Molybdenum zinc tetraoxide

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Molybdenum zinc tetraoxide

EC Number:

237-377-8

EC Name:

Molybdenum zinc tetraoxide

Cas Number:

13767-32-3

Molecular formula:

MoO4Zn

IUPAC Name:

molybdenum(6+) zinc(2+) tetraoxidandiide

Test material form:

solid: particulate/powder

Test animals / tissue source

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.

Test system

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

Results and discussion

In vitro

Results

Results 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

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

1b

Value:

3.35

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.