Diallyl hexahydrophthalate

Endpoint summary

Description of key information

No skin irritation or corrosion was found from either study. Based on the in vitro eye irritation study in the isolated chicken eyes with MDAC, the test item is non-irritant. UN GHS Classification is No Category.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016 - 16 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The draft report was issued later than stated in the study plan, and Szabolcs Gáty became responsible for QA on Feb 15 2017, and no longer a member of Test Facility Management. These deviations had no adverse affect the results or study integrity.

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

yes

Remarks:

The draft report was issued later than stated in the study plan, and Szabolcs Gáty became responsible for QA on Feb 15 2017, and no longer a member of Test Facility Management. These deviations had no adverse affect the results or study integrity.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Osaka Soda Co., Ltd
- Name: MDAC (Chemical name: 1,2-Cyclohexanedicarboxylic acid, di-2-propenyl ester)
- Appearence: Colourless liquid
- Batch No. of test material: 40201
- Expiration date of the batch: 26 January 2019
- Purity test date: 99.2% (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤ 70% Relative Humidity), protected from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility, stability of the test substance in the solvent/vehicle: 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: None, the test item was applied as supplied, no formulation was required.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: source not stated

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity testing in an ECVAM international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431). It was therfore considered as suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM(SM) (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-050, Expiry date: 19 December 2016) a three-dimensional human epidermis model.
- Tissue batch number(s): Batch No.: 16-EKIN-050,
- Date of initiation of testing: 15 December 2016
- Date of completion of testing:16 December 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Incubated for 4 hours (±10 min) at room temperature (22.2-24.3°C) covered with plate lids.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After incubation, all test item treated tissues and positive control tissues were removed and rinsed thoroughly with PBS solution to remove all residual test item or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly. The remaining PBS was removed from the epidermal surface using a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution (0.3 mg/mL). 2 mL of MTT working solution was added to each well below the skin units.
- Incubation time: At 37°C in an incubator with 5% CO2 for 3 hours, protected from light .
- Spectrophotometer: Optical Density (OD) measured using a plate reader (a blank sample containing 2 mL of acidified isopropanol was processed in parallel with MTT solution). The instrument was verified by measuring a verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.
- Wavelength: 570 nm.

- Contamination: No contamination during the study. EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product was assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM).

NUMBER OF REPLICATE TISSUES: Two replicates per test-item were used. Two negative controls and two positive controls were also run in each assay. As the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues : As the test item had an MTT interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.
- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 16-EKIN-033, Expiry Date: 22 August 2016) were placed in a 12 well plate with 2 mL of distilled water, then incubated (37°C with 5% CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation period the water was discarded and the dead epidermis units frozen on 19 August 2016, frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to that of living tissues.
- N. of replicates : Two additional controls on killed epidermis (two test item treated and two negative control treated skin units) were also used to determine the MTT interacting potential of the test item
- Method of calculation used: Additional control samples were used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value was corrected by the result of the additional controls before calculation of viability% as follows:

Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT- ODKNC) / ODNC] × 100
OD KNC: negative control treated killed tissues OD
OD KT: test item treated killed tissues OD
OD NC: negative control OD

If NSMTT is ≤ 50%, then true MTT metabolic conversion (TODTT) had to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues

– The % relative viability (RV%) for each test item replicate was calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item was calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin: if both disks have mean viability of <35% = Corrosive (at the corresponding incubation period), or if the mean value is <35% and the variability is less than 50%.

- The test substance is considered to be non-corrosive to skin: if both disks have mean viability of ≥35% = Non Corrosive, or If the mean value is ≥35% and the variability is less than 50% = Non Corrosive

Exceptions: If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

Classification Packing group Criteria for In Vitro interpretation

UN GHS / EU-CLP Sub Category 1A If corrosive after 3 min exposure
Sub Category 1B If not corrosive after 3 min exposure and corrosive after 1 hour exposure
Sub Category 1C If not corrosive after 1 hour exposure and corrosive after 4 hours exposure

Non corrosive If not corrosive after 4 hours exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume with unit): 50 μL of test item was applied evenly to each of two test units and each additional control skin units.
- Concentration (if solution): The test item was applied as supplied, no formulation was required. No correction for purity of the test item was applied.

NEGATIVE CONTROL
- Amount(s) applied (volume):50 μL of physiological saline was added to each of the two negative control skin units and each of the two negative control treated killed epidermis units.
- Concentration (if solution): 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied (volume): 50 μL of glacial acetic acid was added to each of the two positive control skin units.
- Concentration (if solution): not specifed

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (22.2 - 24.3°C) covered with the plate lids. Following rinsing at the end of the incubation time, MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours, protected from light.

Number of replicates:

In this assay, two replicates per test item were used.Two negative controls and two positive controls were also run in each assay. As the test item had an MTT
interacting potential, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item (MDAC)

Value:

127.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The results indicate the test item (MDAC) is non-corrosive to the skin.

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: 50 μL of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed. After three hours incubation, purple colour in the mixture was detected; therefore additional controls were used in the experiment. Based on the results of the additional controls on killed epidermis, the calculated NSMTT was 0.9%. This was considered to be significant, thus correction with Non-Specific MTT (NSMTT) were made.

- Colour interference with MTT: 10 μL of the test item was added to 90 μL of deionised water and isopropanol. The mixture was shaken for about 15 minutes at room temperature and then colour was checked (unaided visual assessment). As no coloured solution was detected and the test item had no intrinsic colour, no additional colour controls were needed in the experiment to determine the Non Specific Colour (the test item showed no ability to stain the epidermis).
.
DEMONSTRATION OF TECHNICAL PROFICIENCY:
After receipt, the two indicators (pH and temperature) of the delivered kits were checked and based on the observed colours, the epidermis units were acceptable for study use.
The mean OD value of the two negative control tissues was in the recommended range (0.856).
The two positive control treated tissues showed 0.9% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 0.1%.
The difference of viability between the two negative control tissue samples in the MTT assay was 10.6%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with MDAC, the mean cell viability was 127.7% compared to the negative control (after adjustment for non-specific MTT reduction). This is above the threshold of 35%, therefore the test item was considered non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with MDAC (Batch number: 40201), the results indicate that the test item is non-corrosive to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2016 - 16 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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

Remarks:

The draft report was issued later than stated in the Study Plan due to organisational changes (14 Feb 2017) where S. Gáty became responsible for QA (15 Feb 2017). These deviations had no adverse affect on the integrity or validity of the study.

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

Remarks:

The draft report was issued later than stated in the Study Plan due to organisational changes (14 Feb 2017) where S. Gáty became responsible for QA (15 Feb 2017). These deviations had no adverse affect on the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material: MDAC
- Appearence: Colourless liquid
- Source: Osaka Soda Co., Ltd
- Batch/lot No: 40201
- Expiration date of the lot/batch: 26 January 2019
- Purity test date: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70% Relative Humidity (RH)), protected from light
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: MDAC was soluble in physiological saline (30 μL test item in 1 mL saline). Assumed stable for the duration of the study
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út 129., Hungary). Chicken heads were obtained from a veterinary-inspected, commercial slaughter-house, processing chickens for human consumption.
- Number of animal heads: 4/5 chicken heads per box. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box and closed.
- Characteristics of donor animals (e.g. age, sex, weight): Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported at ambient temperature to CiToxLAB Hungary Ltd. at the earliest convenience (i.e. within 2-hours)
- Time interval prior to initiating testing: The heads were received at CiToxLAB and processed within approximately 2 hours of collection. For selected eyes deemed appropriate for testing, acclimatization started and was conducted for approximately 45 to 60 minutes.
- indication of any existing defects or lesions in ocular tissue samples: No, the appropriate number of eyes were examined (with a slit lamp microscope) to ensure that all were in good condition and suitable for study use.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with test substance. The test substance was applied as supplied, no formulation was required.

VEHICLE
- Amount(s) applied (volume or weight with unit): 30 μL of physiological saline was applied to the negative control eye.
- Concentration (if solution): Physiological saline (0.9% (w/v) NaCl)
- Lot number: 62351Y05-2
- Manufacturer: B. Braun Pharmaceuticals SA
- Expiry Date: 31 May 2019
- Storage condition: Room temperature

Duration of treatment / exposure:

The time of application was noted and after an exposure period of 10 seconds, the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item.

Duration of post- treatment incubation (in vitro):

240 minutes. The control eyes and test eyes were evaluated pre-treatment and at ca. 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

Details on study design:

SELECTION OF ISOLATED EYES
After removing the head from the plastic box, it was placed on soft paper. The eyelids were carefully cut away using scissors avoiding any damage to the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. The fluorescein treated cornea was then examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was acceptable for study use, the eyeball was carefully removed from the orbit.

PREPARATION OF ISOLATED EYES
The eye ball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent corneal distortion and subsequent corneal opacity. Once removed, the eye was placed onto damp paper and the nictitating membrane cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain appropriate humidity.

EQUILIBRATION (Acclimitisation) AND BASELINE RECORDINGS
Prepared eyes were placed in thier respective steel clamp with the cornea positioned vertically with the eye in (as per placement within the chicken head). Too much pressure on eyes was avoided when clamping. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding each eye was positioned such that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for any experimental adjustiments or examinations.

The appropriate number of eyes were selected and after being placed in the superfusion apparatus. They were then examined again using a slit lamp microscope to ensure they were acceptable for use. The focus was adjusted to clearly see the physiological saline flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If appropriate for testing, acclimatization started, this was for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were controlled at a temperature (32±1.5°C) during the acclimatisation and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. Cornea thickness should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes of this study. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered suitable for study use. Fluorescein 10% (lot No. 250817F, Manufacturer. Alcon, Expiry date. 31 May 2017) was diluted with physiological saline to a final concentration of 2% (w/v). The formulation was allocated an expiry date of 2 Feb 2017.

QUALITY CHECK OF THE ISOLATED CORNEAS:
The fluorescein treated cornea was examined using a hand-held slit lamp or slit lamp microscope, with the eye still within the chicken head, this ensured the cornea was not damaged. If the cornea was judged adequate for study use the eyeball was carefully removed from the orbit. The appropriate number of eyes was thereafter selected and placed in the superfusion apparatus. They were then examined again to ensure studt acceptability. The focus was adjusted to see clearly the physiological saline flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

NUMBER OF REPLICATES:
Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

NEGATIVE CONTROL USED:
Physiological saline solution (0.9% (w/v) NaCl). Expiry date 31 May 2019.

POSITIVE CONTROL USED:
Benzalkonium chloride solution, 50% in water diluted to acheive a final concentration of 5% (w/v). Allocated expiry date for the positve control formulation was Sep 2017.

APPLICATION DOSE AND EXPOSURE TIME:
30 μL of test item was applied to the entire surface of the cornea and exposed for a period of 10 seconds from the end of applicationn to the cornea surface

TREATMENT METHOD: Closed clamping chamber

POST-INCUBATION PERIOD: yes. 240 minutes. The control eyes and test eyes were evaluated pre-treatment and at Ca.30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test item was rinsed thoroughly with 20 mL physiological saline at ambient temperature. Care was taken to not damage the cornea but attempting to remove all residual test item.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and opacity were measured and scored at all time points (30, 75, 120, 180 and 240 minutes after exposure) Fluorescein
retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to
the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the
investigator.

SCORING SYSTEM:
The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments, as detailed below:

Mean corneal swelling (%). No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes.

Corneal Swelling (%) ICE Class
0 to 5 I
>5 to 12 II
>12 to 18 (>75 min after treatment) II
>12 to 18 (≤75 min after treatment) III
>18 to 26 III
>26 to 32 ( 75 min after treatment) III
>26 to 32 (≤75 min after treatment) IV
>32 IV

Small negative numbers for swelling (0 to -5%) following application are evaluated as class I. Large negative numbers (>12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

- Mean maximum opacity score. No significant corneal opacity change (severity 0.5) was noted on two eyes.

Mean Maximum Opacity Score ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 4.0 IV

- Mean fluorescein retention score at 30 minutes post-treatment. No significant fluorescein retention change (severity 0.5) was observed on one eye. No other corneal effect was observed.

Mean Fluorescein Retention Score at 30 minutes post-treatment ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 3.0 IV

DECISION CRITERIA: In Vitro Irritancy Score (IVIS): The irritation effects of the test item (MDAC) were evaluated according to the OECD No. 438 (26 July 2013). The test as described in OECD 438 is approved by international regulatory agencies as a replacement for the identification of non-irritant, corrosives/severe irritants in the in vivo Rabbit Eye Assay (OECD No. 405).

DECISION CRITERIA: The conclusion on eye irritancy was based on the OECD guideline quantitative assessments. The mean values of treated eyes for maximum corneal thickness, corneal opacity and fluorescein retention are documented in line OECD 438 classification requirements.

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum swelling up to 240 minutes

Value:

ca. 1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum corneal opacity upto 240 minutes

Value:

ca. 0.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean fluorescein retention upto 30 minutes

Value:

ca. 0.17

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The results from all eyes used in the study met the quality control standards. Thenegative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control results were in line with historical data.
- Acceptance criteria met for positive control: The positive control results were in line with historical data.

The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historical data. This experiment was
considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant corneal opacity change (severity 0.5) was noted on two eyes. No significant fluorescein retention change (severity 0.5) was observed on one eye. No other corneal effects were observed.

Based on this in vitro eye irritation in the isolated chicken eyes test with MDAC, the test item is non-irritant. UN GHS Classification is No Category.
.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

This substance is not classified for skin irritation.