MoVNbTe mixed oxide

Endpoint summary

Description of key information

In two GLP compliant OECD TG (439 & 431) the skin irritation and corrosion potency of the test item was evaluated. Further, OECD TG 437 and 405 were performed to evaluate the eye irritation potential.

Within the OECD TG 439, a viability measured via MTT assay of 45.7 % obtained. To to this result, the test item is considered as skin irritant Category 1 or 2 and a subsequent in vitro assay to elucidate the category (OECD TG 431) was performed.

 

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:

16 December 2020 To 29 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 439

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

To evaluate the skin irritation potential of Mo10V3TeNbO42 using Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200- SIT).

GLP compliance:

yes (incl. QA statement)

Remarks:

With GLP Certificate

Specific details on test material used for the study:

Physical Appearance (with color): Solid (black)
Batch No.: EX.14402.600
Date of Manufacture: Oct 2017
Date of Expiry: No change of properties known over time (endless)
Storage Conditions: Ambient (21 to 29ºC)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek In vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic, www.mattek.com; Phone: [phone]; Fax: [phone].

Justification for test system used:

As recommended in OECD Guideline No. 439, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT) has been selected as test system for in vitro skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

The Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT) was used as test system

Control samples:

yes, concurrent negative control

Amount/concentration applied:

Quantity of 30 µL of DPBS (NC) and 5% SDS (PC) were dispensed directly atop the tissue at 1 minute intervals to facilitate rinsing of the NC and PC after exposure.

The tissues were exposed to 25 mg of test item.

Duration of treatment / exposure:

1 hour and 48 hours

Duration of post-treatment incubation (if applicable):

Percentage viability of tissues was measured by performing MTT assay after 60 minutes of treatment and post incubation of 43 hours and 7 minutes.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three replicates

Value:

45.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

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

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results obtained under the laboratory testing conditions, the test item is categorized as irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS (Category 2 or Category 1), as the mean percentage of tissue viability was less than 50% of the negative control after 60 minutes of exposure and 43 hours and 7 minutes post incubation.
Since a subsequent study performed according to OECD TG 431 (see chapter 7.3.1) did not justify a classification as Skin Corrosive Category 1, a justification as Skin Irritant Category in accordance with UN GHS is justified.

Executive summary:

The mean OD of the negative controltissues is 1.273 which is within the range of ≥0.8 and ≤2.8, hence the tissues were considered as viable after shipping and storing procedures and under specific conditions of use.Certificate of analysis, histology report and images of EpiDerm tissues of lot No. 33783 provided by MatTekwas provided as Annexures 3, 4 and 5 respectively. Percentage viability of tissues was measured by performing MTT assay after 60 minutes of treatment and post incubation of 43 hours and 7 minutes.The mean percentage viability of test item treated tissues was 45.7 which were <50% of the negative control, hence the test item is considered as irritantand the mean percentage viability of positive controltreated tissues was 4.4 which were <50% of the negative control clearly represents the irritation potential of positive control.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 December 2021 to 08 June 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 431

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP Certificate

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek in vitro Life Science Laboratories

Justification for test system used:

The Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) was selected as test system to assess the skin corrosion potential of the test item as it represents a recommended in vitro test system according to OECD Guideline No. 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

OECD 431

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of two replicates for 3 min exposure

Value:

100.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 2 replicates of 1 h exposure

Value:

89.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results obtained under the conditions of this study, the test item is considered as non-corrosive in accordance with UN GHS, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Executive summary:

The mean OD of the negative control tissues was 2.537 (3 minutes exposure) and 2.208 (1 hour exposure) which was within the range of ≥0.8 and ≤2.8, hence the tissues were considered as viable after shipping and storing procedures and under specific conditions of use. Percentage viability of tissues was measured by performing MTT assay after 3 minutes and 1 hourof treatment. The mean percentage viability of test item treated tissues was 100.0% after 3 minutes of exposure which were >50% and 100.0% after 1 hour of exposure which were >15% of the negative control hence the test item is considered as non-corrosive.The mean percentage viability of positive controltreated tissues was 2.5% after 3 minutes and 2.1% after 1 hour exposure which were <15% of the negative control clearly represents the irritation potential of positive control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 December 2020 to 04 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Since results of an ex vivo study on eye irritation indicated equivocal results and no stand-alone prediction can be made, a subsequent in vivo study was needed to fulfill the data requirements.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

