3,3,6,6-tetramethoxy-2,7-dioxa-3,6-disilaoctane

Endpoint summary

Description of key information

The test material was identified as non-irritant to the eye in an in vitro testing strategy consisting of a BCOP (according to OECD 437) and an EpiOcular eye irritation test (according to OECD 492).

The test material was identified as irritant to the skin based on findings in the in vitro assays Epiderm, SCT (according to OECD 431) and EpiSkin, EIT (according to OECD 439).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-06-20 until 2018-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 611220160318
- Storage stability: 31 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Stability under test conditions: guaranteed by the sponsor under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: The test item was used as supplied by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- no prior treatment

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Reconstructed Human Epidermis Model Kit

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin from EpiSkin Laboratories, Lyon, France
- Tissue batch number: 18-EKIN-025
- Maintenance Medium lot number: 18-MAIN3-030
- Assay Medium lot number: 18-ESSC-026
- Delivery date: 19 June 2018
- Date of initiation of testing: 20 June 2018

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsing by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item; transferation to maintenance medium afterwards

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL, diluted in assay medium
- Incubation time: in the dark at 37 °C, 5% CO2 in air for 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Tissues Satisfactory: Yes
- Temperature Indicator Color Satisfactory: Yes
- Agar Medium Color Satisfactory: Yes

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
The prediction model is made according to OECD guideline 439 and shown in table 1.

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 μL test item (26.3 μL/cm2) was applied

NEGATIVE CONTROL
- Amount applied: 10 µL DPBS

POSITIVE CONTROL
- Amount applied: 10 µL SDS
- Concentration: 5% (w/v)

Duration of treatment / exposure:

15 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours (37 °C, 5% CO2 in air)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative mean viability

Run / experiment:

mean value after 15 min exposure period and 42 h post-exposure incubation period

Value:

8.3

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:

- 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.

- Colour interference with MTT:
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The mean OD570 for the negative control treated tissues was 1.114 and the standard deviation value of the viability was 4.2%.
The negative control acceptance criteria were therefore satisfied.

- Acceptance criteria met for positive control:
The relative mean tissue viability for the positive control treated tissues was 3.0% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6%.
The positive control acceptance criteria were therefore satisfied.

- Acceptance criteria met for variability between replicate measurements:
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.0%.
The test item acceptance criterion was therefore satisfied.

- Range of historical values if different from the ones specified in the test guideline:

Table 2: Results of the individual and mean OD570 values are given for the test item, negative control item and positive control item.

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.064	1.114	0.047	95.5	100*	4.2
	1.158			103.9
	1.119			100.4
Positive Control Item	0.032	0.033	0.006	2.9	3.0	0.6
	0.039			3.5
	0.027			2.4
Test Item	0.117	0.093	0.022	10.5	8.3	2.0
	0.087			7.8
	0.075			6.7

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item was classified as irritant Cat. 2, (EC) No. 1272/2008 based on the combined knowledge with the findings in the skin corrosion testing.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-17 until 2018-04-20

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 611220160318
- Storage stability: 30 June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (approximately 4°C)
- Stability under test conditions: guaranteed by the sponsor under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: The test item was used as supplied by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- no prior treatment

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Tissues (0.63 cm2), kit A
- Tissue lot number: 25896
- Assay medium lot number: 041218MSA
- Delivery date: 17 April 2018
- Date of initiation of testing: 17 April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 minutes at room temperature and for 35 minutes in the incubator at 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MatTek MTT-100 kit
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation: 1 hours and in the dark
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570 of the negative control between 0.8 and 2.8 and of the positive control (Potassium Hydroxide 8.0N) < 15%
- Coefficient of variance: range 20 to 100% viability, the coefficient of variation between tissue replicates should be ≤ 30%

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
Detailed information are given in table 1 and table 2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL
- Concentration: test item used as supplied

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 min
60 min

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative mean viabilitiy

Run / experiment:

3 minutes

Value:

95.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative mean viability

Run / experiment:

60 minutes

Value:

106.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean OD570 for the negative control treated tissues was 2.070 for the 3-Minutes exposure period and 1.944 for the 60-Minutes exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 2.9% relative to the negative control following the 60-Minutes exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the coefficient of variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Table 1: Results of the epiderm study. Given are the relative mean viabilities for each treatment group.

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minutes	100*	2.6	95.8
60 minutes	100*	2.9	106.2

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

Table 2: Mean OD570 values and viabilities for the negative control item, positive control item and test item

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	2.043	2.070	0.038	1.8	100*
		2.097
	60 Minutes	1.956	1.944	0.017	0.9
		1.932
Positive Control	3 Minutes	0.047	0.054	0.009	na	2.6
		0.060
	60 Minutes	0.066	0.056	0.014	na	2.9
		0.046
Test Item	3 Minutes	1.981	1.983	0.002	0.1	95.8
		1.984
	60 Minutes	2.067	2.064	0.004	0.2	106.2
		2.061

OD = Optical density

* = The mean percentage viability of the negative control tissue is set at 100%

na = Not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-09 until 2019-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 611220160318
- Storage stability: 31 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Stability under test conditions: guaranteed by the sponsor under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: The test item was used as supplied by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- no prior treatment

