive Control	0.042	0.055	0.041	0.046 ± 0.008

 

Table 2: Mean Tissue Viability in the In Vitro Skin Irritation Test

Treatment	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative Control	100	3.4
Test Material	117	3.3
Positive Control	4.6	0.8

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

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

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.

The objective of this study was to evaluate the test material for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM™)). The possible skin irritation potential was tested through topical application for 15 minutes.

Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test material was applied 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 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide 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 treatment with the test material compared to the negative control tissues was 117 %. Since the mean relative tissue viability 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 4.6 % after 15 ± 0.5 minutes 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 < 4 %, indicating that the test system functioned properly.

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

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:

26 March 2018 to 27 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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 the slaughterhouse, where 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 neovascularisation 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: 296.98 to 318.47 mg

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes with sodium fluorecein

Number of animals or in vitro replicates:

3 replicates for each condition (test material, negative control and positive control)

Details on study design:

PREPARATION 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 the minimum of 1 hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
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. Three corneas were selected at random for each treatment group.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of the negative control and 20 % (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test material was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (296.98 to 318.47 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test material on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

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 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.

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 5 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.
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.

DATA 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 IVIS cut-off values for identifying the test material as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are as follows:
- IVIS ≤ 3 = Not classified for irritation
- IVIS > 3; ≤ 55 = No prediction can be made
- IVIS > 55 = Category 1, H318: Causes serious eye damage

ACCEPTABILITY OF THE ASSAY
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.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

A summary of the results can be seen in Table 1.

TEST MATERIAL
The corneas treated with the test material showed opacity values ranging from -2.6 to 0.1 and permeability values ranging from -0.015 to 0.027. The corneas were translucent after the 240 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 -2.2 to -0.1 after 240 minutes of treatment with the test material.

CONTROLS
The individual in vitro irritancy scores for the negative controls ranged from 3.2 to 5.9. The individual positive control in vitro irritancy scores ranged from 111 to 146. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

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 (20 % (w/v) Imidazole) was 133 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.

Table 1: Summary of opacity, permeability and in vitro irritancy scores

Treatment	Mean Opacity	Mean Permeability	Mean In Vitro Irritancy Score
Negative control	4.6	0.021	4.9
Positive control	96	2.515	133
Test material	-1.5	0.002	-1.5

Interpretation of results:

other: Not irritating to eyes according to EU criteria

Conclusions:

Under the conditions of the study, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye irritancy of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 437, under GLP conditions.

During the study the test material was applied as supplied for 240 minutes. Negative and positive controls were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 133 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.  

The negative control gave opacity of 4.6 and permeability 0.021. 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 corneas treated with the test material showed opacity values ranging from -2.6 to 0.1 and permeability values ranging from -0.015 to 0.027. The corneas were translucent after the 240 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 -2.2 to -0.1 after 240 minutes of treatment with the test material.

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 Corrosion (in vitro)

The potential of the test material to cause corrosion to the skin was determined in accordance with the standardised guidelines OECD 431 and EU Method B40.bis under GLP conditions using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

During the study, duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period.

Under the conditions of the study, the positive control had a mean relative tissue viability of 6.3 % 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 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 97 and 104 %, respectively. Because the mean relative tissue viability for 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 was not corrosive in the in vitro skin corrosion test.

Skin Irritation (in vitro)

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B46, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the test material for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential was tested through topical application for 15 minutes.

Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test material was applied 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 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide 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 treatment with the test material compared to the negative control tissues was 117 %. Since the mean relative tissue viability 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 4.6 % after 15 ± 0.5 minutes 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 < 4 %, indicating that the test system functioned properly.

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

Eye Irritation (in vitro)

The eye irritancy of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 437, under GLP conditions.

During the study the test material was applied as supplied for 240 minutes. Negative and positive controls were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 133 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.  

The negative control gave opacity of 4.6 and permeability 0.021. 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 corneas treated with the test material showed opacity values ranging from -2.6 to 0.1 and permeability values ranging from -0.015 to 0.027. The corneas were translucent after the 240 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 -2.2 to -0.1 after 240 minutes of treatment with the test material.

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 corrosion/ irritation or eye irritation.