Reaction mass of butanoic acid, 2-ethyl-2,3,3-trimethyl-, ethyl ester and butanoic acid, 2,3-dimethyl-2-(1-methylethyl)-, ethyl ester and pentanoic acid, 2,2,4,4-tetramethyl-, ethyl ester and ethyl 2,2,3,3-tetramethylpentanoate and ethyl 2,2,3,4-tetramethylpentanoate

Endpoint summary

Description of key information

In an in vitro skin and eye irritation and corrosion test strategy, Neova did not show skin and eye irritation and corrosion potential in a skin irritation test (OECD guideline 439, Reconstructed Human Epidermis Test Method), a skin corrosion test (OECD guideline 431, Reconstructed Human Epidermis Test Method) and an eye irritation test (OECD guideline 492, Reconstructed Human Cornea-like Epithelium). As the results alone were sufficient for a final assessment, further testing within the testing strategies was waived.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: New guidance document on an integrated approach on testing and assessment (IATA) for skin corrosion and irritation, Series on Testing and Assessment No. 203

Version / remarks:

2014-07-11

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis (RhE) model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis and has been validated, considered scientifically valid and adopted as OECD test guideline. The test is part of a Turnkey Testing Strategy to assess the skin corrosion and irritation potential of the test material including transport classification.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Three-dimensional human epidermis model (EpiDerm 200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia)
- Tissue Lot number: 30837

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At room temperature for 25 minutes, then in the incubator at 37°C for 35 minutes.
- Temperature of post-treatment incubation: 37°C for 24±2 hours, then 37°C for another 18 ± 2 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material after start of application.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: filter wavelength 570 nm without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".
- Barrier function: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: No
The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently. If the color of the MTT solution or (in case of water-insoluble test substances) the border to the water-phase turned blue / purple, the test substance was presumed to reduce MTT directly. In case of direct MTT reduction, three freeze-killed control tissues (KC) were treated additionally with each the test article and the negative control in the same way as described for the test item. Based on the result of the pretest, it was judged that application of killed control tissues is not necessary.

PREDICTION MODEL / DECISION CRITERIA
See section "EVALUATION OF RESULTS" in "Any other information on materials and methods incl. tables".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL of undiluted liquid test substance

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% SDS

Duration of treatment / exposure:

25 minutes at room temperature and 35 minutes at 37°C

Duration of post-treatment incubation (if applicable):

24 ± 2 hours + additional 18 ± 2 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 tissues

Value:

99

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:

tissue 1

Value:

104.7

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:

tissue 2

Value:

94.9

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:

tissue 3

Value:

97.5

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:
- Direct MTT reduction: No direct MTT reduction occured.
- Colour interference with MTT: No colour interference occured.

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

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: New guidance document on an integrated approach on testing and assessment (IATA) for skin corrosion and irritation, Series on Testing and Assessment No. 203

Version / remarks:

2014-07-11

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis (RhE) model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis and has been validated, considered scientifically valid and adopted as OECD test guideline. The test is part of a Turnkey Testing Strategy to assess the skin corrosion and irritation potential of the test material including transport classification.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Three-dimensional human epidermis model (EpiDerm 200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia)
- Tissue batch number: 30837

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: for 3 minutes at room temperature or for 1 hour in the incubator at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: filter wavelength 570 nm without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".
- Barrier function: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: No
The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently. If the color of the MTT solution or (in case of water-insoluble test substances) the border to the water-phase turned blue / purple, the test substance was presumed to reduce MTT directly. In case of direct MTT reduction, three freeze-killed control tissues (KC) were treated additionally with each the test article and the negative control in the same way as described for the test item. Based on the result of the pretest, it was judged that application of killed control tissues is not necessary.

PREDICTION MODEL / DECISION CRITERIA
See section "EVALUATION OF RESULTS" in "Any other information on materials and methods incl. tables".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL of undiluted liquid test substance

NEGATIVE CONTROL
- Amount applied: 50 µL

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

Duration of treatment / exposure:

3 minutes at room temperature or 1 hour in the incubator at 37°C

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 2 tissues, exposure period of 3 min

Value:

101

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:

tissue 1, exposure period of 3 min

Value:

98.3

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:

tissue 2, exposure period of 3 min

Value:

103.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of skin corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 2 tissues, exposure period of 1 hour

Value:

98.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of skin corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1, exposure period of 1 hour

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of skin corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2, exposure period of 1 hour

Value:

99.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of skin corrosion

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT reduction: No direct MTT reduction occured.
- Colour interference with MTT: No colour interference occured.

