branched and cross-linked multi-walled carbon nanotubes

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 02 February 2021
Experimental completion date 05 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

branched and cross-linked multi-walled carbon nanotubes

Molecular formula:

C

IUPAC Name:

branched and cross-linked multi-walled carbon nanotubes

Test material form:

solid: nanoform, no surface treatment

Specific details on test material used for the study:

Identification: Athlos™ 200 Carbon Nano Structures
EC Number: 951-407-3
Batch Number: V20169
Purity: >97%
Physical state/Appearance: Black Pellets
Expiry Date: 17 June 2025
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcularTM Human Corneal Model (0.6 cm2)
Supplier : MatTek In Vitro Life Science Laboratories,
Bratislava - Slovakia
Date received : 02 February 2021
EpiOcularTM Tissues Lot Number : 30694
Assay Medium Lot Number : 02012ISA

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

A sufficient volume of the test item, equivalent amount which would adequately cover the tissue surface area
50 μL of the negative and positive controls.

Duration of treatment / exposure:

6 hours ±15 minutes at 37 °C, 5% CO2

Duration of post- treatment incubation (in vitro):

18 hours ±15 minutes at 37 °C, 5% CO2

Number of animals or in vitro replicates:

duplicate

Details on study design:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. Therefore, it was necessary to assess this ability of the test item to directly reduce MTT prior to conducting the assay. This property of the test item is only a problem, if at the time of the MTT test (after the test item has been rinsed off) there is still a sufficient amount of the test item present on (or in) the tissues. In this case the (true) metabolic MTT reduction and the (false) direct MTT reduction can be differentiated and quantified by the procedure described as follows:
A sufficient volume of the test item, equivalent amount which would adequately cover the tissue surface area during the main test, was added to 1 mL of MTT solution and incubated at 37 °C, 5% CO2 for 3 hours. A control (50 μL sterile water in MTT solution) was run concurrently. If the MTT solution turned blue/purple, the test item was presumed to have directly reduced the MTT.
An assessment to determine if the test item was able to directly reduce MTT was inconclusive due to the black color of the test item. Therefore, as a precaution, an additional procedure using non-viable, freeze-killed, tissues was performed for potential direct MTT reduction correction purposes. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of eye irritation potential was performed in parallel on viable and freeze-killed tissues.
This step employs freeze-killed tissues that possess negligible metabolic activity but absorb and bind the test item similar to viable tissues.
Freeze-killed tissues were prepared in-house (outside of the confines of the study) by placing untreated EpiOcularTM tissues in a freezer (-35 to -10 C) overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles). Once killed, the tissues may be stored indefinitely in the freezer. Freeze-killed tissues were thawed for approximately 60 minutes at 37 ±2 °C, 5 ±1% CO2 in air before use.
Each MTT reducing test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues remained untreated (the untreated controls show a small amount of MTT reduction due to residual reducing enzymes within the killed tissue). The entire assay was performed on the frozen tissues in parallel to the viable tissues.

Assessment of Color Interference with the MTT endpoint
Colored test items or those which become colored after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT.
Test items which absorb light and appear red, yellow, green or blue should be considered as intrinsic colorants. A test item which appears black may absorb light and should be
considered as a colorant. Blue, purple and black test items may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue/purple
MTT product. Such test items should also be tested on killed controls because it may not be possible to assess their potential to directly reduce MTT.
Black test items may directly be tested on colorant controls. However, because the test item was insoluble and this characteristic suggested it would not cause a color interference in the main test, an optical density measurement was performed on the test item in isopropanol. The optical density measurement indicated that the test item would not cause significant color interference. However, further consideration was given to the fact that the test item may not completely wash off the tissue culture surfaces at the termination of the exposure period. Therefore it was considered that color correction tissues should be incorporated into the main test as a precaution.
In addition to the normal test procedure, the test item was applied to two viable tissues and treated identically to the main test with the exception that the MTT incubation period was replaced by incubation with assay medium (without MTT). Concurrently, two viable tissues were left untreated to act as a negative color control.

Assessment of Color Interference in non-viable tissues
A third set of controls was included, comprising freeze-killed tissues, in order to prevent a double correction from a colored test item that may also directly reduce MTT.
Colored test items may bind to both living and killed tissues and therefore the non-viable freeze-killed tissues may not only correct for potential direct MTT reduction by the test item, but also for color interference arising from the binding of the test item to killed tissues. This could lead to a double correction for color interference since the viable color interference tissues already correct for color interference arising from the binding of the test item to living tissues.
For each exposure period, the additional group were composed of two test item treated freeze-killed tissues that have undergone the entire testing procedure with the exception that the MTT incubation period was replaced by incubation with assay medium (therefore without MTT). For each exposure period, two freeze-killed tissues remained untreated for negative control purposes. The optical density measurements from these tissues were assessed for possible quantitative correction of the results.

Preparation and Pre-Incubation of EpiOcular Tissues
Upon receipt of the EpiOcularTM tissues, the sealed 24-well plate and the assay medium were
laced into the refrigerator (2 to 10 °C) until the equilibration step. The vial containing the MTT concentrate was placed in the freezer (-35 to -10 °C) and the MTT diluent placed in the refrigerator (2 to 10 °C). The positive control, Methyl Acetate, was stored at room temperature, in the dark.
On the day of receipt the equilibration step (15 minutes at room temperature in the 24-well shipping container) was started. An appropriate volume of EpiOcular™ Assay medium was warmed to approximately 37 °C and 1 mL of the medium aliquoted into the appropriate wells of pre-labeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with ethanol soaked tissue paper. The sterile gauze was removed and each tissue inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping container 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 1 hour in Assay Medium. After 1 hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture
conditions overnight (16 to 24 hours).

Main Test
Application of Test Item and Rinsing
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++ Mg++ free DPBS to mimic the wet condition of the human eye. If the Ca++ Mg++ free DPBS was not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at 37 °C, 5% CO2 for 30 ±2 minutes.
The test item was applied evenly atop duplicate cultures for an exposure period of 6 hours ±15
minutes at 37 °C, 5% CO2 followed by rinsing, a post-treatment immersion and a post-treatment incubation (described below). 50 μL of the negative and positive controls were similarly applied. At the end of the test item exposure period, the test item was removed by extensively rinsing the tissues with Ca++ Mg++ free DPBS at room temperature. Three clean beakers (glass or plastic with minimum 150 mL capacity), containing a minimum of 100 mL each of Ca++ Mg++ free DPBS were used per test item or control with each test item or control item utilizing a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. The tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent paper towel 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 were 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 inserts were then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent paper. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent paper (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labeled 12-well plate for a 25 ±2 minutes immersion incubation (post-treatment immersion) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue.
At the end of the post-treatment immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, the insert was blotted on absorbent paper, and transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 18 hours ±15 minutes at 37 °C, 5% CO2 (post-treatment incubation).

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were placed into the 24-well plate containing 0.3 mL of 1.0 mg/mL MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated at 37 °C, 5% CO2 in air for 3 hours.
A procedure was used which only extracted from beneath the tissue, since residual test item may remain on the tissue and could contaminate the isopropanol. Inserts were removed from the 24-well plate after approximately 3 hours. The bottom of the insert was blotted on absorbent paper and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol flowed into the insert. The plates were sealed with a film sealer (between the plate cover and upper edge of the wells) or a standard plate sealer and stored overnight at 2 to 10 °C in the dark.

Absorbance/Optical Density Measurements
At the end of the extraction period, using a pipette fitted with a 1000 µL tip, the extraction solution was forced vigorously up and down to thoroughly mix. The tissues and empty inserts were discarded.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. 200 μL of isopropanol alone was added to eight wells designated as ‘blanks’. All wells were examined and any air bubbles were removed. The absorbance at 570 nm (OD570) of each well was measured using the LabTech LT-4500 microplate reader and LT-com analysis software.
The plate reader LT-com analysis software was set to correct for blanks and calculate the mean OD570 values of the duplicate wells representing each tissue. The mean OD570 values of the duplicate tissues were manually calculated.

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean

Value:

83.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

It was reported that a small amount of test item adhered to the tissue culture surfaces which
could not be removed by rinsing at the end of the exposure period.

Direct MTT Reduction
An assessment to determine if the test item was able to directly reduce MTT was inconclusive due to the black color of the test item. Therefore, as a precaution, an additional procedure using non-viable, freeze-killed, tissues was performed for potential direct MTT reduction correction purposes. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint
Black test items may directly be tested on colorant controls. However, because the test item was insoluble and this characteristic suggested it would not cause a color interference in the main test, an optical density measurement was performed on the test item in isopropanol: After subtraction of the OD for isopropanol, the OD of the test item solution was 0.002 which
was < 0.080.
The optical density measurement indicated that the test item would not cause significant color interference. However, further consideration was given to the fact that the test item may not completely wash off the tissue culture surfaces at the termination of the exposure period. Therefore it was considered that color correction tissues should be incorporated into the main test as a precaution.
However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

Double Correction Check
The results of the color correction tissues were not used, therefore it was unnecessary to use the results of the killed color correction tissues as no double correction for color interference would have occurred.

Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 24.7% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.661. The negative control acceptance criterion was therefore satisfied.
The difference in viability between the two relating tissues in each treatment group was <20%. This acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	1.705	1.661	102.6	100*	5.3
	1.616		97.3
Positive Control Item	0.404	0.41	24.3	24.7	0.7
	0.415		25
Test Item	1.48	1.39	89.1	83.7	10.8
	1.3		78.3

OD = Optical Density
* = The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classifications apply:
EU CLP and UN GHS No Category

Executive summary:

Introduction
The purpose of this study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

Method
Duplicate tissues were treated with the test item for an exposure period of 6 hours. At the end of the exposure period each tissue was rinsed before incubating for 18 hours.
An assessment to determine if the test item was able to directly reduce MTT was
inconclusive due to the black color of the test item. Therefore, as a precaution, an additional procedure using non-viable, freeze-killed, tissues was performed for potential direct MTT reduction correction purposes. Additionally, due to the black color of the test item and its potential to adhere to the tissue culture surfaces, it was considered necessary to incorporate viable color correction tissues for potential color correction purposes. A third set of controls was included, comprising freeze-killed tissues, in order to prevent a double correction from a
colored test item that may also directly reduce MTT. At the end of the post- exposure incubation period each tissue was taken for MTT-loading. After MTT-loading the tissues was placed into 2 mL of isopropanol for formazan extraction.
At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results
The results obtained showed that negligible interference due to direct reduction of MTT by the test item and no interference due to the color of the test item occurred in the main test. It was therefore considered unnecessary to use the results of the freeze-killed tissues or color correction tissues, for quantitative correction of results.
The relative mean viability of the test item treated tissues was 83.7%.
It was reported that a small amount of test item adhered to the tissue culture surfaces which could not be removed by rinsing at the end of the exposure period.
Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion
The test item was classified as non-irritant. The following classifications apply:
EU CLP and UN GHS No Category