[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation, in vitro: non-irritating (15 minutes exposure/42hour incubation viability = 111%), EPISKIN, OECD TG 439, 2020

Eye irritation, in vitro: non-eye irritating, mean IVIS = 2.4 and mean permeability = 0.005, OECD TG 437, 2019

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-10-2019 to 20-01-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP within minor deviations. All relevant validity criteria were considered to be met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 55%) ; 25 µL test item, negative control, positive controls were applied, respectively rather than 10 µL. These protocol deviations were not considered to affect the reliability of the study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Minor deviation in environmental conditions (humidity minimum 55%) ; 25 µL test item, negative control, positive controls were applied, respectively rather than 10 µL. These protocol deviations were not considered to affect the reliability of the study.

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM Small Model
- Tissue batch number(s): Lot no.: 20-EKIN-003
- Production date: Not reported.
- Shipping date: Not reported. Although the CoA for the tissues QA is provided in the full study report.
- Delivery date: 15-01-2020 ; day prior to test initiation (ca. 24 hours)
- Date of initiation of testing: ca. 16-01-2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.0 °C, 5 ± 0.5% CO2 (actual: 35.6 – 37.4°C)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.0 °C, 5 ± 0.5% CO2; for 42 hours. For further information see below.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. 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. For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells 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 68 hours. The amount of extracted formazan was determined spectrophotometrically.
- Observable damage in the tissue due to washing: Not applicable.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3.0 mg/mL MTT concentrated diluted (x10) to 0.3 mg/mL MTT solution prepared in assay medium
- Incubation time: For the cell viability measurements: cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells 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 72 hours. The amount of extracted formazan was determined spectrophotometrically.
- Spectrophotometer: M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: Without reference filter
- Filter bandwidth: No reported.
- Linear OD range of spectrophotometer: Not reported.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Within NC and PC historic control ranges presented within the report.
- Barrier function: See above.
- Morphology: See above.
- Contamination: Not applicable.
- Reproducibility: The relative mean tissue viability for the positive control treated tissues was 11.0% relative to the negative control following the 15-minute exposure/42-hour incubation period. With standard deviation of 1.9%. The positive control acceptance criterion was therefore satisfied. The negative control triplicate treated tissues mean OD570 was 1.0180 and the standard deviation of viability was 3.1%. The acceptance criterion was therefore satisfied. The test item triplicate treated tissues viability standard deviation was 3.1%. The mean OD570 for both the negative and positive controls were within the historical control ranges.
- Other: As an additional quality control step, the results of the assay are compared to the historical ranges for negative and positive controls generated by the testing laboratory to confirm the functioning of the test system.

NUMBER OF REPLICATE TISSUES: Three (3), triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: In the pretest for colour interference: the solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues. In the pre-test for direct MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT. It was unnecessary to run killed tissues.
- Procedure used to prepare the killed tissues (if applicable): Not applicable.
- N. of replicates : Not applicable.
- Method of calculation used: Not applicable.
- Other: In the assessment of Colour Interference with the MTT endpoint, the solution containing the test item did not become coloured. This was taken to indicate the test item did not have the potential to cause colour interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One (n=1)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is less than or equal to 50%,.
- The test substance is considered to be non-irritating to skin if e.g. the viability after 15-minutes exposure / 42-hours incubation is greater than 50%.
- Cut off points in accordance with OECD TG 439 and the GHS and CLP Classification systems.
- Skin irritation is expressed as the remaining cell viability after exposure to the test substance at exposure times 15-minutes / 42-hours incubation. Where necessary, direct MTT reduction, colour interference modifications were completed.

OTHER:
EpiSkin RHE Small Model (Lot no.: 20-EKIN-003). The test consists of topical application of the test item -one on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly rinsed to remove the test item and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is 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 (MTT) at the end of the treatment. The EPISKIN Small 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. Pre-test checks for Direct MTT reduction and Colour Interference were completed.

Preincubation:
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 23 hours at 37°C. Maintenance medium and Assay medium were supplied by a recognised supplier (documented in the full study report).

