methyl 2-hydroxybut-3-enoate

Endpoint summary

Description of key information

The tests were conducted with the purified and stabilised (registered) form of the test substance. 

 

In an in vitro skin irritation test according to OECD guideline 439, the test item showed irritating properties. In an in vitro skin corrosion test according to OECD guideline 431, the test item was not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. However, in an acute dermal toxicity test according to OECD guideline 402, the test item showed delayed corrosive effects at all concentrations tested (200-2000 mg/kg bw). Thus, in summary, the test item is considered corrosive to skin.

 

In an Isolated Chicken Eye Test according to OECD guideline 438, the test item was categorized as corrosive to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-03-31 to 2021-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

2020-06-26

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: Adult human donors

Justification for test system used:

The Reconstructed Human Epidermis test method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).

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: EpiSkin Small Model (EpiSkin SM), manufactured by EPISKIN Laboratories Lyon, France
- Tissue batch number: 21-EKIN-013 (main experiment), 21-EKIN-010 (killed tissues to check for direct MTT reduction)
- Expiry date: 05 April 2021 (main experiment), 15 March 2021 (killed tissues to check for direct MTT reduction)
- Date of initiation of testing: 31 March 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (23.9-24.5 °C)
- Temperature of post-treatment incubation: 37±1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Units were rinsed thoroughly with approx. 25 mL 1x PBS solution
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 200-1000 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The IC50 of the batch was determined using SDS in the MTT test. An IC50 between 1.5 mg/mL and 3 mg/mL was considered acceptable. The IC50 of the batch was determined to be 2.3 mg/mL and thus within the acceptance range. For HCD see "Attached background material".
- Barrier function: A number of cell layers of 4 or more is considered acceptable. A representative reconstructed epidermis from each batch was stained with HES and showed 7.5 cell layers and was thus considered acceptable.
- Contamination: The product was prepared and packaged under aseptic techniques. The absence of HIV1 and HIV2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs as well as bacteria, fungi or mycoplasma was verified by blood and cellular analysis of donor blood and keratinocytes.
- Reproducibility: Yes, the obtained results for positive and negative controls were within the acceptance criteria of the assay and within the laboratory's HCD (see "Attached background material")

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Three killed test item treated tissues and three killed negative control treated tissues were used for the MTT evaluation in one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues (batch No. of killed epidermis: 21-EKIN-010). The same treatment steps were followed with these tissues and the living tissues.
- Procedure used to prepare the killed tissues: Fresh tissues were placed in a 12 well plate with 2 mL of distilled water and incubated at 37±1 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere for 48 h +/- 1 hours. Killed epidermis were kept in a freezer at -15 to -30 °C until use.
- N. of replicates: 3 (for each test item and negative control treatment)
- Method of calculation used: See "Any other information on materials and methods incl. tables"

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One independent experiment was used with three tissue replicates.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure and 42 hours post incubation is less or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 μL

NEGATIVE CONTROL
- Amount applied: 10 μL

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

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

Three tissue replicates in one independent experiment

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 3 tissue replicates

Run / experiment:

1

Value:

10

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: Yes. During the check-method for possible direct MTT reduction, a colour change was observed after three hours of incubation. The test item interacted with MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 3%. As the NSMTT were below 50%, the true MTT metabolic conversion for all samples was calculated and the correction of viability percentages determined.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is slightly yellow and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

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
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

study cannot be used for classification

Remarks:

This test method cannot resolve between UN GHS Categories 1 and 2, thus classification is only possible in combination with OECD guideline 431.

Conclusions:

In an in vitro skin irritation test according to OECD guideline 439, the test item showed irritating properties.

Executive summary:

An EpiSkin^TM SM test according to OECD guideline 439 and GLP has been performed to predict the irritation potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 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 min) with MTT solution at 37±1 °C in 5±1 % CO₂, ≥95 % humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5 % aq.) and 1×PBS 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. The test item is MTT-reducer, therefore additional controls (three test item treated killed tissues and three negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement. The test item 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 % of 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 corrected relative viability value: 10 %). All obtained test item viability results were far 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. All acceptance criteria of the assay were fulfilled. 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.

Reference 2

Endpoint:

skin irritation: in vivo

Remarks:

Acute dermal toxicity study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-09-20 to 2021-11-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No.: 402. Acute Dermal Toxicity

Version / remarks:

adopted 2017-10-09

Deviations:

yes

Remarks:

The study was stopped due to delayed corrosive effects.