Species:

human

Strain:

other: normal human derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

Description of the cell system used: Reconstructed human cornea-like epithelium
- Model used: EpiOcular model (OCL-200)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 27073
- Assay medium lot number: 100818ISA
- no further details given

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL
- Concentration: undiluted test item was used

BENCHMARK CONTROL, NEGATIVE CONTROL, POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: all controls were applied as supplied by the sponsor

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

tested in duplicates

Details on study design:

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were immersed and swirled three times in each of three beakers filled with sterile PBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation: 3 hours, 37°C, 5% CO2, incubated in the dark
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm, without reference filter

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance can not be predicted based on EU CLP/UN GHS Category 1 and Category 2 if the mean relative tissue viability with a test material is less than 60%.
- The test substance is considered to be non- irritant to the eye if the mean relative tissue viability with a test material is greater than 60%.
- Justification for the selection of the cut-off point is based on OECD TG 492.

ACCEPTANCE CRITERIA
- Negative control: Tissue viability is acceptable if the mean OD570 of the negative control (NC) is ≥ 0.8. The mean OD570 of the NC should not exceed 2.5.
- Positive control: Methyl acetate used as positive control (PC) usually leads to a tissue viability of approx. 25%. A viability of ≤ 50% is acceptable.
- Variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.
- Benchmark control: The assay establishes the acceptance criteria for an acceptable test if the relative mean tissue viability for the benchmark control treated tissues was ≤60% historicrelative to the negative control treated tissues

HISTORICAL DATA : As dicussed before was the benchmark control used to prove the proviciency. No historical data exist to date.

Irritation parameter:

other: relative mean viability

Run / experiment:

one run / mean of 2 individual experiments

Value:

87.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

OD570 = 2.787, greater than the upper limit of 2.5 set out in the acceptance criteria; Further description is given below (other effects).

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The results of the test are given in table 1.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Diethyl toluamide was used as a benchmark control as demonstration of proficiency had not been completed in the laboratory.
Based on an agreement, the benchmark control was tested alongside with the test item and negative and positive controls. Diethyl toluamide was selected based on the OECD 492 due to its expected eye irritancy potential of EU CLP/UN GHS Category 2B.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
At 2.787 the mean optical density (OD570) of the negative control tissues was greater than the upper limit of 2.5 set out in the acceptance criteria.
However, as the results of the controls and test item gave unequivocal conclusions it was considered unnecessary to repeat the test.
Besides, test item tissues were both indicative of a negative response and were not close to the cut-off for irritation.
Therefore, the result of the negative control was considered to have not impacted upon the conclusion of the study and the integrity or validity of the study was not compromised.

- Acceptance criteria met for positive control:
The relative mean tissue viability for the positive control treated tissues was 34.7% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.

- Acceptance criteria met for benchmark control:
The relative mean tissue viability for the benchmark control treated tissues was 31.4% relative to the negative control treated tissues. The benchmark control acceptance criterion was therefore satisfied.

Table 1: Mean OD570 values and percentage viabilities for the negative control item, positive control item, benchmark control item and test item

Item	OD570 of tissues	Mean OD570 of duplicate tissues	Relative individual tissue viability (%)	Relative mean viability (%)	CV of Relative mean viability (%)
Negative Control Item	2.873	2.787	103.1	100*	4.4
	2.701		96.9
Positive Control Item	1.073	0.968	38.5	34.7	15.3
	0.863		31.0
Test Item	2.521	2.448	90.5	87.8	4.2
	2.375		85.2
Benchmark Control	0.864	0.876	31.0	31.4	1.9
	0.888		31.9

Interpretation of results:

GHS criteria not met

Conclusions:

According to the conditions of the test, the test item was classified as non-irritant.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2018-06-28 until 2018-06-28

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:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 611220160318
- Expiration date of the batch: 18 June 2018
- Purity test date: 27 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator
- Stability under test conditions: see above
- Solubility and stability of the test substance in the solvent/vehicle: The test item was used as supplied by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- no prior treatment

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: not specified
- Characteristics of donor animals: adult cattle, 12 to 60 months old, freshly slaughtered animals
- Storage, temperature and transport conditions of ocular tissue: placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL), transported over ice packs on the same day of slaughter
- Time interval prior to initiating testing: cornea prepared immediately on arrival

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 in vitro replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes excised by an abattoir employee after slaughter
- at the test facility, cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling
- iris and lens were peeled away from the cornea
- isolated corneas were immersed in a dish containing Hanks’ Balanced Salt Solution (HBSS) until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders
- anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged
- holders were incubated at 32 ± 1 ºC for 60 minutes
- at end of incubation period each cornea was examined for defects

QUALITY CHECK OF THE ISOLATED CORNEAS
- macroscopically examined before and after dissection
- corneas free of damage were used
- after pre-treatment an opacity reading was taken for each cornea using a calibrated opacitometer