(Section 4: Health Effects), “Acute Eye Irritation/Corrosion” adopted on 09 October 2017 (Corrected on 26 June 2020)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: EX. 14402. 600
- Expiration date of the batch: No change of properties known over time (endless)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Adita Biosys Private Limited
- Age at study initiation: 4 months
- Weight at study initiation: 2.39066 kg to 2.42196 kg
- Housing: stainless steel wire mesh cage L 24 x B 18 x H 18 inches
- Diet (e.g. ad libitum): Altromin Maintenance diet for rabbits – Rich in crude fibre 2123
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: Start: 21 December 2020 End: 31 December 2020

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3°C to 22.8°C
- Humidity (%): 47% to 67%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 26 December 2020 To: 04 January 2021

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (weight with unit): Initial test: 45.2 mg and Confirmatory test: 47.6 mg and 49.1 mg

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

1, 24, 48 and 72 hour

Number of animals or in vitro replicates:

3 (1 for initial test, 2 for confirmatory test)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eye was rinsed using 0.9% w/v normal saline after 1 hour treatment (post treatment)
- Time after start of exposure: 1 hour post test item instillation

SCORING SYSTEM: Cornea, area of cornea involved, iris, conjuctivae and chemoses was scored after 1, 24, 48 and 72 hour post test item instillation all with a maximum score of 4

TOOL USED TO ASSESS SCORE: hand-slit lamp and fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Other effects:

- Lesions and clinical observations: No clinical signs of toxicity and mortality were observed in both initial and confirmatory test animals

In both initial and confirmatory test, treated eye (left) revealed occular lesions like redness [Some blood vessels hyperaemic (injected)] at 1, 24 and 48 hour observation and chemosis (Some swelling above normal) at 24 hour observation. The observed lesion reversed back to normal by 72 hour observation

In both initial and confirmatory test, ocular lesion like conjunctivitis were observed at 24 and 48 hour observation. No ocular lesion was observed at 72 hour observation during slit lamp examination

- Effects of rinsing or washing: no effects on the eye due to washing

             TABLE 1. INDIVIDUAL ANIMAL CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Phase of the Experiment	Dose (mL/animal)	Animal No.	Sex	Clinical Signs of Toxicity and Mortality on Day
				1		2		3		4
				#	*	#	*	#	*	#
Initial Test	0.1	Nb5987	M	N	N	N	N	N	N	N
Confirmatory Test	0.1	Nb5988	M	N	N	N	N	N	N	N
	0.1	Nb5989	M	N	N	N	N	N	N	N

           M: Male; N: Normal; #: First observation; *: Second observation

            TABLE 2. INDIVIDUAL ANIMAL EYE IRRITATION/CORROSION SCORING RECORD

		Chemosis			Opacity			Area
Eyes		LE		RE		LE		RE		LE		RE		LE		RE		LE		RE
1 hr		1		0		0		0		0		0		0		0		-		-
24 hrs		1		0		1		0		0		0		0		0		-		-
48 hrs		1		0		0		0		0		0		0		0		-		-
72 hrs		0		0		0		0		0		0		0		0		-		-
Mean Tissue Score		0.67		0		0.33		0		0		0		0		0		-		-

hr: hour; hrs: hours; LE: Left Eye (Treated Eye); RE: Right Eye (Untreated/Reference control Eye);

Mean Tissue Score = (24 hr+48 hr+72 hr)/3

Redness: 0: Normal; 1: Some blood vessels hyperaemic (injected)

Chemosis:0: Normal; 1: Some swelling above normal

Iris:0: Normal               

Opacity:0: No ulceration or opacity

 

TABLE 2 (Contd...).  INDIVIDUAL ANIMAL EYE IRRITATION/CORROSION SCORING RECORD

Confirmatory Test                           Sex: Male                     Dose: 0.1 mL/animal      Animal No.:Nb5988
Observation Period	Ocular Lesions
	Conjunctiva				Iris		Cornea
	Redness		Chemosis				Opacity		Area
Eyes	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
1 hr	1	0	0	0	0	0	0	0	-	-
24 hrs	1	0	1	0	0	0	0	0	-	-
48 hrs	1	0	0	0	0	0	0	0	-	-
72 hrs	0	0	0	0	0	0	0	0	-	-
Mean Tissue Score	0.67	0	0.33	0	0	0	0	0	-	-

 

 