GLP compliance:

yes (incl. QA statement)

Irritation parameter:

erythema score

Basis:

animal #4

Remarks:

Main study

Time point:

24/48/72 h

Reversibility:

not fully reversible within: 4 days

Remarks on result:

probability of severe irritation

Remarks:

Local signs like erythema, oedema, crust, (bloody) wounds, scrabs and necrosis were detected in the animal treated with 200 mg/kg bw of the test substance in the main test which increased from day 1 on. This animal had to be killed on day 4.

Irritation parameter:

edema score

Basis:

animal #4

Remarks:

Main study

Time point:

24/48/72 h

Reversibility:

not fully reversible within: 4 days

Remarks on result:

probability of severe irritation

Remarks:

Local signs like erythema, oedema, crust, (bloody) wounds, scrabs and necrosis were detected in the animal treated with 200 mg/kg bw of the test substance in the main test which increased from day 1 on. This animal had to be killed on day 4.

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

Range-finding study

Time point:

24/48/72 h

Reversibility:

not fully reversible within: 1 day

Remarks on result:

probability of severe irritation

Remarks:

The animal died on day 1.

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

Range-finding study

Time point:

24/48/72 h

Reversibility:

not fully reversible within: 1 day

Remarks on result:

probability of severe irritation

Remarks:

The animal died on day 1.

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

Range-finding study

Time point:

24/48/72 h

Reversibility:

not fully reversible within: 5 days

Remarks on result:

probability of severe irritation

Remarks:

Local signs like erythema, (bloody) wounds and necrosis were detected in the animal treated with 1000 mg/kg bw of the test substance which increased from day 1 on. This animal had to be killed on day 5.

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

Range-finding study

Time point:

24/48/72 h

Reversibility:

not fully reversible within: 5 days

Remarks on result:

probability of severe irritation

Remarks:

Local signs like erythema, (bloody) wounds and necrosis were detected in the animal treated with 1000 mg/kg bw of the test substance which increased from day 1 on. This animal had to be killed on day 5.

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

Range-finding study

Time point:

24/48/72 h

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Remarks:

Local signs like dry skin, crust, small wounds and very slight erythema were detected in the animal treated with 200 mg/kg bw of the test substance. The symptoms started on day 3 and the animal was free of symptoms on day 14.

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

Range-finding study

Time point:

24/48/72 h

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Remarks:

Local signs like dry skin, crust, small wounds and very slight erythema were detected in the animal treated with 200 mg/kg bw of the test substance. The symptoms started on day 3 and the animal was free of symptoms on day 14.

Executive summary:

In an acute dermal toxicity study according to OECD guideline 402 and GLP, the toxicity of test item was assessed when administered in a single dermal dose to rats at one or more defined dose levels. At first, the range-finding study was performed in one female Han:WIST rat. The starting dose was 200 mg/kg bw. The test item was applied in original form and left in contact with the skin for a 24 hours period in all animals. The animal did not die in this first step, therefore one further rat was treated with 1000 mg/kg body weight in range-finding study. This animal did not die in second step, therefore one further rat was treated with 2000 mg/kg body weight in range-finding study. This animal died on Day 1. The animal treated with 1000 mg/kg bw was however humanely killed on Day 5 as the test item caused with some delay corrosive local signs. Based on this finding one further animal was treated 200 mg/kg bw in main study. This animal was however also humanely killed on Day 4 because of massive skin corrosion developing with time. The study was terminated for animal welfare reasons, because the test item caused massive skin damage up to clear and massive skin corrosion over time at 1000 mg/kg bw, and even at 200 mg/kg bw in the second test animal. This massive skin damage was not to be expected based on the in vitro skin irritation/corrosivity tests classifying the test item as irritant but clearly not as corrosive.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-03-02 to 2021-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No 440/2008 of May 30th, 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

Version / remarks:

2019-07-31

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

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: Adult human donors

Justification for test system used:

The Reconstructed Human Epidermis test method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin Small Model (EpiSkin SM), EPISKIN Laboratories Lyon, France,
- Tissue batch number: 21-EKIN-010 (main experiment), 21-EKIN-008 (killed tissues to check for direct MTT reduction)
- Expiry date: 15 March 2021 (main experiment), 01 March 2021 (killed tissues to check for direct MTT reduction)
- Date of initiation of testing: 10 March 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Units were rinsed thoroughly with approx. 25 mL 1x PBS solution
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 200-1000 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The IC50 of the batch was determined using SDS in the MTT test. An IC50 between 1.5 mg/mL and 3 mg/mL was considered acceptable. The IC50 of the batch was determined to be 2.2 mg/mL and thus within the acceptance range. For HCD see "Attached background material".
- Barrier function: A number of cell layers of 4 or more is considered acceptable. A representative reconstructed epidermis from each batch was stained with HES and showed 7.5 cell layers and was thus considered acceptable.
- Contamination: The product was prepared and packaged under aseptic techniques. The absence of HIV1 and HIV2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs as well as bacteria, fungi or mycoplasma was verified by blood and cellular analysis of donor blood and keratinocytes.
- Reproducibility: Yes, the obtained results for positive and negative controls were within the acceptance criteria of the assay and within the laboratory's HCD (see "Attached background material")

NUMBER OF REPLICATE TISSUES: 2 replicates per test item (for each exposure time), 2 replicates of negative control (for each exposure time) and 2 replicates of the positive control (for the 4 h exposure only) were used. Furthermore, 2 killed test item treated tissues and 2 killed negative control tissues were used for the MTT evaluation (for each exposure time).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Two killed test item treated tissues and two killed negative control treated tissues were used for the MTT evaluation in one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues (batch No. of killed epidermis: 21-EKIN-008). The same treatment steps were followed with these tissues and the living tissues.
- Procedure used to prepare the killed tissues: Fresh tissues were placed in a 12 well plate with 2 mL of distilled water and incubated at 37±1 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere for 48 h +/- 1 hours. Killed epidermis were kept in a freezer at -15 to -30 °C until use.
- N. of replicates: 2 killed test item treated tissues and 2 killed negative control tissues (for each exposure time).
- Method of calculation used: See "Any other information on materials and methods incl. tables"

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One independent experiment was used with two tissue replicates per time point (except for the positive control where only the 4-hour time point was measured).

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin (optional sub-category 1A) if the viability after 3 minutes exposure is less than 35%.The test substance is considered to be corrosive to skin (optional sub-category 1B and 1C) if the viability after 3 minutes exposure is greater than or equal to 35% but less than 35% after 1 hour of exposure or if the viability after 1 hour exposure is greater than or equal to 35% but less than 35% after 4 hours of exposure
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 mL

NEGATIVE CONTROL
- Amount applied: 50 mL
- Concentration: 9 g/L

POSITIVE CONTROL
- Amount applied: 50 mL
- Concentration: Pure (≥99.8 % purity)

Duration of treatment / exposure:

4 hours (±10 min), 1 hour (±5 min) and 3 minutes (test item and negative controls were tested with each exposure time; the positive control was tested only for the 4h exposure time)

Number of replicates:

2 replicate tissues per test item (for each exposure time), 2 replicate tissues of the negative control (for each exposure time) and 2 replicate tissues of the positive control (for the 4 h exposure only) were used in a single independent experiment. Furthermore, 2 killed test item treated tissues and 2 killed negative control tissues were used for the MTT evaluation (for each exposure time).

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean viability from 2 tissue replicates after 4 hours of exposure

Run / experiment:

1

Value:

57.5

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

Remarks:

Mean viability from 2 tissue replicates after 1 hour exposure

Run / experiment:

1

Value:

86.5

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

Remarks:

Mean viability from 2 tissue replicates after 3 min exposure

Run / experiment:

1

Value:

117

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: Yes. During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with MTT, therefore additional controls and data calculations were necessary. The non-specific MTT reduction (NSMTT) was determined to be 7.5 %, 4.1 % and 4.9 % at the 4h, 1h and 3 min exposure respectively. As the NSMTT were below 50 % in each case, the true MTT metabolic conversion for all samples was calculated and the correction of viability percentages determined.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is slightly yellow and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was demonstrated in a separate study using the twelve 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

Interpretation of results:

study cannot be used for classification

Remarks:

This test method cannot resolve between UN GHS Categories 1 and 2, thus classification is only possible in combination with OECD guideline 439.

Conclusions:

In an in vitro skin corrosion test according to OECD guideline 431, the test item was not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. In conclusion, the test item can be classified as non-corrosive.

