Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

Endpoint summary

Description of key information

In a OECD and GLP compliant in vitro skin irritation assay, the registered substance was concluded to be non irritant to skin.

Based on in vivo and in vitro OECD and GLP compliant eye irritation assays, the registered substance was concluded to be non irritant to eyes .

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

Study period:

From 10 August to 10 October 2017

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable (The test item was applied as supplied, no formulation was required)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable (The test item was applied as supplied)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified (Adult donors)

Source strain:

other: No applicable (human)

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Assessment of the effects of the test on on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and
conversion into a blue formazan salt that is quantitatively measured after extraction from tissues.
- Quality control for skin discs: The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (23.2-27.6°C).
- Temperature of post-treatment incubation (if applicable): 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: one with PBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none
DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] MTT (Concentration of the working solution: 0.3 mg/mL)
- Spectrophotometer:Thermo Fisher Scientific
- Wavelength: 570 nm.
- Filter: Not applicable
- Filter bandwidth: Not applicable
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be irritant to skin (Category 2) or corrosive (Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
- The test item is considered to be noni-irritant to skin (Not classified in GHS), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is above (>) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 μL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 5% (w/v) in distilled water

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h) at 37°C

Number of replicates:

Three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay.

Irritation / corrosion parameter:

% tissue viability

Value:

70.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- Direct-MTT reduction: After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT.
- Colour interference with MTT: As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Negative and positive controls as well as variability between replicate measurements met the acceptability criteria, therefore the study was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18. The mean OD value of the three negative control tissues was in the recommended range (0.751). Standard deviation of the viability results for negative control samples
was 3.8%.
- Acceptance criteria met for positive control: The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18. The positive control treated tissues showed 8.0% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.3%.
- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.6%.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with INNROAD PROTECT, the mean cell viability was 70.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKINTM (SM) model test with INNROAD PROTECT (Batch number: CY71353002), the results indicate that the registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is non-irritant to skin.

Executive summary:

An in vitro skin irritation test of INNROAD PROTECT test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) t o 5 0% o f t he negative control, the test item is considered to be irritant to skin.

Following exposure with INNROAD PROTECT, the mean cell viability was 70.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with INNROAD PROTECT (Batch number: CY71353002), the results indicate that

the registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is non-irritant to 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 vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 September to 2 November 2017

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable (The test item was applied as supplied, no formulation was required)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable (The test item was applied as supplied)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Species:

other: Bovine cattle (Bos Taurus)

Strain:

other: Not appplicable

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): bovine cattle were up to 12 months old (typically, 5 to 8 months old).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
- Time interval prior to initiating testing: Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible.
- indication of any existing defects or lesions in ocular tissue samples: careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded.
- Indication of any antibiotics used: As the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

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): 750 μL (± 8 μL)
- Concentration (if solution): Not applicable (the test item was tested undiluted).

VEHICLE: Not Applicable (As the test item was a non-surfactant liquid, it was tested undiluted (i.e. in its original form)).

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes) in a water bath at +32°C (± 1°C).

Number of animals or in vitro replicates:

The test item, the negative and positive control were tested on three corneas each.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.

QUALITY CHECK OF THE ISOLATED CORNEAS
The corneas were carefully examined macroscopically before their assembly in the holders, in order to
detect the presence of any defects. Any corneas with defects were discarded.

NUMBER OF REPLICATES:The test item, the negative and positive control were tested on three corneas each.

NEGATIVE CONTROL USED: 0.9% Sodium Chloride

POSITIVE CONTROL USED: Absolute Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 μL (± 8 μL) applied for 10 minutes (± 30 seconds)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes. If YES please specify duration: 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: On completion of the treatment period, the test item was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured).
Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry / OD490)
- Others (e.g, pertinent visual observations, histopathology): After permeability determination, the corneas were removed from the holders and observed for opaque
spots, other irregularities and any separation of the epithelium.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Value:

32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
No notable opaque spots or irregularities were observed on the negative control-treated corneas.
Opacity and fluorescein fixation were observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The individual and mean opacity and permeability values for the test item, positive control and negative control fulfilled all acceptance criteria.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

other:

Remarks:

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, INNROAD PROTECT, could not be predicted. Theregistered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to the OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, INNROAD PROTECT, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item, applied undiluted, and both negative and positive controls were applied in a single experiment, using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed

for opaque spots and other irregularities.

Macroscopic examination:

Opacity and fluorescein fixation were observed on the three corneas treated with the test item.

In Vitro Irritancy Score:

All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 32.

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, INNROAD PROTECT, could not be predicted.The registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to the OECD Guideline 437, further testing should be

conducted for classification and labeling purposes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 April to 31 May 2018

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:18CDL008V
- Expiration date of the lot/batch: 13 March 2019
- Analytical purity: 99.7%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable (The test item was applied as supplied, no formulation was required)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable (The test item was applied as supplied)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Species:

human

Strain:

other: Reconstructed human Cornea-like Epithelium (tissues)

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
The design of this study was based on the protocol published by MatTek Corporation: "EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals" (adopted version,June 2015). The principle of this test is based on the hypothesis that the level of ocular irritation is related to the extent of initial injury, and that regardless of the processes leading to tissue damage, the extent of initial injury is the principal factor determining the outcome of ocular irritation (Maurer et al. 2002).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcular tissue construct is a non-keratinized epithelium composed of stratified normal human keratinocytes in a three-dimensional structure. It models the cornea epithelium with progressively stratified, but not cornified cells. Its use for eye irritation testing involves a topical application of test items on the surface of the cornea epithelial construct for different exposure incubations.
Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in the study files.

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): Not applicable (the test item was tested undiluted).

VEHICLE: Not Applicable (The test item was used in its original form, which was noted as colourless liquid).

Duration of treatment / exposure:

30 (± 2) minutes

Duration of post- treatment incubation (in vitro):

2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

- Details of the test procedure used:
The principle of this test is based on the hypothesis that the level of ocular irritation is related to the extent of initial injury, and that regardless of the processes leading to tissue damage, the extent of initial injury is the principal factor determining the outcome of ocular irritation (Maurer et al. 2002). The EpiOcular tissue construct is a non-keratinized epithelium composed of stratified normal human keratinocytes in a three-dimensional structure. It models the cornea epithelium with progressively stratified, but not cornified cells. Its use for eye irritation testing involves a topical application of test items on the surface of the cornea epithelial construct for different exposure incubations. Cell viability determination is based on cellular mitochondrial succinate dehydrogenase activity measured (within the mitochondria of viable cells) by the reduction and the conversion of a yellow dye, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], into a blue formazan salt. The formazan precipitate is then extracted using isopropanol and quantified by spectrophotometry. For each test item, the mean Optical Density of two treated tissues is determined and expressed as a relative percentage of viability of the negative control.
- RhCE tissue construct used, including batch number: Reconstructed human Cornea-like Epithelium (tissues) from MatTek (Bratislava, Slovak Republic). Batch number and any information relating to the characterization and integrity of the test system are documented in a certificate of analysis, which is archived in the study files.
- Doses of test chemical and control substances used: as such

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): see above.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 50 μL of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 50 μL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 10 minutes).
Then the colour of both solutions obtained was evaluated. If the MTT solution colour turns blue/purple when compared to the negative control, the test item was presumed to reduce MTT.
As a test item may be coloured or become coloured in contact with water and/or isopropanol, it is necessary to test its potential interference with the MTT determination in these two conditions. The maximum amount of test item, 50 μL was added to 1 mL of water and incubated for at least 1 hour at +37°C, 5% CO2 and 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 to 3 hours at room temperature. After that, the presence and intensity of the colouration were evaluated. If the solution changes colour significantly, additional controls were performed on viable tissues in parallel to the main test. Otherwise, no additional tissue controls were used.
- Number of tissue replicates used per test chemical and controls (positive control, negative control): duplicate

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm using a plate reader

- Description of the method used to quantify MTT formazan: At the end of the formazan extraction period, tissues (test item, negative and positive control-treated tissues) were pierced.
The extract solution was mixed using a pipette and two 200 μL aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate.
One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate), and a separate 96-well plate was used for test item-treated tissues.
For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: A test substance is predicted as ocular irritant, if the mean relative tissue viability (%) of two tissues exposed to the test item is ≤ 60%.

- Positive and negative control means and acceptance ranges based on historical data: The results of the study were considered acceptable if the following criteria are fully met:
. the mean cOD of the negative controls is between 0.8 and 2.5,
. relative mean viability of the positive control is < 50% of the relative mean viability of the negative control, .
. the difference of viability between the two tissue replicate is < 20%.

- Acceptable variability between tissue replicates for positive and negative controls: < 20%

- Acceptable variability between tissue replicates for the test chemical: < 20%

Irritation parameter:

other: Cell viability (%)

Run / experiment:

Run 1

Value:

62

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: unconclusive

Irritation parameter:

other: Cell viability (%)

Run / experiment:

Run 2

Value:

61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: unconclusive

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

PRELIMINARY TESTS:

Test for direct MTT reduction with the test item:

The MTT solution containing the test item did not turn blue/purple when compared with the negative control. The test item was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on freeze-dead tissues in parallel to the main test.

Additionally, it was noted that the bottom of the plastic well used to run the preliminary assay for possible direct MTT reduction seemed altered at the end of the incubation period

Test for the detection of the colouring potential of the test item:

During this test, as both water and isopropanol solutions containing the test item did not change colour, the test item was found not to have a colouring potential. As a result, no additional controls were used in parallel to the main test.

Additionally, it was noted that the bottom of the plastic wells used to run the preliminary assay for possible colouring potential seemed altered at the end of the incubation period.

MAIN TEST:

First main test:

Evaluation of the colouration of tissues at the end of the MTT incubation period:

In this experiment, the test item-treated tissues appeared blue which was considered to be indicative of viable tissues.

Evaluation of the MTT results:

All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the first main test was considered to be valid.

The relative mean viability of the tissues treated with the test item was 62% (with a difference of 4% between duplicate tissues). Individual viability values were spread at the cut-off of 60% for one tissue, and slightly above for the second one, with values of 60% and 64% for tissue No. 01 and No. 02, respectively.

As the mean viability of the test item-treated tissues was borderline (within 60 ± 5 % viability), no conclusion was made on the first experiment, and a second experiment was performed.

Second main test:

Evaluation of the colouration of tissues at the end of the MTT incubation period:

In this experiment, the test item-treated tissues appeared blue which was considered to be indicative of viable tissues.

Evaluation of the MTT results:

All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the second main test was considered to be valid.

The relative mean viability of the tissues treated with the test item was 61% (with a difference of 1% between duplicate tissues). Individual were very almost identical, with rounded values of 61% for each test item-treated tissue.

As the mean viability of the test item-treated tissues was again borderline (within 60 ± 5% viability), no conclusion was made on the second main test.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of this study, the eye irritant potential of the test item, INNROAD PROTECT, was considered equivocal when tested in vitro using Reconstructed human Cornea-like Epitheliums.
Based on the results of this study, further testing is required for determining of the test item classification with regard to eye irritation according to criteria laid down in GHS (2015) and Regulation (EC) No. 1272/2008.

Executive summary:

The purpose of this study was to predict the acute eye irritation potential of the test item, INNROAD PROTECT, by measurement of its cytotoxic effect on the EpiOcularTM cornea epithelial model. The study was conducted according to OECD Test Guideline no. 492 in compliance with Citoxlab France standard operating procedures and the principles of Good Laboratory Practice.

Methods:

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test. Two main tests were performed in the study due to unclear outcome obtained at completion of the first main test. During both main tests, the test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 30 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 2 hours

at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

Results:

Preliminary tests:

In the preliminary tests, the test item was neither found to have direct MTT reducing properties, nor colouring potential.

It was also noted that the bottom of the plastic wells used to run the preliminary assays seemed to be altered by contact with the test item.

Main test:

All acceptance criteria for the negative and positive controls were fulfilled in each main test. The study was therefore considered to be valid.

The relative mean viability of the tissues treated with the test item in the first experiment was:

- 62% (with a difference of 4% between duplicate tissues). As borderline results were obtained (within the viability range 55 - 65%), a second main test was performed.

The relative mean viability of the tissues treated with the test item in the first experiment was:

- 61% (with a difference of 1% between duplicate tissues).

As the mean viability of the test item-treated tissues was borderline (i.e. within 60 ± 5%) for both experiments, it was considered that running a third main test would not have led to a clear outcome. As a result, together with observations made during the preliminary assays, the applicability of the test item to

the test method was questionable and the eye irritant potential of the test item was considered equivocal.

In conclusion, under the experimental conditions of this study, the eye irritant potential of the test item, INNROAD PROTECT, was considered equivocal when tested in vitro using Reconstructed human Cornea-like Epitheliums.

Based on the results of this study, further testing is required for determining of the test item classification with regard to eye irritation according to criteria laid down in GHS (2015) and Regulation (EC) No. 1272/2008.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 to 16 September 2018

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY81353001
- Expiration date of the lot/batch: 15 May 2020
- Analytical purity: 99.5%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable (The test item was applied as supplied, no formulation was required)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable (The test item was applied as supplied)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary.
- Age at study initiation: 11 weeks old (young adult)
- Weight at study initiation: 2963 g to 3100 g
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet (e.g. ad libitum): Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út 0399, Hungary, ad libitum.
- Water (e.g. ad libitum): The animal received tap water, fit for human consumption, ad libitum, from the automatic system supplied by the communal water network.
- Acclimation period: At least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 – 21.5°C
- Humidity (%): 46 – 77%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 11 To: 16 September 2019

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A single amount of 0.1 ml of the test item as such was administered to the left eye of each animal.

Duration of treatment / exposure:

A single instillation of the test item

Observation period (in vivo):

The eyes were examined at approximately 1, 24, 48, 72 hours after treatment

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No test item remained in the eye sac of any animals at 1 hour after the application, therefore no washing was performed during the study

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (09 October 2017)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

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:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

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:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Other effects:

There was no mortality observed during the study.
The body weight of the animals was considered to be within the normal range of variability.
There were no clinical signs observed that could be related to treatment.

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed.

Animal 1 (No. 7929) clinical observation
At 1 hour after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 2) were noted in the rabbit. No test item remained in the eye sac.
Animal 2 (No: 7931) clinical observation
At 1 hour after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 2) were noted in the rabbit. No test item remained in the eye sac.
Animal 3 (No: 7939) clinical observation
At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 2) were noted in the rabbit. No test item remained in the eye sac.
As no clinical signs were observed, the experiment was terminated after 72 hours observation of the second and third rabbits. During the experiment, the control eye of each animal was symptom-free. Washing of the treated eyes was not necessary during the study. The general state and behaviour of animals were normal throughout the study period.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Innroad Protect, applied to rabbit eye mucosa, caused conjunctival effects at 1 hour after the treatment which were fully reversible within 24 hours. There were no effects on cornea and iris observed during the study.

Executive summary:

The purpose of this study was to assess the acute eye irritation potential of the test item INNROAD PROTECT (batch No. CY81353001), in New Zealand White rabbits, according to the OECD test guideline No. 405 (2017). The study was conducted in compliance with Citoxlab Hungary standard operating procedures and the principles of Good Laboratory Practice.

Three rabbits were treated with the test item. Prior to test item application, the animals were treated with analgesic and anaesthetic as per the regulatory guideline.

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 ml of the test item was administered to the left eye of each animal.

The eyes were examined at 1, 24, 48 and 72 hours after application.

Clinical observations:

No mortality occurred during the study. The general state and behaviour of animals were normal throughout the study period.

The bodyweights of all rabbits were considered to be within the normal range of variability.

As no clinical signs were observed, the experiment was terminated after 72 hours observation of the second and third rabbits.

Examination of ocular reactions:

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed.

At 1 hour after the application, conjunctival redness (score 1 or 2), chemosis (score 1) and discharge (score 2) were noted in the three rabbits. No test item remained in the eye sac.

At 24, 48 and 72 hours after the application, no clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) was 0 for the three rabbits.

During the experiment, the control eye of each animal was symptom-free. Washing of the treated eyes was not necessary during the study.

In conclusion, the test item Innroad Protect, applied to rabbit eye mucosa, caused conjunctival effects at 1 hour after the treatment which were fully reversible within 24 hours. There were no effects on cornea and iris observed during the study.

According to Regulation (EC) No 1272/2008 and GHS-UN, Innroad Protect does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

One Klimisch score 1 study is available to assess the skin irritating potential of the registered substance and was used as key study:

In this study, performed according to OECD guideline no. 439 and in compliance with GLPs, the irritating potential of the registered substance was tested through topical application for 15 minutes on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM)).

The relative mean tissue viability obtained after 15 minutes treatment with the registered substance compared to the negative control tissues was 70.8%. Since the mean relative tissue viability was above 50%, the registered substance is considered to be non-irritant to skin.

Three Klimisch score 1 studies are available to assess the eye corrosive and irritating potential of the registered substance:

In the first study, performed according to OECD guideline no. 437 (BCOP test) and in compliance with GLPs, a volume of 750 µl of the registered substance (as such) was applied for 10 minutes on isolated bovine corneas. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 32. As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted and further testing had to be conducted for classification and labeling purposes.

In the second study, performed according to OECD guideline no. 492 and in compliance with GLPs, the registed substance was applied topically on duplicate tissues of Reconstructed human Cornea-like Epithelium ( EpiOcular cornea epithelial model) and incubated at +37°C for 30 minutes.

The relative mean viability of the tissues treated with the test item, compared to the negative control tissues, was 61% in the first experiment and 62% in the second experiment. As the mean viability of the test item-treated tissues was borderline (i.e. within 60 ± 5%) for both experiments, it was considered that running a third main test would not have led to a clear outcome. As a result, the applicability of the test item to the test method was questionable and the eye irritant potential of the test item was considered equivocal. An in vivo study was therefore conducted do conclude definitely on the classification of the test item.

In a third study, performed according to OECD guideline no. 405 and in compliance withe GLPs, the registered substance was tested in three New Zealand White rabbits. The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 ml of the test item was administered to the left eye of each animal. The eyes were examined at 1, 24, 48 and 72 hours after application. discharge (score 2) were noted in the three rabbits. No test item remained in the eye sac. At 24, 48 and 72 hours after the application, no clinical signs and no ocular reactions (conjunctivae, cornea and iris) were observed. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) was 0 for the three rabbits.

Based on the results of the in vivo rabbit study,the registered substance does not require classification as an eye irritant according to GHS UN and CLP regulations.

Justification for classification or non-classification

Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is not classified for skin irritation according to the Regulation (EC) 1272/2008 (CLP) based on a negative response in a in vitro skin irritation test.

 

Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is not classified for eye irritation according to the Regulation (EC) 1272/2008 (CLP) based on a negative response in a in vivo eye irritation test.