Confirmatory Test                           Sex: Male                     Dose: 0.1 mL/animal      Animal No.:Nb5989
Observation Period	Ocular Lesions
	Conjunctiva				Iris		Cornea
	Redness		Chemosis				Opacity		Area
Eyes	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
1 hr	1	0	0	0	0	0	0	0	-	-
24 hrs	1	0	1	0	0	0	0	0	-	-
48 hrs	1	0	0	0	0	0	0	0	-	-
72 hrs	0	0	0	0	0	0	0	0	-	-
Mean Tissue Score	0.67	0	0.33	0	0	0	0	0	-	-

hr: hour; hrs: hours; LE: Left Eye (Treated Eye); RE: Right Eye (Untreated/Reference control Eye)

Mean Tissue Score = (24 hr+48 hr+72 hr)/3

Redness: 0: Normal; 1: Some blood vessels hyperaemic (injected)

Chemosis:0: Normal; 1: Some swelling above normal

Iris:0: Normal               

Opacity:0: No ulceration or opacity

                                       TABLE 3.  INDIVIDUAL ANIMAL SLIT LAMP EXAMINATION RECORD

Initial Test                   Sex: Male                       Dose: 0.1 mL/animal                         Animal No.: Nb5987
	Day 2 (24 hours)		Day 3 (48 hours)		Day 4 (72 hours)
Eye	LE	RE	LE	RE	LE	RE
Lids	13	N	13	N	N	N
Ducts	N	N	N	N	N	N
Cornea	N	N	N	N	N	N
Pupil	N	N	N	N	N	N
Sclera	N	N	N	N	N	N
Ciliary Bodies	N	N	N	N	N	N
Iris	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N
Lens	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N

LE: Left Eye; RE: Right Eye; N: Normal/No Abnormality Detected; 13: Conjunctivitis

 

  

TABLE 3 (Contd…). INDIVIDUAL ANIMAL SLIT LAMP EXAMINATION RECORD

Confirmatory Test                   Sex: Male                       Dose: 0.1 mL/animal            Animal No.: Nb5988
	Day 2 (24 hours)		Day 3 (48 hours)		Day 4 (72 hours)
Eye	LE	RE	LE	RE	LE	RE
Lids	13	N	13	N	N	N
Ducts	N	N	N	N	N	N
Cornea	N	N	N	N	N	N
Pupil	N	N	N	N	N	N
Sclera	N	N	N	N	N	N
Ciliary Bodies	N	N	N	N	N	N
Iris	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N
Lens	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N

 

 

Confirmatory Test                   Sex: Male                       Dose: 0.1 mL/animal            Animal No.: Nb5989
	Day 2 (24 hours)		Day 3 (48 hours)		Day 4 (72 hours)
Eye	LE	RE	LE	RE	LE	RE
Lids	13	N	13	N	N	N
Ducts	N	N	N	N	N	N
Cornea	N	N	N	N	N	N
Pupil	N	N	N	N	N	N
Sclera	N	N	N	N	N	N
Ciliary Bodies	N	N	N	N	N	N
Iris	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N
Lens	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N

LE: Left Eye; RE: Right Eye; N: Normal/No Abnormality Detected; 13: Conjunctivitis

 

   TABLE 4.   INDIVIDUAL ANIMAL BODY WEIGHT (kg) AND PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1

Phase of the Experiment	Dose (mL/animal)	Animal No.	Sex	Body Weight (kg) on Day		Percent Change in Body Weight with Respect to Day
				1	4	1 to 4
Initial Test	0.1	Nb5987	Male	2.47619	2.51406	1.52937
Confirmatory Test	0.1	Nb5988	Male	2.49617	2.52473	1.14415
	0.1	Nb5989	Male	2.51403	2.54009	1.03658
			Mean	2.50510	2.53241	1.09037
			±SD	0.01263	0.01086	0.07606
			n	2	2	2

 SD: Standard deviation; n: Number of animals

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the above results of the experiment and under the experimental conditions employed, it is concluded that the mean score of the test item does not meet classification criteria and hence not classified or categorized as per the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

The test item was evaluated for Acute Eye Irritation/Corrosion in New Zealand White Rabbits.

The study was performed in two phases i.e., initial and confirmatory tests. Both the eyes of each experimental animal was examined within 24 hours before the treatment. The animals with absence of signs of eye irritation, ocular defects or pre-existing corneal injury were used for the treatment. The pH of test item was 3.08 as per in-house pH determination.

