4,8-Dimethyl-2,5,7,10-tetraoxaundecane

Endpoint summary

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:

25 October 2016 to 01 November 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie - 1604211010R
- Purity, including information on contaminants, isomers, etc.: 99.97%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

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): 16-EKIN-043
- Production date: 25 October 2016
- Delivery date: 26 October 2016
- Date of initiation of testing: 25 October 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 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. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT solution
- Incubation time: tissues were incubated for 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: without a reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: 21.8
- IC 50 determination: 1.7 mg/ml
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: Absence of bacteria, fungus and mycoplasma

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure followed by the 42-Hour post-exposure incubation period is less than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µl
- Concentration (if solution): Unknown

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µl
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 1: 102.4%
replicate 2: 102.4%
replicate 3: 98.4%

Value:

101.1

Negative controls validity:

valid

Remarks:

replicate 1: 104.1% replicate 2: 97.3% replicate 3: 98.7%

Positive controls validity:

valid

Remarks:

replicate 1: 7.2% replicate 2: 2.6% replicate 3: 5.7%

Interpretation of results:

GHS criteria not met

Remarks:

UN GHS Not classified for Irritation (category 3 can not be determined)

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.
Method
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
Results
The relative mean viability of the test item treated tissues was 101.1% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.
Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.
Conclusion
The test item was classified as non-irritant. The following classification criteria apply:
EU DSD and CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 September 2016 to 29 September 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie - 1604211010R
- Purity, including information on contaminants, isomers, etc.: 99.97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C in the dark under Nitrogen

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek corporation. Exact source not indicated in the certificate of analysis

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was used as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm Reconstructed Human Epidermis Model
- Tissue batch number(s): 23359
- Production date: 28 September 2016
- Shipping date:28 September 2016
- Delivery date:28 September 2016
- Date of initiation of testing:28 September 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 60 minutes

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.612 +/- 0.155
- Barrier function: 6.64h
- Morphology: presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers
- Contamination: sterile

NUMBER OF REPLICATE TISSUES: 2


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

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µl

Duration of treatment / exposure:

3 and 60 min

Duration of post-treatment incubation (if applicable):

3-Hour MTT incubation

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1 (duration of exposure 3 minutes): 90.2% viability
Run 2 (duration of exposure 60 minutes): 70.4% viability

Value:

70.4

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: non corrosive

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction
The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.
Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.
Methods
Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.
At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).
Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
Results
The relative mean viabilities for each treatment group were as follows:

Exposure period	Percentage Viability
	Negative control	Positive control	Test item
3 minute	100*	3.5	90.2
60 minute	100*	3.2	70.4

*The mean viability of the negative control tissues is set at 100%
Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.
Conclusion
The test item was considered to be non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 September 2021 to 08 October 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie - 2102121010R
- Purity, including information on contaminants, isomers, etc.: 99.93%

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: approximately 11-14 weeks old
- Weight at study initiation: 3025 to 3520 g
- Housing: On arrival and following assignment to the study, animals were housed individually in labeled
cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. These housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room in which the animals were kept were documented in the study records. Each cage was clearly labeled.
- Diet (e.g. ad libitum): Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Switzerland) was provided once daily throughout the study. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was available during the study period.
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C
- Humidity (%): 50 to 73%. The value that was outside the targeted mean humidity range occurred for a single occasion and was without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

IN-LIFE DATES: From: 14 September 2021 To: 08 October 2021

Vehicle:

unchanged (no vehicle)

Remarks:

The other eye remained untreated and served as the reference control.

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): undiluted

Duration of treatment / exposure:

not rinsed - Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage

Observation period (in vivo):

24 hours

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no washing

SCORING SYSTEM: corneal irritation, iris, conjunctival irritation scored as indicated in the OECD TG 405

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: Only chemosis observed after 1 hour but fully reversible after 24 hours

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

other: Only redness observed after 1 hour but fully reversible after 24 hours

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:

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:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: Only chemosis visible after 1 hour but fully reversible after 24 hours

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

other: Only conjunctivae redness visible after 1 hour but fully reversible within 24 hours

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:

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:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

other: Only a score of 1 after 1 hour, reversible within 24 hours

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:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results, 4,8-Dimethyl-2,5,7,10-tetraoxaundecane does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this acute eye irritation study was to assess the possible irritation or corrosion potential when a single dose of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane was placed in the conjunctival sac of the rabbit eye.
The study was carried out in compliance with the guidelines described in:
• OECD No.405 (2021) "Acute Eye Irritation / Corrosion".
• EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion".
• EPA, OPPTS 870.2400 (1998), "Acute Eye Irritation".
• JMAFF Guidelines (2000), including the most recent revisions.
Single samples of 0.1 mL of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation.
Instillation of the test item resulted in minimal irritation of the conjunctivae, which consisted of redness, chemosis and/or discharge. The irritation had completely resolved within 24 hours in all animals.
Based on these results, 4,8-Dimethyl-2,5,7,10-tetraoxaundecane does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 May 2021 to 28 May 2021

Reliability:

1 (reliable without restriction)

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)

Version / remarks:

June 18, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie - 2101081010R and 2102121010R
- Purity, including information on contaminants, isomers, etc.: 99.96% and 99.93%

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 31789 kit C)
- Cell line used, its source: normal human keratinocytes; keratinocyte strain: 4F1188 from MatTek corporation
- RhCE tissue, including batch number: 31789
- In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution): as supplied

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test item together with a negative control
and positive control.

Details on study design:

The liquid test item was applied undiluted (50 μL) directly on top of the tissue.

Application/Treatment of the Test Item:
The test was performed on a total of 2 tissues per test item together with a negative control
and positive control. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free- DPBS. The tissues were incubated at standard culture conditions for minimal 30 minutes.
Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively.
Fifty μL of the undiluted test item was added into the 6-well plates on top of the tissues.
After the exposure period with the test item (30 ± 2 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.
After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.

Cell Viability Measurement:

After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface. Formazan was extracted with 2 mL isopropanol refrigerated for 18 ± 2 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

replicate 1: 0.248 (OD570)
replicate 2: 0.224 (OD570)

Value:

16

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.9

Interpretation of results:

other: no prediction can be made

Conclusions:

In conclusion, 4,8-Dimethyl-2,5,7,10-tetraoxaundecane is identified as no prediction can be made regarding the classification in the EpiOcular™ test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 4,8-Dimethyl-2,5,7,10- tetraoxaundecane. For this purpose 4,8-Dimethyl-2,5,7,10-tetraoxaundecane was topically applied on the Reconstructed Human EpiOcular™ Model.
The possible eye hazard potential of the test item was tested through topical application for 30 minutes.
The study procedures described in this report were based on the most recent OECD guideline (2017).
Batches 2101081010R and 2102121010R of the test item were clear colourless liquids. The test item was applied undiluted (50 μL) directly on top of the tissue for 30 ± 2 minutes.
After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.
The positive control had a mean cell viability of 7.9% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 5%, indicating that the test system functioned properly.
Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 16%. Since the mean relative tissue viability for the test item was below or equal to 60% after 30 ± 2 minutes treatment it is
considered to be potentially irritant or corrosive to the eye.
In conclusion, the test item is identified as no prediction can be made regarding the classification in the EpiOcular™ test under the experimental conditions described in this report.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2016 - 18 October 2016

Reliability:

1 (reliable without restriction)

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)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie - 1604211010R
- Purity, including information on contaminants, isomers, etc.: 99.97%

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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.
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL
- Selection and preparation of corneas: 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 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): used as supplied

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3

Details on study design:

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 0.75 ml - 10 minutes

POST-INCUBATION PERIOD: yes 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured: optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.
- Others (e.g, pertinent visual observations, histopathology): The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Irritation parameter:

cornea opacity score

Run / experiment:

replicate 1: 0.683 (corrected OD value)
replicate 2: 0.702 (corrected OD value)
replicate 3: 0.689 (corrected OD value)
IVIS 37.0

Value:

37

Negative controls validity:

valid

Remarks:

Mean OD value: 0.026 - IVIS 0.4

Positive controls validity:

valid

Remarks:

Mean corrected OD value: 1.170 - IVIS: 44.5

Other effects / acceptance of results:

The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤2.9 and permeability ≤0.103. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:

other: No prediction of eye irritation can be made

Conclusions:

No prediction of eye irritation can be made

Executive summary:

Introduction
The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.
The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.
Method
The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).
Data Interpretation
The test item is classified according to the prediction model as follows:

IVIS	Classification
≤ 3	No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

Results
The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	37.0
Negative Control	0.4
Positive Control	44.5

Conclusion
No prediction of eye irritation can be made

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)