Dimolybdenum trizinc nonaoxide

Endpoint summary

Description of key information

For the skin irritation endpoint a reliable GLP in vitro study is available providing a negative response for skin irritation. For the eye irritation parameter, two GLP in vitro studies are available both providing inconclusive results. As such, an in vivo eye irritation study was conducted in accordance the the relevant GLP guideline which provided a negative result for eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

Cell source:

other: adult human-derived epidermal keratinocytes

Source strain:

not specified

Details on animal used as source of test system:

The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

Standard as per OECD Guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 18-EKIN-032
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: 07 August 2018 (expiry date 13 August 2018)
- Date of initiation of testing: Pre-incubation commenced on day of tissue arrival.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++.
Rinsing was achieved 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. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well.
- Observable damage in the tissue due to washing: None specified
- Modifications to validated SOP: Not applicable

MTT LOADING/FORMAZAN EXTRACTION
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ADSORBANCE/OPTICAL DENSITY
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: Triplicate tissues treated with the test item for an exposure period of 15 minutes.

MTT INTERFERANCE
A test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results. A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 mg of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability is - The test substance is considered to be a non-irritant to skin if the relative mean tissue viability is >50%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm2).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): DPBS used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): SDS
- Concentration (if solution): Applied at 0.3 mg/mL in assay medium

Duration of treatment / exposure:

Tissues were exposed for 15 minutes ahead of rinsing

Duration of post-treatment incubation (if applicable):

42 hours post exposure incubation

Number of replicates:

Tissues were tested in triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

107

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: Not specified
- 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 colourless. It was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.704 and the standard deviation value of the viability was 5.5%. 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 5.8% relative to the negative control treated tissues and the standard deviation value of the viability was 1.6%. 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 6.5%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Mean OD570 values and Viabilities for the Negative Control Item, Positive Control Item and Test 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	0.660	0.704	0.039	93.8	100	5.5
	0.718			102.0
	0.734			104.3
Positive Control Item	0.052	0.041	0.011	7.4	5.8	1.6
	0.030			4.3
	0.040			5.7
Test Item	0.784	0.753	0.046	111.4	107.0	6.5
	0.700			99.4
	0.774			109.9

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance did not induce a relative mean tissue viability of

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: In house stock supply
- Age at study initiation: 39 to 40 weeks old
- Weight at study initiation: 3.34 to 4.35 Kg
- Housing: Each animal was housed individually in a plastic cage with perforated floors.
- Diet (e.g. ad libitum): 150 g of standard laboratory rodent diet per day. A dietary supplement of hay was offered during acclimatization and throughout the study observation period.
- Water (e.g. ad libitum): Provided ad libitum
- Acclimation period: 21 to 27 weeks prior to the start of study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 20°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Not specified.
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycles.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

Three rabbits each received a singular ocular instillation of 0.1 g of the test substance

Observation period (in vivo):

Up to 1 week after instillation

Number of animals or in vitro replicates:

Three rabbits. A single animal was treated in advance; in the absence of a severe effect in this animal two further animals were committed to the study.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Eyes were flushed with saline to remove residual test substance at 24 and/or 48 hours after instillation as required per animal.

SCORING SYSTEM: Draize as per the OECD 405 guideline.

TOOL USED TO ASSESS SCORE: An ophthalmoscope or pencil beam torch was available for use to facilitate inspection of the eyes.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Iritis, a crimson-red conjunctival appearance, slight or moderate chemosis and moderate discharge were evident in the treated eye of all animals one hour after instillation. Injection of the conjunctival blood vessels or a crimson-red conjunctival appearance and slight or moderate chemosis were apparent during the next three days; slight discharge was evident in two animals 24 hours after instillation and in one animal at the 48 hour examination. The treated eye of all animals was overtly normal one week after instillation. Instillation of the test item gave no initial pain response.

Other effects:

There was no sign of toxicity or ill health in any rabbit during the observation period.

Mean values for ocular lesions:

24, 48 and 72 hours after instillation of Zinc Molybdate

 

Animal number and sex	Corneal Opacity	Iridial Lesions	Redness of Conjunctiva	Chemosis
1F#	0.0	0.0	1.7	1.7
2F	0.0	0.0	1.3	1.0
3F	0.0	0.0	1.7	1.3

#             Sentinel animal

F             Female

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was shown to be negative for eye irritation and no classification under EU CLP (EC No. 1272/2008) is required.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

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:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% aqueous
- Lot/batch no. (if required): 3012487

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes after application of sodium fluorescein

Number of animals or in vitro replicates:

Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS :
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

TREATMENT OF CORNEAS:
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed at least 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

PERMEABILITY DETERMINATIONS:
After incubation the medium in the posterior chamber of each holder was decanted and retained.