 

The initial test was conducted using single male rabbit and confirmatory test was conducted using two male rabbits. The systemic analgesic (tramadol hydrochloride injection 2.5 mg/kg) and two drops of topical ocular anaesthetic (0.5% tetracaine hydrochloride) were given to each animal sixty minutes and five minutes prior to test item instillation respectively. The 0.1 mL (Initial test: 45.2 mg and Confirmatory test: 47.6 mg and 49.1 mg) of test item was instilled into the conjunctival sac of the left eye and right eye served as control. The eyes were scored approximately at 1, 24, 48 and 72 hours. Slit lamp examination was carried out using fluorescein strips (fluorescein sodium ophthalmic strip USP) and were scored approximately at 24, 48 and 72 hours for both initial and confirmatory test.

 

All the animals (initial test and confirmatory test) were observed twice daily for clinical signs of toxicity and mortality. No treatment related clinical signs of toxicity and mortality were observed in all the animals (initial and confirmatory tests) after the test item instillation.

 

In both initial and confirmatory test, treated eye (left) revealed occular lesions like redness at 1, 24 and 48 hour; chemosis at 24 hour observation. The observed lesion reversed back to normal by 72 hour observation.

 

In both initial and confirmatory test, ocular lesion like conjunctivitis were observed at 24 and 48 hour observation. No ocular lesion was observed at 72 hour observation during slit lamp examination.

 

The body weight was recorded on the day of receipt, on the day of treatment (prior to instillation of test item) and at termination of the experiment. No changes were noted in body weight and percent change in body weight with respect to day 1 in both initial and confirmatory test. All the animals revealed physiologically normal increase in body weight in both initial and confirmatory tests.

 

All the animals were sacrificed by intravenous administration of sodium thiopentone and carcass was disposed.

 

For initial test and confirmatory test, the mean score calculated across 3 scoring times (approximately 24, 48 and 72 hours after test item instillation) for cornea, iris and conjunctival redness and conjunctival chemosis were 0, 0, 0.67 and 0.33 respectively

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03 November 2020 to 02 December 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Study No. BIO-ALT 047, entitled “Bovine Corneal Opacity and Permeability Test of Mo10V3TeNbO42” was performed in compliance with the OECD Principles of Good Laboratory Practice [C(97)186/Final]

Species:

cattle

Details on test animals or tissues and environmental conditions:

Justification for selection of Test system: Cornea of the cattle is the recommended test system in the guideline.

Vehicle:

water

Remarks:

Name : Distilled water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

G1 Vehicle Control 750 µL of distilled water -
G2 Positive Control 750 µL of Imidazole 20% w/v
G3 Test Item 750 µL of test item 20% w/v

Duration of treatment / exposure:

3 hours and 55 minutes

Duration of post- treatment incubation (in vitro):

3 hours and 55 minutes

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: water

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 20 % w/v, 3 h 55 min

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: with EMEM (with phenol red) until no visual evidence of the test item was apparent. Subsequent washing step with MEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry at 49 0nm
- Other: Histopathological evaluation

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: IVIS ≤ 3 considered as UN GHS no category. IVIS score, >3 and ≤ 55 considered as equivocal, subsequently testing with any other adequate method needed. IVIS > 55 considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.

Irritation parameter:

cornea opacity score

Run / experiment:

mean of three replicates

Value:

44.89

Vehicle controls validity:

valid

Remarks:

Distilled water

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Imidazole

Remarks on result:

positive indication of irritation

Irritation parameter:

fluorescein leakage

Remarks:

Corrected Permeability Value

Run / experiment:

Mean of 3 replicates

Value:

0.009

Vehicle controls validity:

valid

Remarks:

distilled water

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Imidazole

Remarks on result:

no indication of irritation

Irritation parameter:

histopathological observations

Run / experiment:

observations in thrre replicates

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The results indicated an increase in the endpoint “opacity” but not “permeability”. As the test item resulted in IVIS >3; ≤ 55, it is considered as equivocal and classified as UN GHS no stand-alone prediction can be made. Hence, further testing is needed.

• SUMMARY OF IN VITRO OPACITY SCORE

               Refer Appendix - 2, 3 & 4

Group & Treatment	Initial Opacity Reading (I)	Opacitometer Reading (After Treatment)	Change in Opacity Value	Corrected Opacity Value	Average of Permeability Value	Corrected Permeability Value	Individual IVIS Value	Mean of Corrected Opacity Value	Corrected Permeability Value	IVIS Value
G1 & Vehicle Control	6.073	6.164	0.091	1.094	0.060	0.061	-	-	-	-
	6.256	6.905	0.649		0.062
	6.119	8.662	2.543		0.061
G2 & Positive  Control	6.301	132.116	125.815	124.720	1.636	1.575	148.3	120.34	1.573	143.9
	5.983	113.696	107.713	106.619	1.634	1.573	130.2
	5.983	136.752	130.769	129.675	1.632	1.571	153.2
G3 &Test Item	6.439	48.667	42.228	41.133	0.051	-0.010	41.0	44.89	-0.009	44.8
	6.717	52.204	45.487	44.393	0.053	-0.008	44.3
	6.393	56.641	50.248	49.153	0.052	-0.009	49.0

IVIS: In Vitro Irritancy Score.

