Cyclohexanamine, 4,4’-methylenebis[N-(2,2-dimethyl-3-(4-morpholinyl)propylidene)]-

Endpoint summary

Description of key information

Skin irritation

In an in vitro skin irritation test according to OECD 439 using the EPISKIN model, the test item was shown to be irritant (UN GHS Category 2) and/or corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2.

 

In an in vitro skin corrosion test (EpiDerm model) according to OECD 431 with the test item, the results indicate that the test item is not corrosive. According to the UN GHS classification systems, the test item has been categorized as “Non-corrosive”.

 

Eye irritation

In an ICET according to OECD guideline 438, the overall ICE classes of the test item were once I, once II and once IV. According to the guideline OECD 438, the test item is categorized as “Non prediction can be made”.

 

In an in vitro eye irritation test according to OECD guideline 492, using the EpiOcular™ model, the test item was eye irritant (UN GHS Category 2) and/or eye corrosive (UN GHS Category 1). However, this test method cannot resolve between UN GHS Categories 1 and 2.

 

Conclusion: Combining both tests for skin irritation/corrosion, the test item is irritating to skin. Combining both tests for eye irritation/corrosion, the test item is irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-08-11 to 2021-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: In Vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT), For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDermTM

Version / remarks:

2014-11-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No 440/2008, Annex Part B, B.40bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, amended by Commission Regulation (EU) 2019/1390 of 31 July 2019.

Version / remarks:

2019-07-31

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: human adult donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This method is approved by international regulatory agencies as a replacement for the identification of corrosives in the in vivo Rabbit skin assay (OECD 404) and is specifically approved as a replacement for the in vivo skin corrosivity test within OECD 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200, MatTek, Ashland, MA, USA and Bratislava, Slovakia)
- Tissue batch number: 34188
- Expiry date: 2021-08-20
- Date of initiation of testing: 2021-08-18

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (3 min treatment), 37±1 °C, (1 hour treatment)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The units were rinsed thoroughly with 1x DPBS sterile solution to remove all of the test material from the epidermal surface. The constant soft stream of DPBS was used from approximately 1.5 cm distance, and filling and emptying the tissue insert was completed 20 times.The rest of the DPBS was decanted onto the absorbent material.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL of 1 mg/mL MTT per well
- Incubation time: 3 hours (± 15 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability, barrier function and reproducibility: The quality of the final product is assessed via the MTT cell viability test using the cytotoxic test compound Triton X-100 (1.0%)
- Contamination: The absence of HIV1 and 2 antibodies, Hepatitis C antibodies and Hepatitis B antigens HBs as well as funghi, bacteria and mycroplasma were verified beforehand.

NUMBER OF REPLICATE TISSUES: 3 replicate tissues per test item and negative/positive control (for each exposure time)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues: The test item did not show direct interaction with MTT. Using of additional fresh tissue controls was not necessary.
- Killed tissues: The test item did not show direct interaction with MTT. The use of additional killed tissue controls was not necessary.
- Method of calculation used: See "Any other information on materials and methods, incl. tables"

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 min exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%. If a substance identified as corrosive shows a viability of less than 25% after 3 min exposure, it can be assigned to sub-category 1A. If a substance identified as corrosive shows a viability of equal or higher than 25% after 3 min exposure, it can be assigned to a combination of sub-categories 1B and 1C.
- The test substance is considered to be non-corrosive to skin if the viability after 3 min exposure is higher or equal to 50% or if the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: Approx. 50 μL

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
- Concentration: 8N KOH solution

Duration of treatment / exposure:

3 min and 1 hour

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

3 replicate tissues per test item, negative control and positive control (for each exposure time) were tested in a single independent experiment.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure (mean of 3 tissue replicates)

Value:

84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure (mean of 3 tissue replicates)

Value:

70

Vehicle controls validity:

not applicable

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: No
- 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 (Study No.: 392-431-5488) using the ten Proficiency Chemicals according to OECD Test Guideline No. 431.

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
- Range of historical values if different from the ones specified in the test guideline: See "Attached background material"

Interpretation of results:

study cannot be used for classification

Remarks:

This test method (OECD 431) must be considered together with a suitable test method for identifying skin irritants. The final classification is chosen based on the results from both test guidelines will be described in the endpoint summary.

