Molybdenum zinc tetraoxide

Endpoint summary

Description of key information

Skin irritation/corrosion:

Key study: OECD 431. GLP study. After 3 minutes and 1 hour treatment, the mean values of relative tissue viability of the test item were increased to 102.2% and 98.2%, respectively. These values value are well above the threshold for corrosivity (50%) and therefore, the test item is considered as non-corrosive to skin.

Key study: OECD 439: GLP study: After the treatment with the test item, the mean value of relative tissue viability was reduced to 83.1 %. This value is above the threshold for skin irritation potential (50%). Therefore, the test item was considered non- irritant to skin.

Based on both results, the test item is considered non-irritant to skin.

Eye irritation/corrosion:

Key study: OECD 437. GLP study. The calculated IVIS was 3.35 in experiment 1b and 10.02 in experiment 1c. Being >3 and ≤ 55, the substance is not classified as Eye damage Category 1 but no further predictions can be made.

Key study: OECD 492. GLP study. The mean value of relative tissue viability was reduced to 53.7 %. This value is below the threshold for eye irritation potential (≤ 60%) and thus, the test item is considered either eye irritant or inducing serious eye damage.

Based on both results, the test item is considered as eye irritant.

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:

14 May 2018 - 17 May 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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This study was performed in order to evaluate the skin corrosion potential of Zinc molybdate to human skin in an in vitro study. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cyto-toxic to the underlying cell layers and its use is recommended by the relevant OECD guideline for corrosion testing (OECD No. 431).

Vehicle:

water

Remarks:

Demineralised water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM
- Tissue batch number(s): 28610
- Production date: MatTek In Vitro Life Science Laboratories, Brati-slava
- Delivery date: 15 May 2018
- Date of initiation of testing: 15 May 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C (and 5.0 ± 0.5% CO2)
- Temperature of post-treatment incubation: 37 ± 1°C (and 5.0 ± 0.5% CO2)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: DPBS, once
- Observable damage in the tissue due to washing: No.
- Modifications to validated SOP: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL (1 mg/mL in DPBS)
- Pre-incubation time: 1h
- Incubation time: Treatment: 3 minutes, 1 hour.
- MTT incubation time: 3h
- Spectrophotometer: Microtiter plate photometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
The values for negative control and for positive control were within the range of historical data of the test facility.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : No interference was detected.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Tissue 1: 25.1 mg (3 min); 27.1 mg (1h)
Tissue 2: 25.7 mg (3 min); 27.2 mg (1h)
- Concentration (if solution): 25 μL demineralised water.

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 μL
- Lot/batch no. (if required): Batch no: 20180221

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): Solution in demineralised water (8 M)

Duration of treatment / exposure:

3 minutes and 1 hour.

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

102.2

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean value of 2 tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

98.2

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean value of 2 tissues

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: No.
- Colour interference with MTT: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.7 (3 minutes) and 1.6 (1 hour).
- Acceptance criteria met for positive control: Yes . The positive control showed clear corrosive effects. The mean value of relative tissue viability was 6.7% (1 h).
- Acceptance criteria met for variability between replicate measurements: Yes. The RSD were 0.9% (3 min treatment) and 0.0% (1h treatment), being <30%.
- Range of historical values:
Optical density negative control - 3 min: 1.197 - 3.077
Optical density negative control - 1 h: 1.377 - 2.571
% Tissue variability positive control 3 min: 9.6 - 57.3%
% Tissue variability positive control 1h: 4.1 - 24.2%

Absorbance values (OD 570 nm):

Incubation	Negative Control		Test Item		Positive Control
	Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
3 min	1.694	1.691	1.726	1.707	0.427	0.407
	1.686	1.695	1.741	1.720	0.430	0.409
	1.688	1.678	1.740	1.713	0.433	0.411
1 h	1.723	1.643	1.652	1.647	0.141	0.154
	1.707	1.661	1.658	1.665	0.141	0.156
	1.744	1.644	1.660	1.661	0.145	0.155

Mean absorbance values - 3 minutes:

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.651	1.697	0.391
Mean – blank (tissue 2)	1.649	1.675	0.370
Mean	1.650	1.686	0.381
RSD	0.1%	0.9%	3.9%

Mean absorbance values - 1 hour:

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.686	1.618	0.104
Mean – blank (tissue 2)	1.611	1.619	0.116
Mean	1.648	1.619	0.110
RSD	3.2%	0.0%	8.1%

Tissue viability:

Test Item	Positive Control	Incubation
102.2%	23.1%	3 min
98.2%	6.7%	1 h

Interpretation of results:

GHS criteria not met

Conclusions:

After 3 minutes and 1 hour treatment, the mean values of relative tissue viability of the test item were increased to 102.2% and 98.2%, respectively. These values value are well above the threshold for corrosivity (50%) and therefore, the test item is considered as non-corrosive to skin.

Executive summary:

An in-vitro skin corrosion test was performed according to the OECD Guideline 431 (GLP study). Two tissues of the human skin model EpiDermTM were treated with the test item for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size. Demineralised water was used as negative control and 8 M KOH was used as positive control. After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution. The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.7 (3 minutes) and 1.6 (1 hour). The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 6.7%. The values for negative control and for positive control were within the range of historical data of the test facility. The mean value of relative tissue viability of the test item was increased to 102.2% after 3 minutes treatment. This value is above the threshold for corrosivity (50%). After 1 hour treat-ment, the mean value of relative tissue viability of the test item was reduced to 98.2%, lying above the threshold for corrosivity (15%). Therefore, the test item is considered as non-corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 June 2018 - 15 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This in vitro study was performed in order to evaluate the potential of the test item to evoke skin irritation in a reconstructed human epidermis (RhE) test method. Its use is recommended by the relevant OECD guideline for corrosion testing (OECD No. 431).

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT)
- Tissue batch number(s): 28623
- Delivery date: 12. June 2018
- Date of initiation of testing: 12 June 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C (and 5.0 ± 0.5% CO2)
- Temperature of post-treatment incubation: 37 ± 1°C (and 5.0 ± 0.5% CO2)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: DPBS
- Observable damage in the tissue due to washing: No.
- Modifications to validated SOP: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL (1 mg/mL)
- Treatment: 1
- Incubation time: 23h and 20 min
- Post-incubation time: 19h and 10 min of post-incubation
- MTT incubation time: 3h.
- Spectrophotometer: Microtiter plate photometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
The values for negative control and for positive control were within the range of historical data of the test facility.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : No interference.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure is less than 50% of the negative control.
- The test substance is considered to be irritant to skin if the viability after exposure is greater than or equal to 50% of the negative control.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Tissue 1: 27.0 mg
Tissue 2: 27.1 mg
Tissue 3: 26.5 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 μL

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight): 30 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SDS-solution in demineralised water
- Lot/batch no.: 040418SVA.

Duration of treatment / exposure:

1h

Duration of post-treatment incubation (if applicable):

42h and 30 min

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

78.6

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

90.4

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

80.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

83.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: No.
- Colour interference with MTT: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The OD of negative control was 1.5 (≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: % tissue viability of positive control SDS was 2.8% (≤ 20% of negative control)
- Acceptance criteria met for variability between replicate measurements: SD of mean viability of the tissue replicates (%) were 4.6% (negative control), 0.5% (positive control) and 6.3% (test item), being ≤ 18%.
- Range of historical values:
Negative Control (OD): 0.476 – 2.471
Positive Control (% OD compared to Negative Control): 1.7 – 17.1%

Absorbance values (OD 570 nm):

Designation	Measurement	Negative Control	Zinc molybdate	Positive Control
Tissue 1	1	1.532	1.241	0.068
	2	1.528	1.234	0.082
Tissue 2	1	1.636	1.426	0.078
	2	1.652	1.407	0.082
Tissue 3	1	1.516	1.266	0.097
	2	1.519	1.265	0.083

Mean absorbance values:

Designation	Negative Control	Zinc molybdate	Positive Control
Mean – blank (tissue 1)	1.491	1.199	0.036
Mean – blank (tissue 2)	1.605	1.378	0.041
Mean – blank (tissue 3)	1.479	1.227	0.051
Mean of the three tissues	1.525	1.268	0.043

% Tissue viability:

Designation	Zinc molybdate	Positive Control
% Tissue viability (tissue 1)	78.6%	2.4%
% Tissue viability (tissue 2)	90.4%	2.7%
% Tissue viability (tissue 3)	80.5%	3.3%
% Tissue viability (mean)	83.1%	2.8%
± SD of mean tissue viability (%)	6.3%	0.5%

Interpretation of results:

GHS criteria not met

Conclusions:

The mean value of relative tissue viability was reduced to 83.1%. This value is above the threshold for skin irritation (50%). Therefore, the test item Zinc molybdate is considered as non- irritant to skin.

Executive summary:

An in-vitro skin irritation test was performed according to the OECD Guideline 439 (GLP study). Three tissues of the human skin model EpiDermTM were treated with Zinc molybdate for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.5. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.8% (≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 83.1 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non- irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2018 - 21 May 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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according 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 (incl. QA statement)

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Müller Fleisch GmbH slaughterhouse. Enzstr. 2-4, 75217 Birkenfeld, Germany
- Characteristics of donor animals (e.g. age, sex, weight): The cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were transported to the test facility in Hanks’ Balanced Salt Solu-tion with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour 20 minutes (exp. 1b) or 1 hour 5 minutes (exp. 1c).
- Time interval prior to initiating testing: Fresh bovine eyes were obtained on the day of the test
- indication of any existing defects or lesions in ocular tissue samples: No.
- Indication of any antibiotics used: No.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20% concentration in HBSS.

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES : 3

SOLVENT CONTROL USED: Yes:
HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in de-min. water (1:10), batch no.: 20180515 (exp. 1b) and 20180524 (exp. 1c)

POSITIVE CONTROL USED : Yes:
Imidazole solution: 20% C3H4N2 (CAS-No. 288-32-4), dissolved in HBSS, batch no.: 20180515 (exp. 1b) and 20180524 (exp. 1c)

APPLICATION DOSE AND EXPOSURE TIME :

TREATMENT METHOD: open chamber
The test item was given directly on the epithelium in such a manner that as much as possible of the cornea was covered with test item. Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.

After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

POST-INCUBATION PERIOD: No.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

DECISION CRITERIA:The decision criteria as indicated in the TG was used.
≤ 3 - No category
> 3 and ≤ 55 - No prediction can be made
> 55 - Eye damage Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1a

Negative controls validity:

not valid

Remarks on result:

not determinable

Remarks:

Study invalid: opacity values of the negative control were too high

Irritation parameter:

in vitro irritation score

Run / experiment:

1b

Value:

3.35

Vehicle controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

1c

Value:

10.02

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Annually, proficiency chemicals are tested in order to ensure the accuracy and reliability of the test method over time.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean IVIS of the negative control has to show an IVIS ≤ 3 (1.68 (exp. 1b) and 2.36 (exp. 1c)).
- Acceptance criteria met for positive control: Yes. IVIS of positive control 20% imidazole solution falled within two standard deviations of the current historical mean, i.e. between 69.37 – 157.73 (98.54 (exp. 1b) and 123.48 (exp. 1c)).
- Range of historical values if different from the ones specified in the test guideline:
Range of IVIS (validity) - Negative control HBSS: ≤ 3
Range of IVIS (validity) - Positive control 20% imidazole solution: 69.37 – 157.73
Range of opacity - Negative control HBSS: -1.86 – 4.08
Range of opacity - Positive control 20% imidazole solution: 41.72 – 133.11
Range of Permeability - Negative control HBSS: -0.02 – 0.10
Range of Permeability - Positive control 20% imidazole solution: 0.75 – 5.89

The first experiment was aborted during the second opacity measurement, because the opacity values of the negative control were too high. Therefore the experiment was declared invalid and repeated. The second experiment (experiment 1b) was valid, but the test item result was equivocal, because 2 of the 3 replicates gave discordant prediction from the mean. This is why a fur-ther run was performed (experiment 1c). As the result of experiment 1c corroborated the prediction of the experiment 1b (based upon the mean IVIS value), a final decision could be achieved without further testing.

Opacity and Permeability Values of Experiment 1b:

Illuminance values:

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	1008	997	996	1015	1018	1013	1017	1015	1051
(I) Measured values after exposure	958	975	974	953	853	927	326	356	421

Opacity values negative control:

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.10	3.56	3.61
Opacity after exposure	5.31	4.53	4.58
Opacity Difference	2.22	0.97	0.97
Mean Opacity Difference	1.39

Opacity values test item and positive control:

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.80	8.68	2.89	2.72	2.80	1.36
Opacity after exposure	5.55	10.82	6.81	92.04	80.96	62.37
Opacity Difference	2.75	8.14	3.92	89.32	78.16	61.02
Opacity Differencecorrected	1.36	6.76	2.54	87.93	76.77	59.63
Mean Opacity Diff. corr.	3.55			74.78

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.044
2. Measurement	0.044
3. Measurement	0.047
Mean	0.045

Optical density at 492 nm of negative control, test item and positive control:

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1. Measurement	0.091	0.048	0.054	0.071	0.049	0.034	1.469	1.434	2.065
2. Measurement	0.092	0.047	0.055	0.071	0.050	0.032	1.471	1.433	2.033
3. Measurement	0.090	0.049	0.057	0.073	0.051	0.033	1.476	1.429	2.034
1. Measurement – blank	0.0460	0.0030	0.0090	0.0260	0.0040	-0.0110	1.4240	1.3890	2.0200
2. Measurement – blank	0.0470	0.0020	0.0100	0.0260	0.0050	-0.0130	1.4260	1.3880	1.9880
3. Measurement – blank	0.0450	0.0040	0.0120	0.0280	0.0060	-0.0120	1.4310	1.3840	1.9890
Mean of each replicate	0.0460	0.0030	0.0103	0.0267	0.0050	-0.0120	1.4270	1.3870	1.9990
Mean of the 3 replicates	0.0198			--			--
Corrected	--	--	--	0.0069	-0.0148	-0.0318	1.4072	1.3672	1.9792
Corrected mean of the 3 replicates	--			-0.0132			1.5846

IVIS:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	2.91	1.68	63.12%
	1.01
	1.13
Test Item	1.46	3.35	82.66%
	6.53
	2.06
Positive Control 20% imidazole solution	109.04	98.54	10.07%
	97.28
	89.32

Opacity and Permeability Values of Experiment 1c:

Illuminance values:

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	993	995	1024	996	1015	994	986	991	986
(I) Measured values after exposure	919	972	997	732	823	815	308	319	302

Opacity values negative control:

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.74	3.65	2.43
Opacity after exposure	7.21	4.67	3.56
Opacity Difference	3.48	1.02	1.13
Mean Opacity Difference	1.88

Opacity values test item and positive control:

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.61	2.80	3.69	4.04	3.83	4.04
Opacity after exposure	19.13	12.65	13.16	99.72	94.92	102.48
Opacity Difference	15.52	9.85	9.47	95.67	91.10	98.44
Opacity Differencecorrected	13.64	7.97	7.59	93.80	89.22	96.56
Mean Opacity Diff. corr.	9.74			93.19

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.037
2. Measurement	0.045
3. Measurement	0.044
Mean	0.042

Optical density at 492 nm of negative control, test item and positive control:

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1. Measurement	0.057	0.117	0.049	0.045	0.037	0.196	0.547	1.542	2.186
2. Measurement	0.057	0.116	0.049	0.042	0.039	0.201	0.548	1.540	2.206
3. Measurement	0.054	0.118	0.050	0.044	0.041	0.195	0.539	1.525	2.171
1. Measurement – blank	0.0150	0.0750	0.0070	0.0030	-0.0050	0.1540	0.5050	1.5000	2.1440
2. Measurement – blank	0.0150	0.0740	0.0070	0.0000	-0.0030	0.1590	0.5060	1.4980	2.1640
3. Measurement – blank	0.0120	0.0760	0.0080	0.0020	-0.0010	0.1530	0.4970	1.4830	2.1290
Mean of each replicate	0.0140	0.0750	0.0073	0.0017	-0.0030	0.1553	0.5027	1.4937	2.1457
Mean of the3 replicates	0.0321			--			--
Corrected	--	--	--	-0.0304	-0.0351	0.1232	2.4812*	1.4616	2.1136
Corrected mean of the 3 replicates	--			0.0192			2.0188

IVIS:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	3.69	2.36	52.38%
	2.14
	1.24
Test Item	13.19	10.02	29.06%
	7.45
	9.44
Positive Control 20% imidazole solution	131.02	123.48	8.72%
	111.14
	128.27

Interpretation of results:

other:

Conclusions:

The calculated IVIS was 3.35 in experiment 1b and 10.02 in experiment 1c. Being >3 and ≤ 55, the substance is not classified as Eye damage Category 1 but no further predictions can be made.

Executive summary:

An ex-vivo eye damage test was performed according to the OECD Guideline 437 (GLP study). Three experiments were performed. The first experiment was aborted during the second opacity measurement, because the opacity values of the negative control were too high. Therefore the experiment was declared invalid and repeated. The second experiment (experiment 1b) was valid, but the test item result was equivocal, because 2 of the 3 replicates gave discordant prediction from the mean. This is why a further run was performed (experiment 1c). The result of experiment 1c corroborated the prediction of the experiment 1b (based upon the mean IVIS value), and a a final decision could be achieved. In all experiments bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old. The test item was brought onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) was 1.68 in experiment 1b and 2.36 in experiment 1c. 20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS was 98.54 in experiment 1b and 123.48 in experiment 1c. Under the conditions of this study, the test item showed effects on the cornea of the bovine eye in both valid experiments. The calculated IVIS was 3.35 in experiment 1b and 10.02 in experiment 1c. The calculated IVIS was 3.35 in experiment 1b and 10.02 in experiment 1c. Being >3 and ≤ 55, the substance is not classified as Eye damage Category 1 but no further predictions can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June 2018 - 21 June 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability :
The method used in the present in-vitro test, allows the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye
irritation or serious eye damage in accordance with UN GHS. It makes use of reconstructed human cornea-like epithelium (RhCE) which closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The EpiOcular™ Eye Irritation Test (EIT) is a validated test method and it is included in the OECD Guideline 492.

- Description of the cell system used:

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):
Tissue 1: 52.5 mg
Tissue 2: 50.4 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used :
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 29 minutes. After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate in one- minute- intervals. At the beginning of each experiment, a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minuteintervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in
pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT assay was performed.

- RhCE tissue construct used, including batch number :
The EpiOcular tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

EpiOcular tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 19. Jun. 2018
Batch no.: 27046

- Doses of test chemical and control substances used
Test item: 52.5 mg (Tissue 1), 50.4 mg (Tissue 2)
Negative control: 50 μL
Positive control: 50 μL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Treatment incubation: 6hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
Post-exposure immersion: 25 minutes
Post-treatment incubation: 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light. The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate. Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
% Viability > 60% - Non eye irritant
% Viability ≤ 60 % - At least eye irritant (no prediction can be made - Category 1 or 2).

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria :
Optical density negative control: 1.610 ± 0.275 (1.047-2.340)
Relative tissue viability positive control: 34.5 ± 7.5% (21.1-53.9%)

Irritation parameter:

other: % Viability

Run / experiment:

1

Value:

55.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % Viability

Run / experiment:

2

Value:

51.5

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % Viability

Run / experiment:

Mean

Value:

53.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. OD of negative control was 2.0 (> 0.8 and < 2.5).
- Acceptance criteria met for positive control: Yes. % mean relative viability of positive control was 38.2 (< 50% of negative control).
- Acceptance criteria met for variation within replicates: Yes. They were 1.6% (negative control), 5.0% (positive control) and 4.4% (test item) (<20%).
- Range of historical values if different from the ones specified in the test guideline:
Optical density negative control: 1.610 ± 0.275 (1.047-2.340)
Relative tissue viability positive control: 34.5 ± 7.5% (21.1-53.9%)

Absorbance values (OD at 570 nm):

Designation	Measurement	Negative Control	Positive Control	Zinc molybdate
Tissue 1	1	1.986	0.831	1.136
	2	1.994	0.847	1.141
Tissue 2	1	2.009	0.742	1.053
	2	2.035	0.741	1.049

Mean absorbance:

Designation	Negative Control	Positive Control	Zinc molybdate
Mean – blank (Tissue 1)	1.952	0.801	1.101
Mean – blank (Tissue 2)	1.984	0.704	1.013

% Viability:

Designation	Positive Control	Zinc molybdate
% Viability (Tissue 1)	40.7%	55.9%
% Viability (Tissue 2)	35.7%	51.5%
% Viability Mean	38.2%	53.7%

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The mean value of relative tissue viability was reduced to 53.7 %. This value is below the threshold for eye irritation potential (≤ 60%) and thus, the test item is considered either eye irritant or inducing serious eye damage.

Executive summary:

An in-vitro eye damage test was performed according to the OECD Guideline 492 (GLP study). The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and <2.5, OD was 2.0. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 38.2 % (< 50%).

Variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 53.7 %. This value is below the threshold for eye irritation potential (≤ 60%) and thus, test items that induce values below the threshold are either eye irritant or inducing serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Skin irritation/corrosion: Based on available data, the substance is not classified for skin irritation according to the CLP Regulation (EC) no. 1272/2008.

Eye irritation/damage: Based on available data, the substance is classified as Eye irritant, Category 2 (H319) according to the CLP Regulation (EC) no. 1272/2008.