 

Note:

Change in Opacity Value = Opacity reading (after treatment) - initial opacity reading (I).

Corrected Opacity Value = Change in opacity value of positive/test item – mean change in opacity value of vehicle control.

Corrected Permeability Value = Average of permeability value of positive/test item - mean Average of permeability value of vehicle control.

• INDIVIDUAL DATA OF ANIMAL AGE AND SEX

Sl. No.	Approximate Age (Years)	Sex
1	4.0	Female
2	4.5	Male
3	3.5	Female
4	4.0	Male
5	4.5	Male
6	3.5	Female
7	4.0	Male
8	4.5	Male

• INDIVIDUAL DATA OF SELECTION AND DISTRIBUTION OF CORNEAS

Initial Reading of Empty Cornea Holder with Media

Cornea Holder No.		Opacitometer I0 Value			Mean I0 Value
1		1134			1145
2		1180
4		1174
5		1105
6		1167
7		1168
8		1177
9		1204
10		1168
12		1189
14		1108
15		1192
17		1094
18		1010
20		1109
Opacity Reading (Before Treatment)
Corneal Holder No.	Initial Opacitometer Reading (I)		Opacity=I0/I	Opacity Value [= (I0/I-b)/a]
1	998		1.148	6.3
2	1005		1.140	6.0
4	854		1.341	14.0
5	742		1.543	22.1
6	1002		1.143	6.1
7	995		1.151	6.4
8	586		1.954	38.4
9	989		1.158	6.7
10	698		1.641	26.0
12	1003		1.142	6.1
14	657		1.743	30.0
15	996		1.150	6.4
17	1005		1.140	6.0
18	999		1.146	6.3

APPENDIX 2 (Contd…) INDIVIDUAL DATA OF SELECTION AND DISTRIBUTION OF CORNEAS Selection and Distribution of Cornea - Before Treatment
Corneal Holder No.	Group & Treatment	Initial Opacitometer Reading (I) Before Treatment	Opacity=I0/I	Opacity Value  (= (I0/I-b)/a)	Mean Opacity Value
12	G1 & Vehicle Control	1003	1.142	6.0734	6.1
18		999	1.146	6.2555
6		1002	1.143	6.1188
1	G2 & Positive  Control	998	1.148	6.3013	6.1
2		1005	1.140	5.9828
17		1005	1.140	5.9828
7	G3 & Test Item	995	1.151	6.4391	6.5
9		989	1.158	6.7173
15		996	1.150	6.3931

 a=0.0251, b=0.9894, I₀=1145

• INDIVIDUAL DATA OF OPACITY READINGS

Group & Treatment	Corneal  Holder No.	Opacitometer Reading (After Treatment)	Opacity=I0/I	Opacity Value            (= (I0/I-b)/a)	Mean Opacity Value
G1 & Vehicle Control	12	1001	1.144	6.164	7.24
	18	985	1.163	6.905
	6	949	1.207	8.662
G2 & Positive  Control	1	266	4.306	132.116	127.52
	2	298	3.843	113.696
	17	259	4.422	136.752
G3 &Test Item	7	518	2.211	48.667	52.50
	9	498	2.300	52.204
	15	475	2.411	56.641

  I₀=1145 a=0.0251, b=0.9894

• INDIVIDUAL PERMEABILITY VALUES

After Treatment

 

Group & Treatment	Replicate	Permeability OD490 value			Average  Permeability
G1 & Vehicle Control	R1	0.059	0.062	0.060	0.060
	R2	0.061	0.061	0.064	0.062
	R3	0.062	0.059	0.062	0.061
G2 & Positive  Control	R1	1.632	1.628	1.648	1.636
	R2	1.638	1.632	1.633	1.634
	R3	1.636	1.638	1.623	1.632
G3 & Test Item	R1	0.050	0.052	0.052	0.051
	R2	0.053	0.054	0.051	0.053
	R3	0.053	0.052	0.052	0.052

    R=Replicates, IVIS= Mean opacity Value + (15×Mean Permeability OD₄₉₀ value)