Conclusions:

In this in vitro skin corrosion test in EpiDerm model according to OECD 431 and GLP with the test item, the results indicate that the test item is not corrosive. According to the UN GHS classification systems, the test item has been categorized as “Non-corrosive”.

Executive summary:

A GLP-compliant EpiDerm test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 18 June 2019. Disks of EpiDermTM (three units / chemical / incubation time) were treated with the test item and incubated for 60 minutes (+ 3 min) at standard culture condition (37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere) and 3 min at room temperature. Exposure of test material was terminated by rinsing with DPBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using MTT-100-EXT (isopropanol) and quantified spectrophotometrically. Potassium hydroxide (KOH) 8N solution and sterile deionized water treated (three units / positive and negative control) epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 60 minutes of exposure is above or equal 15 % and the mean relative viability after 3 minutes of exposure is above or equal 50 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 60 minutes and 3 minutes of exposure. The average test item treated tissue mean relative viability was 70 % at 60 minutes of exposure and the mean relative viability was 84 % at 3 minutes of exposure. Positive and negative controls showed the expected optical density (OD) and cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. In conclusion, in this in vitro skin corrosion test in EpiDermTM model (OECD 431), the results indicate that the test item is not corrosive. According to the UN GHS classification systems, the test item has been categorized as “Non-corrosive”.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-10-25 to 2021-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 131: EpiSkinTM Skin Irritation Test 15 min – 42 hours

Version / remarks:

2012-06-09

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-07-31

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2021-06-14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: Adult human donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. 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: EpiSkin SM (EPISKIN SNC Lyon, France)
- Tissue batch number: 21-EKIN-043
- Expiry date: 2021-11-01
- Date of initiation of testing: 2021-10-27

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: 37 ± 1 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The units were rinsed with approx. 25 mL PBS. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging the epidermis).
- Observable damage in the tissue due to washing: 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 (±15 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability, barrier function and reproducibility: The quality of the final product is assessed via the MTT cell viability test using the cytotoxic test compound sodium dodecyl sulphate (SDS)
- Contamination: The absence of HIV1 and 2 antibodies, Hepatitis C antibodies and Hepatitis B antigens HBs as well as funghi, bacteria and mycroplasma were verified beforehand.

NUMBER OF REPLICATE TISSUES: 3 replicate tissues per test item, negative and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues: The test item showed no direct interaction with MTT. The use of additional fresh tissue controls was not necessary.
- Killed tissues: The test item did not show direct interaction with MTT. The use of additional killed tissue controls was not necessary.
- N. of replicates: 3 replicates per test item, 3 replicates per controls (negative, positive controls) and 2 replicates of colour control (NSCliving) were used.
- Method of calculation used: See "Any other information on materials and methods, incl. tables"

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant or corrosive to skin (category 2 or 1) if the viability after 15 min exposure is equal or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 min exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: Approx. 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5% SDS

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h)

Number of replicates:

3 replicate tissues per test item, negative and positive control were tested in a single independent experiment.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (mean of 3 tissue replicates)

Value:

17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency in a separate study (Study No.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

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
- Range of historical values: See "Attached background material"

Interpretation of results:

study cannot be used for classification

Remarks:

This test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2 and must be considered together with a suitable test method for identifying skin corrosives and severe irritants. The final classification is chosen based on the results from both test guidelines will be described in the endpoint summary.

Conclusions:

In an in vitro skin irritation test according to OECD 439, using the EPISKIN model, with the test item indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2.