Executive summary:

The EpiSkin^TMSM test with 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 EPISKIN (two units / chemical / incubation time) were treated with the test item and incubated for 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 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 acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement. 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 4 hours of exposure is above or equal 35 % of the negative control. Every test item treated tissue viability was above 35 % of the mean negative control value after 4 hours, 1 hour and 3 minutes exposure. The average test item treated tissue relative viabilities were 57.5 % at 4 hours, 86.5 % at 1 hour and 117.0 % at 3 minutes of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. In conclusion, the results of the in vitro skin corrosion test in the EPISKIN model (OECD 431) showed that the test item is not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. In conclusion, the test item can be classified as non-corrosive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

2021-04-12 to 2021-05-13

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 Ocular Corrosives and Severe Irritants)

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: Not indicated, but all eyes used in the assay were from the same groups of eyes collected on one specific day.
- 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 that can be closed (4-5 heads/box). The heads were then transported to the laboratory approx. within 2 hours from collection. The temperature was between 19.3 ºC and 20.2 ºC during the transport.
- Indication of any existing defects or lesions in ocular tissue samples: One small drop of fluorescein solution 2 (w/v) % was applied 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 slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged.
- Selection and preparation of corneas: Eyelids were carefully cut away with scissors, avoiding damaging the cornea. If the cornea was in good condition (as determined by fluorescein examination), the eyeball was carefully removed from the orbit. The eyeball was carefully removed from the orbit by holding the nictitating membrane with 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.
- Quality check of the isolated corneas: After being placed in the superfusion apparatus, the selected eyes were examined again 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 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: 30 μL

Number of animals or in vitro replicates:

Three eyes were used for the test substance and positive control treatment and one eye was used for the negative control treatment.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: If the corneas were in good condition (as determined by fluorescein staining), the eyeball was carefully removed from the orbit. The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. 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 prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). 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/minutes.

EQUILIBRATION AND BASELINE RECORDINGS: If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization 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.

NUMBER OF REPLICATES: Three eyes were used for the test substance and positive control treatment and one eye was used for the negative control treatment.

NEGATIVE CONTROL USED: NaCl (9 g/L saline)

POSITIVE CONTROL USED: Benzalkonium chloride solution (5%)

APPLICATION DOSE AND EXPOSURE TIME: The test item was applied unchanged at a volume of 30 µL for 10 seconds.

OBSERVATION PERIOD: Eyes were evaluated at approx. 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE:
- Volume and washing procedure after exposure period: After treatment, the eyes were immediately rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The cornea opacity was measured at all time points. Eyes were examined with a slit lamp microscope.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse. Eyes were examined with a slit lamp microscope.
- Swelling: The cornea thickness was measured at all time points. Eyes were examined with a depth measuring device on the slit lamp microscope (slit-width setting: 9½, equaling 0.095 mm).
- Macroscopic morphological damage to the surface: If morphological effects were observed, the classification of these findings were done by study director (e.g. pitting or loosening of the epithelium.
- Other: Corneas were stored in preservative fluid (4% formaldehyde) for potential histopathology after all measurements were done. Histopathology of the corneas was not performed as a clear result was obtained.

SCORING SYSTEM:
- Mean corneal swelling (%): See "Any other information on materials and methods incl. tables"
- Mean maximum opacity score: See "Any other information on materials and methods incl. tables"
- Mean fluorescein retention score at 30 minutes post-treatment: See "Any other information on materials and methods incl. tables"

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

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean of three eyes

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Fluorescein retention of 0.7

Irritation parameter:

cornea opacity score

Run / experiment:

Mean of three eyes

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Corneal opacity of 3.7

Irritation parameter:

corneal swelling 

Run / experiment:

Mean of three eyes

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Corneal swelling of 35% within 240 minutes

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 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:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In an Isolated Chicken Eye Test according to OECD guideline 438, the test item is categorized as corrosive to the eyes (Category 1).

Executive summary:

The Isolated Chicken Eye Test (ICET) was conducted according to OECD guideline 438 and GLP and aimed 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 μL/eye) 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/CLP)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. 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 item exposure) were determined at each of the above time points. 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 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 overall ICE classes of the test item were once II (based on the fluorescein retention of 0.7) and twice IV (based on the cornea swelling of 35 % within 240 minutes and corneal opacity score of 3.7). The positive control was classed as corrosive/severely irritating, UN GHS/CLP Classification: Category 1. The positive and negative controls showed the expected results and were within the respective historical control ranges. The negative control had not significant effects on the eye and the positive control showed the expected effects for a Category 1 substance (three times ICE class IV) (The experiment was therefore considered to be valid.