NUMBER OF REPLICATES
- 3

CONTROLS USED
- as described above

APPLICATION DOSE AND EXPOSURE TIME
- as described above

TREATMENT METHOD:
- not specified

POST-INCUBATION: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times at the end of the exposure
- POST-EXPOSURE INCUBATION: holders were incubated, anterior chamber facing forward, at 32 ºC for 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading
- correction by subtracting the average change in opacity observed for the negative control corneas
- mean opacity value of each treatment group calculated by averaging the corrected opacity values of each cornea for that treatment group
- Corneal permeability:
- corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea
- OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group
- 1 in 5 dilution was performed where the OD492 was ≥1.3, all calculations were taken from the second run with the dilutions being performed

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA:
- classified according to the prediction model by UN GHS
- IVIS <=3 results in "No category", IVIS >3; <55 results in "no prediction can be made", IVIS>55 results in "category 1"

- acceptable test according to positive and negative control criteria:
- neat ethanol as positive control: test acceptable if the positive control produced an IVIS within two standard deviations of the historical mean collated during 2017 for this testing facility (29.6 - 65.1)
- sodium chloride 0.9% w/v as negative control: test acceptable if the negative control produced an In IVIS less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control (opacity ≤2.3 and for permeability ≤0.041)

Irritation parameter:

in vitro irritation score

Run / experiment:

test Item

Value:

6.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
The corneas treated with the test item were clear post treatment and slightly cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.
Further details are given in table 4.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.3 and permeability ≤0.041. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control IVIS was within the range of 29.6 to 65.1. The positive control acceptance criterion was therefore satisfied.

Table 2: Results of the in vitro irritancy

Treatment	In VitroIrritancy Score
Test Item	6.5
Negative Control	0.4
Positive Control	50.8

Table 3: Individual and mean opacity and permeability measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation -Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	6	6	5	0		0.002
	2	2	3	3	1		0.006
	3	3	3	3	0		0.000
					0.3*		0.003 $		0.4
Positive Control	4	5	29	29	24	23.7	2.015+	2.012
	5	4	24	27	23	22.7	2.015+	2.012
	6	3	28	30	27	26.7	1.270	1.267
						24.3 %		1.764 %	50.8
Test Item	16	2	3	9	7	6.7	0.028	0.025
	18	3	5	10	7	6.7	0.005	0.002
	19	4	5	10	6	5.7	0.000	0.000
						6.3 %		0.009 %	6.5

OD = Optical density * = Mean of the post-incubation − pre-treatment values $ = Mean permeability % = Mean corrected value

+ = A 1 in 5 dilution was performed. All calculations were taken from the second run with the dilutions being performed.

Table 4: Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	1	Clear	Clear
	2	Clear	Clear
	3	Clear	Clear
Positive Control	4	Cloudy	Cloudy
	5	Cloudy	Cloudy
	6	Cloudy	Cloudy
Test Item	16	Clear	Slightly Cloudy
	18	Clear	Slightly Cloudy
	19	Clear	Slightly Cloudy

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction for eye irritation or serious eye damage can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

EpiDerm SCT, OECD 431, 2018

A corrosion test on the EpiDerm human skin model was performed according to OECD 431 with treatment periods of 3 and 60 minutes. 

Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200mL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. Theoptical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minutes	100*	2.6	95.8
60 minutes	100*	2.9	106.2

The quality criteria required for acceptance of results in the test were satisfied.

The test item was considered to be non-corrosive to the skin.

EpiSkin SIT, OECD 439, 2018

An irritation test on the EpiSkin reconstructed human epidermis model was performed according to OECD 439. The treatment period was 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into microtubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

The relative mean viability of the test item treated tissues was 8.3% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

The quality criteria required for acceptance of results in the test were satisfied.

The test item was classified as irritant.

Eye irritation

BCOP, OECD 437, 2018

The study is performed to assess the potential of the test item to induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage.

The Bovine Corneal Opacity and Permeability (BCOP) according to OECD 437 test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1.

Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as follows: 

IVIS	UN GHS
≤ 3	No Category
>3; ≤ 55	No prediction can be made
> 55	Category 1

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items 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).

 

Treatment	In Vitro Irritancy Score
Test Item	6.5
Negative Control	0.4
Positive Control	50.8

No prediction of eye irritation can be made.

EpiOcular, OECD 492, 2018

An eye irritation study was performed according to OECD 492 with the purpose to evaluate the eye irritation potential of the test item using the reconstructed human EpiOcular model after a treatment period of 30 minutes followed by a post-exposure incubation period of 2 hours.

The principle of the assay was based on the measurement of cell viability following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Duplicate tissues were treated with the test item for an exposure period of 30 minutes. A benchmark control was tested alongside the test item to provide additional confidence in the assay results. At the end of the exposure period each tissue was rinsed before being immersed for 12 minutes in assay medium. Following the post-treatment immersion the tissues were incubated for 2 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each solution was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 87.8% after the 30 minute exposure period and 2 hour post-exposure incubation period.

The quality criteria required for acceptance of results in the test were considered to be satisfied although the negative control quality criterion was slightly exceeded.

This was reported as a deviation but it was considered unnecessary to repeat the test.

According to the conditions of the test, the test item was classified as non-irritant.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480. As a result the substance is considered to cause skin irritation, Cat. 2 under Regulation (EC) No. 1272/2008.