360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

DATA EVALUATION:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT:
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IVIS SCORING:
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

22

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤ 2.3 and permeability ≤ 0.44. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.

In Vitro Irritancy Scores:

Treatment	In Vitro Irritancy Score
Test item	22
Negative control	1.7
Positive control	129.3

 

Interpretation of results:

study cannot be used for classification

Conclusions:

The IVIS of 22 fell within the range of > 3 and ≤ 55. In accordance with the OECD guideline no prediction with regards to the CLP/UN GHS classification for the substance can be made (inconclusive).

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Version / remarks:

For the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits were purchased from MatTek Corporation. The EpiOcular™ tissue consists of normal, human-derived epidermal 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 which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts.

EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) overnight (about 17 hours).

Vehicle:

water

Controls:

yes

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 50 mg of the test substance
- Concentration (if solution): Not applicable

VEHICLE
- Amount(s) applied (volume or weight with unit): Approximately 50 µL of the vehicle (deionised water).

Duration of treatment / exposure:

After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca2+Mg2+free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.

After the 30 minute Ca2+Mg2+free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) for 6 hours.
At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature).

The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts are mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker.

The culture was then rinsed in the second and third beaker of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.

Since it was not possible to remove the visible test item completely, this was noted in the study file. No further rinsing was done.

After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.

Duration of post- treatment incubation (in vitro):

At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay medium. The tissues were incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

Number of animals or in vitro replicates:

Duplicate tissue replicates with two wells per control, positive control and test item.

Details on study design:

Application: Approximately 50 mg of the test item were tested topically on duplicate EpiOcular™ tissues.

Assessment of direct MTT reduction by the test item:
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT. Since the MTT solution colour did not turn blue/purple, the test item was not presumed to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.

Assessment of coloured or staining materials:
Coloured test items or test items which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement, if the colourant binds to the tissue and is extracted together with MTT. Therefore, each test item has to be checked for its colouring properties.

Additional tests had to be performed to assess, if the non-coloured Test Item becomes coloured after contact with water or isopropanol. For this purpose, approximately 50 mg of the test item were added to 2.0 mL of isopropanol (glass tube) and shaken for 3 hours at room temperature. 2.0 mL of isopropanol was used as control. Additionally 50 ± 2 mg of the test item were added to 1.0 mL of deionised water (glass tube) and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for at least 1 hour. 1 mL of deionised water was used as control. Two 200 µL aliquots of isopropanol solutions and of pure isopropanol were transferred to a 96-well plate and the absorbance is measured with a plate reader at the MTT measurement wavelength. Since the test item was not completely soluble in isopropanol, it was centrifuged, and 200 µL aliquots of the supernatant were used for measurement. Since the test item did not become coloured either in water or isopropanol and the OD of the test item solution was < 0.08, it was not considered as possibly interacting with the MTT measurement and an additional test with viable tissues (with medium instead of MTT addition) did not have to be performed.

MTT assay:
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.

Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) and were immediately extracted (shaken for 2 to 3 hours at room temperature). For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate. The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Prediction model:
If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is labeled irritant.

Assay acceptability:
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: Tissue viability (%)

Run / experiment:

1

Value:

57.63

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Met validity criteria

Positive controls validity:

valid

Remarks:

Met validity criteria

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None noted

DEMONSTRATION OF TECHNICAL PROFICIENCY: Laboratory technical proficiency with the test system according to OECD 492 was demonstrated at the testing lab in a specified project.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (1.925 and 1.991).
- Acceptance criteria met for positive control: The tissue viability of the positive control is below 50% of the negative control viability (20.54%).
- Range of historical values if different from the ones specified in the test guideline: Values within historical control ranges.

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.

Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue/purple colour. Therefore, an additional test with freeze-killed tissues was not necessary.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the test, the substance possesses borderline eye irritating potential, and no conclusive prediction on the lack of eye irritation potential can be made.

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

In the available in vitro skin irritation assay the test substance did not induce a relative mean tissue viability of </= 50% and accordingly provided a negative result.

 

For the eye irritation parameter, two GLP in vitro studies are available both providing inconclusive results. As such, an in vivo eye irritation study was conducted in accordance the the relevant GLP guideline which provided a negative result for eye irritation. No data is available to assess potential respiratory irritation effects of the substance.

 

Based on all the available data, the substance is not classified for eye or skin irritant/corrosive effects in accordance with the CLP Regulation (EC No. 1272/2008, as amended).