 

In this ICET, the overall ICE classes were once II (based on the fluorescein retention of 0.7) and twice IV (based on corneal swelling of 35% within 240 minutes and the opacity score of 3.7). According to the guideline OECD 438, the test item is categorized as corrosive (Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro, RL1

An EpiSkin^TM SM test according to OECD guideline 439 and GLP has been performed to predict the irritation potential of the test item by measurement of its cytotoxic effect, as reflected in the MTT assay. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 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 min) with MTT solution at 37±1 °C in 5±1 % CO₂, ≥95 % humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5 % aq.) and 1×PBS 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. The test item is MTT-reducer, therefore additional controls (three test item treated killed tissues and three negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement. The test item 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 % of 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 corrected relative viability value: 10 %). All obtained test item viability results were far 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. All acceptance criteria of the assay were fulfilled. 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, RL1

The EpiSkin^TMSM test with 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 EPISKIN (two units / chemical / incubation time) were treated with the test item and incubated for 4 hours (±10 min), 1 hour (±5 min) and 3 min at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 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 acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. The test item is a MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test item interference with the viability measurement. 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 4 hours of exposure is above or equal 35 % of the negative control. Every test item treated tissue viability was above 35 % of the mean negative control value after 4 hours, 1 hour and 3 minutes exposure. The average test item treated tissue relative viabilities were 57.5 % at 4 hours, 86.5 % at 1 hour and 117.0 % at 3 minutes of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. In conclusion, the results of the in vitro skin corrosion test in the EPISKIN model (OECD 431) showed that the test item is not corrosive to skin after 4 hours, 1 hour and 3 minutes of exposure time. In conclusion, the test item can be classified as non-corrosive.

 

Acute dermal toxicity, rat, RL2

The objective of the study was to assess the toxicity of test item when administered in a single dermal dose to rats at one or more defined dose levels.  At first, the range-finding study was performed in one female Han:WIST rat. The starting dose was 200 mg/kg bw. The test item was applied in original form and left in contact with the skin for a 24 hours period in all animals. The animal did not die in this first step, therefore one further rat was treated with 1000 mg/kg body weight in range-finding study. This animal did not die in second step, therefore one further rat was treated with 2000 mg/kg body weight in range-finding study. This animal died on Day 1. The animal treated with 1000 mg/kg bw was however humanely killed on Day 5 as the test item caused with some delay corrosive local signs. Based on this finding one further animal was treated 200 mg/kg bw in main study. This animal was however also humanely killed on Day 4 because of massive skin corrosion developing with time. The study was terminated for animal welfare reasons, because the test item caused massive skin damage up to clear and massive skin corrosion over time at 1000 mg/kg bw, and even at 200 mg/kg bw in the second test animal. This massive skin damage was not to be expected based on the in vitro skin irritation/corrosivity tests classifying the test item as irritant but clearly not as corrosive.

 

Eye corrosion ex vivo, RL1

The Isolated Chicken Eye Test (ICET) was conducted according to OECD guideline 438 and GLP and aimed 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 μL/eye) 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/CLP)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. 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 item exposure) were determined at each of the above time points. 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 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 overall ICE classes of the test item were once II (based on the fluorescein retention of 0.7) and twice IV (based on the cornea swelling of 35 % within 240 minutes and corneal opacity score of 3.7). The positive control was classed as corrosive/severely irritating, UN GHS/CLP Classification: Category 1. The positive and negative controls showed the expected results and were within the respective historical control ranges. The negative control had not significant effects on the eye and the positive control showed the expected effects for a Category 1 substance (three times ICE class IV) (The experiment was therefore considered to be valid.

 

In this ICET, the overall ICE classes were once II (based on the fluorescein retention of 0.7) and twice IV (based on corneal swelling of 35% within 240 minutes and the opacity score of 3.7). According to the guideline OECD 438, the test item is categorized as corrosive (Category 1).

 

Overall conclusion: In summary, the test substance is classified as corrosive to skin based on the delayed massive skin damage observed in an acute dermal toxicity study. Based on an Isolated Chicken Eye Test, the test substance is classified as corrosive to the eyes.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin and eye irritation, the test item is classified and labelled for skin and eye damage (Category 1) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.