Executive summary:

The purpose of this OECD 439 and GLP compliant study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 ± 0.5 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 ± 1 °C for a post-incubation period of 42 ± 1 hours (h) in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours ± 5 minutes with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified by means of the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item showed significantly reduced cell viability in comparison to the negative control (mean viability: 17 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The mean OD value of the blank samples was below 0.1. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2021-10-18 to 2021-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2017-02-14

Deviations:

no

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:

2018-06-25

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: TARAVIS KFT., 9600 Sárvár, Rábasömjéni, utca 129. Hungary
- Number of animals: Number not specified, but eyes used in the assay were from the same groups of eyes collected on one specific day
- Characteristics of donor animals: Approx. 7 weeks old healthy animals, weighting 1.5 – 2.5 kg
- Storage, temperature and transport conditions of ocular tissue: After collection, the heads were wrapped with paper moistened with saline, then placed in a plastic box (4-5 heads/box).The heads were transported to the testing laboratory approx. within 2 hours from collection. The transport temperature was between 19.3 ºC and 20.2 ºC.
- Time interval prior to initiating testing: Approx. 2 hours
- Indication of any existing defects or lesions in ocular tissue samples: Cornea integrity was checked by applying one small drop of fluorescein 2% (w/v) solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged.
- Selection and preparation of corneas: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. If the corneas were in good condition, the eyeballs were carefully removed from the orbit. The nictitating membrane was held 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 in order to prevent distortion of the cornea and subsequent 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 so that the appropriate humidity was maintained. The enucleated eye was placed in a steel clamp, avoiding too much pressure on the eye by the clamp. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
- Quality check of the isolated corneas: The enucleated eyes were examined again in the superfusion apparatus with the slit lamp microscope to ensure that they were in good condition. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or a high corneal opacity score (i.e. > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope. Only corneas were used which did not show a change in cornea thickness by more than ± 5-7 % within approximately 45 to 60 minutes.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 μL of test item was applied via a micropipette onto the center of the cornea

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The control and test item treated eyes stayed in the chambers until measurement were finished (approx. 240 minutes after the post-treatment rinse, minor variations within ± 5 minutes were considered acceptable).

Number of animals or in vitro replicates:

The treatment group and concurrent positive control group consisted of three eyes. The negative control group consisted of one eye.

Details on study design:

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. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment.

NUMBER OF REPLICATES
Three replicate eyes (positive control and treatment group) and one replicate eye (negative control)

OBSERVATION PERIOD
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove the entire residual test item if possible. The cornea surface of negative and positive control treated corneas was also rinsed thoroughly after an exposure period of 10 seconds with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

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

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

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

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean of three replicate eyes

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

corneal swelling 

Run / experiment:

Mean of three replicate eyes

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Mean of three replicate eyes

Value:

4

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the laboratory demonstrated the technical proficiency in a separate study (Study Code: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

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

Interpretation of results:

study cannot be used for classification

Remarks:

The final classification is chosen based on the results from both OECD 438 and OECD 492 guidelines and is described in the endpoint summary.

Conclusions:

In this ICET according to OECD guideline 438, the overall ICE classes of the test item were once I, once II and once IV. According to the guideline OECD 438, the test item is categorized as “Non prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) according to OECD guideline 438 and GLP was to evaluate the potential ocular corrosivity or severe irritancy of the test item its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. The test item SIKA Hardener MD (SIKA Härter MD), the positive control (5% solution of Benzalkonium chloride), and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way that the test and control items evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~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 classes of the test item were once I (based on the fluorescein retention of 0.0) and once II (based on the cornea swelling of 17 % within 240 minutes) and once IV (based on the corneal opacity score of 2.7). The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. So, the positive and negative controls showed the expected results. The experiment was considered to be valid. In this ICET, the overall ICE classes of the test item were once I, once II and once IV. According to the guideline OECD 438, the test item is categorized as “Non prediction can be made”.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-11-30 to 2022-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

2021-02-02

Deviations:

no

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:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The three-dimensional RhCE tissue construct is produced using primary human epidermal keratinocytes (EpiOcular™ OCL-200). The EpiOcular™ OCL-200 RhCE tissue construct is similar to the in vivo corneal epithelium three-dimensional structure. For certificate of authenticity and mycoplasma status see "attached background material".
- RhCE tissue used, including batch number: EpiOcular™ OCL-200, MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia. The batch no of the tissues was 34969 .

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL (or a sufficient amount to cover uniformly the entire tissue surface)
- Concentration: The test item was applied in its original form, no formulation was required.