Application of test item and rinsing:
Test item: Tissues were treated with 25 µL test item for 15 minutes exposure period. Negative control: 25 µL PBS (negative control) was similarly applied. Positive control: 25 µL SDS (5% w/v) was respread after 7 minutes contact time to maintain distribution of the SDS - for the remainder of the contact period. This was not deemed necessary in the negative control or test item definitive test group. Minor deviation : 25 µL test item, negative control, positive controls were applied (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2). These protocol deviations were not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. 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. All incubations, with the exception of the test item 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 55 – 94%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): undiluted
- Other: Minor deviation : 25 µL test item, negative control, positive controls were applied (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2). These protocol deviations were not considered to affect the reliability of the study

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): Not applicable. (Phosphate buffered saline, pre-diluted)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5% SDS (pre-diluted)

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature, after which the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

Triplicate; treatment and concurrent negative control and positive control groups

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (n=3)

Value:

111

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Basis: mean. Time point: 15 minute exposure. Remarks: n=3; SD = 7.0% ; Score in terms of percentage of negative control.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data are presented in the full study report. All values for the control groups were within the ranges of the current Historic Control Data. Historic Control Data (n=126): the mean OD of the positive control was 0.11 ; range 0.023 to 0.449. In this same period the mean OD of the negative control was 0.98 ; range 0.422 – 1.547.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.0218	1.0180	0.0311	100*	3.1
	1.0469
	0.9851
Positive Control Item	0.1057	0.1096	0.0709	11	1.9
	0.0924
	0.1308
Test Item	1.1019	1.1300	0.0195	111	7.0
	1.0773
	1.2106

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%.

Values are corrected for background absorption (0.043). Isopropanol was used to measure background adsorption.

Negative control: Phosphate buffered saline (PBS)

Positive control: 5% (aq.) Sodium dodecyl sulphate (SDS)

 

The relative mean tissue viability for the positive control treated tissues was 11.0% relative to the negative control treated tissues and the standard deviation value of the viability was 1.9%. The mean OD570 for the negative control treated tissues was 1.0180 and the standard deviation value of the viability was 3.1%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 7.0%. All assay acceptance criteria were met.

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be irritating to the skin.

Executive summary:

The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added (with a pipette) 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. Use of 25 µL test item, negative control and positive controls (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2) protocol deviation was not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. 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. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 11.0% after 15 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 relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 111%. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 7%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is not considered to be irritant. Under the conditions of this study the test item is not irritant to the 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:

16-07-2019 to 15-11-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

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

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): > 9 months old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. Post-excision, the isolated eyes were stored in physiological saline in the cooled slaughter-house until transportation to the laboratory using a suitable container. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: < 24 hours. Corneas were prepared for testing immediately on same day arrival.
- indication of any existing defects or lesions in ocular tissue samples: None. Only corneas free from damage utilised (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches were discarded).
- Indication of any antibiotics used: None reported.

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): 0.75 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 minutes at 32 ± 1ºC.

Duration of post- treatment incubation (in vitro):

The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

Number of animals or in vitro replicates:

Three (3) per test item, or negative or positive controls, respectively.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The isolated corneas were stored in a petri dish with cMEM (Eagle’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) of MC2 (Clermont-Ferrand, France) 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. 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 an air 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.
SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The isolated corneas were stored in a petri dish with cMEM (Eagle’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, details on cornea holder in the full study report) 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. 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 (details in the full study report). The opacity of each cornea was read against an air 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.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches) and where necessary discarded. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline solution (recognised supplier ; documented in the full study report)

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: neat Ethanol (recognised supplier ; documented in the full study report)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 ± 1 minutes at 32 ± 1ºC.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: Opacity, Permeability and In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: 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). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (n=3)

Value:

2.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

IVIS scores ranged from 1.7 to 3.2 (n=3) with mean 2.4 after 10 minutes treatment ; individual scores = 2.3, 3.2 and 1.7, respectively

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None. No pH effect of the test item was observed on the rinsing medium.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. 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. 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. This deviation had no impact on the study since two negative control eyes were available and yielded results within the historical control range.
- Acceptance criteria met for positive control: Yes. The mean in vitro irritancy score of the positive control neat ethanol was 55 and was within the historical positive control data range (HCD range : 24.0 to 89.6 and mean = 51.31 ; n=108).
- Range of historical values if different from the ones specified in the test guideline: The mean in vitro irritancy score of the negative control were less than the upper limits of the laboratory historical range and for the positive control neat ethanol at 55..0 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively. It was noted 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. This deviation had no impact on the study since two negative control eyes were available and yielded results within the historical control range..