• MICROSCOPIC OBSERVATIONS OF BOVINE CORNEAS

Group	Group Description (Treatment)	Corneal Holder No.	Observations
G1	Vehicle Control (Distilled water)	6	NAD
		12	NAD
		18	NAD
G2	Positive Control (Imidazole)	1	Epithelium:	·      Epithelial cell loss, Squamous layer, mf, 3 ·      Cytoplasmic coagulation, Squamous and Wing layer, diffuse, 4 ·      Cytoplasmic vacuolization, Wing and basal layer, diffuse, 3
			Stroma:	·      Collagen matrix expansion, upper stroma, mf, 2
			Endothelium:	·      NAD
		2	Epithelium:	·      Epithelial cell loss, Squamous layer, mf, 3 ·      Cytoplasmic coagulation, Squamous and Wing layer, diffuse, 4 ·      Cytoplasmic vacuolization, Wing and basal layer, diffuse, 3
			Stroma:	·      Collagen matrix expansion, upper stroma, mf, 2
			Endothelium:	·      NAD
		17	Epithelium:	·      Epithelial cell loss, Squamous layer, mf, 3 ·      Cytoplasmic coagulation, Squamous and Wing layer, diffuse, 4 ·      Cytoplasmic vacuolization, Wing and basal layer, diffuse, 3
			Stroma:	·      Collagen matrix expansion, upper stroma, mf, 2
			Endothelium:	·      NAD
G3	Test Item (Mo10V3TeNbO42)	07	NAD
		09	NAD
		15	NAD

 NAD: No abnormalities detected, mf: Multifocal, 2: Mild, 3: Moderate; 4: Marked.

Interpretation of results:

study cannot be used for classification

Remarks:

With in the Bovine Corneal Opacity and Permeability Test, the test item induced an IVIS of 44.8 after 3 hours and 55 minutes of treatment. The results indicated an increase in the endpoint “opacity” but not “permeability”. As the test item resulted in IVIS >3; ≤ 55, it is considered as equivocal and classified as UN GHS no stand-alone prediction can be made.

Conclusions:

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item induced an IVIS of 44.8 after 3 hours and 55 minutes of treatment. The results indicated an increase in the endpoint “opacity” but not “permeability”. As the test item resulted in IVIS >3; ≤ 55, it is considered as equivocal and classified as UN GHS no stand-alone prediction can be made

Executive summary:

Summary

The test item was evaluated for ocular corrosivity or severe irritancy in Bovine cornea.

Eyes of cattle were collected from slaughter house ad eye balls free of defects were selected for the experiment. Empty cornea holder’s opacity was measured and the mean opacity value obtained was determined as I₀. Cornea holders with selected Corneas were equilibrated at 32±1ºC for 1 hour and baseline opacity was recorded for each cornea. Corneas with opacity units less than 7 were selected and used for the study.

Quantity of 750 µL 20% w/v of test item, distilled water (vehicle control) and 20% w/v imidazole (positive control) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1ºC for 3 hours and 55 minutes. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD₄₉₀).

The mean corrected opacity and mean corrected permeability values of test item are 44.89 and -0.009 respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 44.8 considered as equivocal, no stand-alone prediction can be made.

The mean corrected opacity and mean corrected permeability values of positive control are 120.34 and 1.573 respectively. The in vitro Irritancy Score (IVIS) of positive control resulted in 143.9, indicating corrosivity or severe irritancy to Bovine corneas.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The OECD TG 431 revealed a mean percentage of viability of 100 % after 3 minutes and 1 hour. 

In conclusion, the combination of both OECD TG studies identified the test material as skin irritant category 2.

In regard of eye irritation, OECD TG 437 induced an ICIS of 44.8 after 3 h 55 min, The results indicated an increase in the endpoint "opacity" but not "permeability". As the test item resulted in IVIS > 3 <= 55, it is considered as equivocal and classification according to GHS got not be made as stand alone assay. Hence, a OECD TG 405 was performed. Within this study, both inital and confirmatory test revealed iccular lesions like redness at 1, 24 and 48 h as well as chemosis at 24 h. All effects were reversible after 72 h. Further, noth tests revealed ocular lesion like conjuctivitis at 24 and 48 h, which was reversible after 72 h as well. All in all the mean scoring for cornea, iris and conjuctival redness as well as conjuctival chemosis were 0, 0, 0.67, and 0.33 respectively. Due to the low degree of scores and reversible effects, no classification according to GHS is justified.