Duration of treatment / exposure:

30 minutes (± 2 min)

Duration of post- treatment incubation (in vitro):

120 ± 15 minutes

Details on study design:

- Details of the test procedure used: After arrival of the tissues, they were equilibrated to room temperature for about 15 minutes. Afterwards, the tissues were pre-incubated in pre-warmed medium at standard culture condition (37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere) overnight (16-24 hours) including replacing the medium after one hour incubation. After the overnight incubation, the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS for 30 ± 2 minutes. Afterwards, 50 µL of the test item, positive or negative control was applied topically on the tissues. The plates with the treated tissue units were then incubated for the exposure time of 30 minutes (± 2 min) at standard culture conditions. After the incubation time, the units were removed and rinsed thoroughly with Ca++Mg++ free-DPBS. Because the test substance adhered strongly to the tissue, three clean beakers (glass with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++Free-DPBS was used per test item and controls. The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues were dipped into the first beaker of DPBS and were swirled in a circular motion in the liquid for approximately 2 seconds and thereafter were lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed five additional times case of the test item treated tissues and two additional times case of the controls in their first beaker. The tissue was rinsed in the second and third beakers of DPBS six times case of the test item treated tissues and three times case of the control treated tissues each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. The tissues were then immersed in 5 mL of pre-warmed assay medium for a 12 ± 2 minute post-soak incubation. At the end of the post-soak immersion, the inserts were transferred to new plates and incubated in assay medium for 120 ± 15 minutes post-treatment incubation period. The units are then transferred into MTT ready to use solution-filled plates (300 μL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 15 min). Afterwards, the inserts are transferred into isopropanol and stored overnight or are immediatedly extracted on an orbital plate shaker (~120 rpm) for 2 to 3 hours. 200 μL sample(s) from each tube are placed into the wells of a 96-well plate and Absorbance / Optical Density is read at the wavelength of 570 nm using isopropanol solutions as the blank.
- Doses of test chemical and control substances used: The test item was applied in its original form, no formulation was required. As negative control, sterile deionized water was used. ≥99.0% methyl acetate was used as positive control.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Exposure: 30 minutes (± 2 min) at 37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere
Post-exposure immersion: 12 ± 2 minutes post-soak incubation at room temperature
Post-exposure incubation: 120 ± 15 minutes post-treatment incubation at 37±2 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
Check-method for possible direct MTT reduction with test item: Approximately 50 μL test item were added to 1 mL MTT 1 mg/mL solution and the mixture was incubated for 3 hours (±15 min) in the dark at 37±2°C, 5±1% CO2, ≥95% humidified atmosphere. As no colour change was observed, no additional controls were necessary.
Assessment of coloured or staining materials: Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. 50 μL test item were added to 1 mL of water and the mixture was incubated in the dark at 37 ± 2 °C, 5 ± 1 % CO2, ≥ 95 % humidified atmosphere for at least one hour and then colour checked (unaided eye assessment). Furthermore, approximately 50 μL test article were added to 2 mL isopropanol, the same amount as used for formazan extraction, and is incubated on an orbital plate shaker (120 rpm) for 2 to 3 hours at room temperature and then colour checked (unaided eye assessment).As no colour change was observed, no additional controls are necessary.
- Number of tissue replicates used per test chemical and controls: Two tissue replicates are used for test item, positive and negative controls.
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: Absorption is measured at 570 nm using a Varioskan™ LUX Type 3020 spectrophotometer equipped with a Xenon flash lamp (100Hz) and a photodiode detector with a wavelength range of 200-1000 nm.
- Description of the method used to quantify MTT formazan: Following the formazan extraction, 200 μL sample(s) from each tube are placed into the wells of a 96-well plate and absorption/ optical density of the samples are determined in the spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control. The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. If the viability values of two individual tissue units are highly variable, causing different classifications, the test item may be retested with two more tissues and the mean viability of the four tissues will be taken to classify as above or below 60%. 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.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: See "Attached background material"
- Complete supporting information for the specific RhCE tissue construct: See "Attached background material"
- Reference to historical data of the RhCE tissue construct: See "Attached background material"
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Prior to routine use of the method, the laboratory demonstrated the technical proficiency in a separate study (study no.: 392-492-1722) using the fifteen proficiency chemicals according to OECD Test Guideline No. 492.
- Positive and negative control means and acceptance ranges based on historical data: See "Any other information on materials and methods incl. tables"
- Acceptable variability between tissue replicates for positive and negative controls: See "Any other information on materials and methods incl. tables"
- Acceptable variability between tissue replicates for the test chemical: See "Any other information on materials and methods incl. tables"

Irritation parameter:

mean percent tissue viability 

Run / experiment:

1 (mean of 2 tissue replicates)

Value:

58

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No visible test item was observed on the surface of the test item treated tissues after the end of the rinsing process.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the laboratory demonstrated the technical proficiency in a separate study (study no.: 392-492-1722) using the fifteen proficiency chemicals according to OECD Test Guideline No. 492.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was: 2.161. The difference of viability between two tissue replicates ranged from 0.5 % to 0.8 %. All validity criteria were within acceptable limits in the experiment.
- Acceptance criteria met for positive control: The positive control result showed 4 % viability at 30 minutes exposure. The difference of viability between two tissue replicates ranged from 0.5 % to 0.8 %. All validity criteria were within acceptable limits in the experiment.

Interpretation of results:

study cannot be used for classification

Remarks:

This test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2 and must be considered together with a suitable test method for identifying ocular corrosives and severe irritants.

Conclusions:

The results obtained from this in vitro eye irritation test according to OECD guideline 492, using the EpiOcular™ model, with the test item SIKA Hardener MD (SIKA Härter MD) indicate that the test item is Eye Irritant (UN GHS Category 2) and/or Eye Corrosive (UN GHS Category 1). According to the guideline OECD 492, SIKA Hardener MD (SIKA Härter MD) is categorized as “No prediction can be made”.

Executive summary:

The purpose of this study according to OECD guideline 492 and GLP was to determine the acute ocular irritation potential of the test item SIKA Hardener MD (SIKA Härter MD) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro. Before treatment, the tissues were pre-wetted with approximately 20 μL of Ca⁺⁺Mg⁺⁺Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with the test item (50 µL/unit) and incubated for 30 ± 2 minutes at standard culture conditions (37 ± 2 °C in an incubator with 5 ± 1 % CO₂, ≥ 95 % humidified atmosphere). Exposure with the test item was terminated by rinsing the disks with Ca⁺⁺Mg⁺⁺Free-DPBS solution. After rinsing, the tissues were incubated for 12 ± 2 minutes immersion incubation (post-soak) at room temperature. At the end of the post-soak immersion test items treated tissues were incubated for 120 ± 15 minutes at standard culture conditions (post-treatment incubation). Fresh assay medium was used during the post-soak and post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours
(± 15 minutes) with MTT solution at 37 ± 2 °C in an incubator with 5 ± 1 % CO₂ protected from light, ≥ 95 % humidified atmosphere. The formazan precipitated was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues (two units / control) were used as negative and positive controls, respectively and were treated the same way as the test item-treated disks. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60 % of the negative control. Depending on the regulatory framework in member countries, the test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %. The test item SIKA Hardener MD (SIKA Härter MD) showed reduced cell viability in comparison to the negative control (mean relative viability: 58 %). All obtained test item viability results were below 60 % when compared to the viability values obtained from the negative control. Positive control viability results (mean tissue viability: 4 %) were below 50 % when compared to the viability values obtained from the negative control. Furthermore, the mean optical density (OD) value of the two negative control tissues (mean OD value: 2.161) were between 0.8 and 2.8. So, the positive and negative controls showed the expected values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item SIKA Hardener MD (SIKA Härter MD) indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1) to eyes. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. According to the guideline OECD 492, SIKA Hardener MD (SIKA Härter MD) is categorized as “No prediction can be made”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro

The purpose of this OECD 439 and GLP compliant study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 ± 0.5 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 ± 1 °C for a post-incubation period of 42 ± 1 hours (h) in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours ± 5 minutes with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan crystals were extracted with acidified isopropanol and quantified by means of the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item showed significantly reduced cell viability in comparison to the negative control (mean viability: 17 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The mean OD value of the blank samples was below 0.1. The experiment was considered to be valid. The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2.

 

Skin corrosion in vitro

A GLP-compliant EpiDerm test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 18 June 2019. Disks of EpiDermTM (three units / chemical / incubation time) were treated with the test item and incubated for 60 minutes (+ 3 min) at standard culture condition (37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere) and 3 min at room temperature. Exposure of test material was terminated by rinsing with DPBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using MTT-100-EXT (isopropanol) and quantified spectrophotometrically. Potassium hydroxide (KOH) 8N solution and sterile deionized water treated (three units / positive and negative control) epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 60 minutes of exposure is above or equal 15 % and the mean relative viability after 3 minutes of exposure is above or equal 50 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 60 minutes and 3 minutes of exposure. The average test item treated tissue mean relative viability was 70 % at 60 minutes of exposure and the mean relative viability was 84 % at 3 minutes of exposure. Positive and negative controls showed the expected optical density (OD) and cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. In conclusion, in this in vitro skin corrosion test in EpiDermTM model (OECD 431) with the test item, the results indicate that the test item is not corrosive. According to the UN GHS classification systems, the test item has been categorized as “Non-corrosive”.

 

Eye irritation in vitro

The purpose of this study according to OECD guideline 492 and GLP was to determine the acute ocular irritation potential of the test item SIKA Hardener MD (SIKA Härter MD) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro. Before treatment, the tissues were pre-wetted with approximately 20 μL of Ca⁺⁺Mg⁺⁺Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with the test item (50 µL/unit) and incubated for 30 ± 2 minutes at standard culture conditions (37 ± 2 °C in an incubator with 5 ± 1 % CO₂, ≥ 95 % humidified atmosphere). Exposure with the test item was terminated by rinsing the disks with Ca⁺⁺Mg⁺⁺Free-DPBS solution. After rinsing, the tissues were incubated for 12 ± 2 minutes immersion incubation (post-soak) at room temperature. At the end of the post-soak immersion test items treated tissues were incubated for 120 ± 15 minutes at standard culture conditions (post-treatment incubation). Fresh assay medium was used during the post-soak and post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 minutes) with MTT solution at 37 ± 2 °C in an incubator with 5 ± 1 % CO₂ protected from light, ≥ 95 % humidified atmosphere. The formazan precipitated was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues (two units / control) were used as negative and positive controls, respectively and were treated the same way as the test item-treated disks. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60 % of the negative control. Depending on the regulatory framework in member countries, the test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %. The test item SIKA Hardener MD (SIKA Härter MD) showed reduced cell viability in comparison to the negative control (mean relative viability: 58 %). All obtained test item viability results were below 60 % when compared to the viability values obtained from the negative control. Positive control viability results (mean tissue viability: 4 %) were below 50 % when compared to the viability values obtained from the negative control. Furthermore, the mean optical density (OD) value of the two negative control tissues (mean OD value: 2.161) were between 0.8 and 2.8. So, the positive and negative controls showed the expected values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, with the test item SIKA Hardener MD (SIKA Härter MD) indicate that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1) to eyes. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2. According to the guideline OECD 492, SIKA Hardener MD (SIKA Härter MD) is categorized as “No prediction can be made”.

 

Eye corrosion ex vivo

The purpose of this Isolated Chicken Eye Test (ICET) according to OECD guideline 438 and GLP was to evaluate the potential ocular corrosivity or severe irritancy of the test item its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. The test item, the positive control (5% solution of Benzalkonium chloride), and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way that the test and control items evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~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 classes of the test item were once I (based on the fluorescein retention of 0.0) and once II (based on the cornea swelling of 17 % within 240 minutes) and once IV (based on the corneal opacity score of 2.7). The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. So, the positive and negative controls showed the expected results. The experiment was considered to be valid. In this ICET, the overall ICE classes of the test item were once I, once II and once IV. According to the guideline OECD 438, the test item is categorized as “Non prediction can be made”.

 

Conclusion: Combining both tests for skin irritation/corrosion, the test item is irritating to skin. Combining both tests for eye irritation/corrosion, the test item is irritating to eyes

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, the test item is classified for skin and eye irritation (category 2) according to Regulation (EC) No 1272/2008 (CLP) as amended for the eighteenth time in Regulation (EU) 2022/692.