Table 1.0 - Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score #1, #2
Negative control	1.6	-0.040	1.6
Positive control	24	2.060	55.0
Test item	2.3	0.005	2.4

#1 Calculated using the negative control (corrected) mean opacity and mean permeability values for the positive control and test item

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2.0 - Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity #1	Negative control corrected Final Opacity #2	Mean Final Opacity
Negative control	3.6	6.3	2.7		1.6
	4.7	4.3	0.5
	4.6 #3	9.8 #3	5.2 #3
Positive control	2.7	25.4	22.8	21	24
	4.3	28.2	23.9	22
	4.1	35.3	31.2	30
Test item	4.3	8.2	3.9	2.3	2.3
	3.7	8.4	4.7	3.1
	2.3	5.5	3.2	1.6

#1 Final Opacity = Opacity after treatment – Opacity before treatment.

#2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

#3 Excluded from analysis (above historic control range)

#4 Calculations are made without rounding off

 

Table 3.0 - Permeability score individual values (uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD		Mean final negative control
Negative control	1	-0.005	-0.005	-0.005	-0.005		-0.005	-0.040
	1	-0.004	-0.001	-0.004	-0.003		-0.003
	1	-0.0028 #2	0.030 #2	0.029 #2	0.029 #2		0.029 #2
Positive control	6	0.368	0.365	0.364	0.366		2.194
	6	0.423	0.430	0.431	0.428		2.568
	1	1.369	1.361	1.368	1.366		1.366
Test item	1	-0.004	-0.004	-0.004	-0.004		-0.004
	1	0.002	0.004	0.008	0.005		0.005
	1	0.005	0.001	0.001	0.002		0.002

#1 Calculations are made without rounding off.

#2 Excluded from analysis (above historic control range)

 

Table 4.0 - In vitro irritancy score

Treatment	Final Opacity #1	Final OD4902	In vitro Irritancy Score #2
Negative control	2.7	-0.005	2.6
	0.5	-0.003	0.5
	5.2 #3	0.029 #3	5.7 #3
Positive control	21	2.218	54
	22	2.592	61
	30	1.370	50
Test item	2.3	0.000	2.3
	3.1	0.009	3.2
	1.6	0.006	1.7

#1 Positive control and test item are corrected for the negative control.

#2 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

#3 Excluded from analysis (above historic control range)

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be irritant in the in vitro Bovine Corneal Opacity and Permeability model. The in mean vitro irritancy score (IVIS) was < 3.0 in the prediction model.

Executive summary:

The study was performed according to OECD TG 437 to assess the eye irritancy potential of the item in isolated bovine corneas in accordance with GLP. The purpose of this test was to evaluate the test item for its potential to induce eye damage/ocular irritation. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. A total of three corneas per treatment group were used. A volume of 750 microlitres of the item was placed the cornea. The negative control group received physiological saline and the positive control group received neat Ethanol. For each group the corneas were incubated for 10 ± 1minutesat 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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. 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. This deviation had no impact on the study since two negative control eyes were available and yielded results within the historical control range. The mean in vitro irritancy score of the positive control (neat ethanol) was 55.0 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment. Under the conditions of this study, the item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation :

Key study : in vitro, OECD TG 439, 2020 : The study was performed to OECD TG 439 and EU Method B.46 to assess the skin irritation potential of the test item in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added (with a pipette) 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. Use of 25 µL test item, negative control and positive controls (ca. 65 µL/cm2), respectively rather than 10 µL (ca. 26 µL/cm2) protocol deviation was not considered to affect the reliability of the study. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. 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. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 11.0% after 15 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 relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 111%. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 7%, indicating that the test system functioned properly. Since the mean relative tissue viability for the test item was above 50% after 15 minutes treatment it is not considered to be irritant. Under the conditions of this study the test item is not irritant to the skin.

 

Eye irritation/corrosion :

Key study : in vitro, OECD TG 437 2019 : The study was performed according to OECD TG 437 to assess the eye irritancy potential of the item in isolated bovine corneas in accordance with GLP. The purpose of this test was to evaluate the test item for its potential to induce eye damage/ocular irritation. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. A total of three corneas per treatment group were used. A volume of 750 microlitres of the item was placed the cornea. The negative control group received physiological saline and the positive control group received neat Ethanol. For each group the corneas were incubated for 10 ± 1minutesat 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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. 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. This deviation had no impact on the study since two negative control eyes were available and yielded results within the historical control range. The mean in vitro irritancy score of the positive control (neat ethanol) was 55.0 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment. Under the conditions of this study, the item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.

Respiratory Irritation:

No data available.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for skin irritation

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation