N-carbamoylmethyliminodi(acetic acid)

Endpoint summary

Description of key information

The test item is considered to be non-irritant to skin in a OECD 439 study (reference 7.3.1 -1).

In an experimental study according to OECD test guideline 438 an overall ICE score of 1xI and 2xII was determined for the test item (reference 7.3.2 -1).

In an experimental study according to OECD test guideline 492 the test substance showed no eye irritation potential (reference 7.3.2 -2).

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:

2016-06-29 to 2016-07-01

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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors, not further detailed.

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTM SM), manufactured by EPISKIN SNC Lyon, France
- Tissue batch number: 16-EKIN-026
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Expiry date: 04 July 2016
- Date of initiation of testing: 2016-06-29

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 step using 25 mL PBS 1x solution
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Histology scoring: Historical control value: >= 19.5, 22.7 +/- 0.5 for batch used
- IC50: Historical control value: 1.5 mg/mL <= IC50 <= 3 mg/mL, 2.6 mg/mL for batch used
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spnous and granular layers and a thick stratum corneum was satisfactory in the batch used.
- Contamination: Blood of donor: absence of HIV 1+2, hepatitis C anitbodies and hepatitis B antigen HB; epidermal cells of donor: absence of bacteria, fungus and mycoplasma

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritating to skin if the viability after exposure is <= 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after exposure is greater than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg (to moistened skin)

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq.

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

18 %

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES (mean OD of 0.762 in the range from 0.6 to 1.5; SD of 10.23 < 18)
- Acceptance criteria met for positive control: YES (mean viability of 18 % in the range from 0 to 40 %; SD of 7.55 < 18)
- Acceptance criteria met for variability between replicate measurements: YES (test substance: SD of 7.11 < 18)
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered to be non-irritant to skin.

Executive summary:

An EpiSkinTM SM test with the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015. Triplicates of the human skin model EPISKIN (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with test item and incubated for 15 minutes at room temperature. Exposure of the reconstructed human epidermis skin units with test item was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 91 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be 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:

weight of evidence

Study period:

2016-06-22

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)

Version / remarks:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

08 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughter house, TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: heads were transported approximately within 2 hours from collection, 19.4 to 20.2 ºC, heads were wrapped with paper moistened with saline, placed in a plastic box that can be closed (4-5 heads/box)
- Indication of any existing defects or lesions in ocular tissue samples: Eyes with defects before treatment were rejected.
- Indication of any antibiotics used: not specified
- Selection and preparation of corneas: Non-damaged eyes were removed from the heads. The nictitating membrane was cut away with other connective tissue.
- Quality check of the isolated corneas: Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g (per eye)

CONTROLS
- Amount applied: 0.03 g imidazole; 30 µL NaCl (in saline)

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

30, 75, 120, 180 and 240 minutes after post-treatment rinse (minor variations +/- 5 minutes were considered acceptable)
Cornea thickness and opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and at 30 min.

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3 eyes (test substance and positive control)
1 eye (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
With the eyes still in the chicken heads, eyelids were carefully cut away with scissors from the chicken eyes. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and then rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye, which may lead to corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain the appropriate humidity.

EQUILIBRATION AND BASELINE RECORDINGS
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. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After 10 seconds the cornea surface was rinsed thoroughly with approx. 20 mL saline solution at ambient temperature. Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole and test item treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes, using a slit lamp microscope
- Damage to epithelium based on fluorescein retention: yes, using a slit lamp microscope
- Swelling: depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm
- Macroscopic morphological damage to the surface: yes, using a slit lamp microscope

SCORING SYSTEM:
- Mean corneal swelling (%): according to TG
- Mean maximum opacity score: according to TG
- Mean fluorescein retention score at 30 minutes post-treatment: according to TG

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

Irritation parameter:

percent corneal swelling

Run / experiment:

mean maximum up tp 240 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

28

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

mean maximum

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

4

Irritation parameter:

fluorescein leakage

Run / experiment:

mean maximum

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

3

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Interpretation of results:

study cannot be used for classification

Conclusions:

Using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xI, 2xII.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. The Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 2xII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-10-24 to 2016-10-28

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)

Version / remarks:

28. Jul. 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: Keratinocyte strain: 4F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcularTM Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the human cornea model. Within a testing strategy, the EpiOcularTM EIT is used as a replacement of the Draize Eye Irritation Test. Solids are within the applicability domain of the test method.
- Description of the cell system used: EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories (Mylnské Nivy 73, 82105 Bratislava, Slovakia) Designation of the kit: OCL-212-EIT, Day of delivery: 26. Oct. 2016, Batch no.: 23742, certificate was provided, Tissue was checked for potential biological contaminants and none were detected.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 52.1 and 52.4 mg

Controls: 50 μL/tissue

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

17h and 49 minutes

Number of animals or in vitro replicates:

2 tissues per test or control item

Details on study design:

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity; 25 minutes post soak at room temperature; post-treatment incubation 17 hours and 49 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity
- Description of any modifications to the test procedure : none
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: In a pre-test, no indication for direct MTT reduction or colouring by the test substance was observed. Therefore, no such controls were conducted.
- Wavelength used for quantifying MTT formazan: 570 nm (device used: 96-well-plate photometer Anthos Reader)
- Description of the method used to quantify MTT formazan: spectrophotometer
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : The evaluation criteria of the OECD TG 492, i.e. a threshold of 60 % to discriminate non eye irritants from eye irritants, were used.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Both the responses of the negative and the positive control were within the range of the historical data.
- Complete supporting information for the specific RhCE tissue construct used: see above
- Reference to historical data of the RhCE tissue construct: Acceptable OD range of negative control: 1.000 - 2.024; acceptable relative viability range of the positive control: 14.5 - 49.3 %
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not reported
- Positive and negative control means and acceptance ranges based on historical data: Acceptable OD range of negative control: 1.000 - 2.024; acceptable relative viability range of the positive control: 14.5 - 49.3 %
- Acceptable variability between tissue replicates for positive and negative controls: < 20 %)
- Acceptable variability between tissue replicates for the test chemical: < 20 %

Irritation parameter:

other: percent tissue viability  (migrated information)

Run / experiment:

mean of 2 tissues

Value:

83.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

39.5

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, 0.1 % was in line with < 20 %
- Acceptance criteria met for positive control: yes, 7.5 % was in line with < 20 %
- Acceptance criteria met for the test substance: yes (SD of 17.6 %, i.e. < 20 %; individual values: 92.2 % and 74.6 %)

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered as not eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

An in vitro eye irritation study according to OCED 492 was conducted. One valid experiment was performed. The solid test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. Demineralised water was used as negative control and Methyl acetate was used as positive control. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 39.5 % (< 50 %). Variation within tissue replicates was acceptable (range ≤ 20 %). After treatment with the test item, the relative absorbance values were reduced to 83.4 %. This value is above the threshold for eye irritation potential (≤ 60 %). Under the conditions of the test, the test item is considered as not eye irritant in the EpiOcularTM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An EpiSkinTM SM test with the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015. Triplicates of the human skin model EPISKIN (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with test item and incubated for 15 minutes at room temperature. Exposure of the reconstructed human epidermis skin units with test item was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean value: 91 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin.

 

Eye irritation

To determine the corrosion or irritation potential of the test substance for the eye a stepwise weight of evidence approach was done. An Isolated Chicken Eye Test (ICET) according to OECD 438 (version 2013) was conducted resulting in “No prediction can be made” and therefore, neither classification in Category 1 (UN GHS) nor no classification (UN GHS: No Category) was triggered. Therefore an eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 (version 2015) was conducted in accordance with the top down approach described in the Guidance on an integrated approach on testing and assessment (IATA) for serious eye damage and eye irritation (Series on testing and assessment No. 263).

 

OECD 438

The purpose of this Isolated Chicken Eye Test (ICET, OECD 438) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. The Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 30 mg/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 2xII. Positive and negative controls showed the expected results. The experiment was considered to be valid.

 

OECD 492

An in vitro eye irritation study according to OCED 492 was conducted. One valid experiment was performed. The solid test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. Demineralised water was used as negative control and Methyl acetate was used as positive control. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8≤mean OD≤2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 39.5 % (< 50 %). Variation within tissue replicates was acceptable (range≤20 %). After treatment with the test item, the relative absorbance values were reduced to 83.4 %. This value is above the threshold for eye irritation potential (≤60 %). Under the conditions of the test, the test item is considered as not eye irritant in the EpiOcularTM Eye Irritation Test.

 

Conclusion

Based on the result from the Isolated Chicken Eye Test according to OECD 438 (version 2013) a classification as No category according to UN GHS was not possible due to the result of “No predication can be made”. The second in vitro study, the eye irritation study using Reconstructed human Cornea-like Epithelium according to OECD 492 (version 2015), determined that the test item showed no eye irritation potential.

OECD guideline 492 (version 2015) determined that a classification into No Category is suitable for the test item. The result “No predication can be made” of the OECD 438 (version 2013) study does not contradict the determination of no classification for eye irritation of the test item. Furthermore, in the updated guideline version of OECD TG 438 (27th June 2018) the overall ICE class of 1xI, 2xII is considered as “No category” (previously “No predication can be made”). The prediction of “No category” OECD TG 492 (updated: 18th June 2019) remains.

Based on the study results and using the top down approach described in the Guidance on IATA for serious eye damage and eye irritation an overall conclusion for the test item can be reached based on the initial and updated guidelines. The weight of evidence with the two conducted studies determined that the test substance does not cause eye irritation and no classification is required (No Category according to UN GHS).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin irritation, the test item does not require classification for skin irritation or corrosion according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.

 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on the potential to cause damage to the eye, the test item does not require classification for eye irritation or damage according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.