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

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

2019-06-18

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on integrated approaches to testing and assessment (IATA) for serious eye damage and eye irritation, Series on Testing and Assessment No. 263

Version / remarks:

2019-07-05

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: This Test Guideline describes an in vitro procedure allowing the identification of chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. The reconstructed human cornea-like epithelium (RhCE) closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium and has been validated, considered scientifically valid and adopted as OECD test Guideline.

- Description of the cell system used: The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived, epidermal keratinocytes used to model the human corneal epithelium. The EpiOcularTM tissues are cultured on cell culture inserts.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume or weight with unit): 50 µL of undiluted test substance

VEHICLE
- Amount applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

Pre-treatment of tissues: 30 minutes with PBS
Application of the test substance/controls: 30 minutes

Duration of post- treatment incubation (in vitro):

12 minutes post-soak immersion and 2 hours post-incubation period

Number of animals or in vitro replicates:

2 tissues

Details on study design:

- Details of the test procedure used
- RhCE tissue construct used: EpiOcula model (OCL-200), MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia (Lot No: 30637)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Exposure for 30 minutes at 37°C, post-exposure immersion for 12 minutes in pre-warmed medium and post-incubation period for 2 hours at 37°C, incubation with MTT solution for 3 hours at 37°C.

- Indication of controls used for direct MTT-reducers: The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently. As no MTT reduction occurs, no freeze-killed control tissues (KC) are included in the main test.

- Number of tissue replicates used per test chemical and controls: 2 tissues were treated with each the test substance, positive and negative control

- Wavelength used for quantifying MTT formazan: 570 nm (optical density) without reference filter using a SunriseTM Absorbance Reader

- Description of the method used to quantify MTT formazan: Optical density, for data evaluation see section "DATA EVALUATION" in "Any other information on materials and methods incl. tables".

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: See "Any other information on materials and methods incl. tables" section.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".

- Positive and negative control means and acceptance ranges based on historical data: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".

- Acceptable variability between tissue replicates for positive and negative controls: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".

- Acceptable variability between tissue replicates for the test chemical: See section "ACCEPTANCE CRITERIA" in "Any other information on materials and methods incl. tables".

Irritation parameter:

other: Relative tissue viability [% of NC]

Run / experiment:

mean of 2 tissues

Value:

79

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Relative tissue viability [% of NC]

Run / experiment:

tissue 1

Value:

78.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Relative tissue viability [% of NC]

Run / experiment:

tissue 2

Value:

79.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

The test substance is not able to reduce MTT directly.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

In the chosen key study for skin irritation/corrosion according to OECD TG 431/439 and GLP (BASF SE 2020; 70V0493/19A063), three assays were considered for an in vitro skin irritation and corrosion test strategy, i.e. the Skin Corrosion Test (SCT), the Skin Irritation Test (SIT) and the In vitro Membrane Barrier Test (Corrositex®), since a single in vitro assay is not considered sufficient to cover the full range of a skin irritating/corrosion potential including a transport classification decision. However, in the case of testing Neova, the results derived with the SIT and SCT alone were sufficient for a final assessment, without further testing.

In the SCT, the potential to cause skin corrosion was assessed by a single topical application of 50μL undiluted Neova to a reconstructed three-dimensional, human epidermis model (EpiDerm™), so that the surface of the epidermis model was completely covered with the test substance. Two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. In addition, 50μL per tissue of a negative control (deionized water) and of a positive control (8 N KOH) were applied to two tissues each per exposure period. The relative mean viability of the tissues treated with Neova determined after an exposure period of 3 minutes was 101.0%, and it was 98.6% after an exposure period of 1 hour. The tissues treated with the positive control (8 N KOH) showed a relative mean viability of 9.3% (3-minute exposure) and 4.6% (1-hour exposure) reflecting the expected sensitivity of the tissues. Based on the results observed and by applying the evaluation criteria, it was concluded that Neova does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test method under the test conditions chosen.

In the SIT, the potential to cause dermal irritation was assessed by a single topical application of 30μL undiluted Neova to a reconstructed three-dimensional, human epidermis model (EpiDerm™), so that the surface of the epidermis model was completely covered with the test substance. Three EpiDerm™ tissues were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition, 30μL per tissue of a negative control (PBS) and of a positive control (5% SDS) were applied to three tissues each. The relative mean viability of the tissues treated with Neova determined after an exposure period of 1 hour with an about 42-hour post-incubation was 99.0%. The tissues treated with the positive control (5% SDS) showed a relative mean viability of 2.6% reflecting the expected sensitivity of the tissues. Based on the results observed and by applying the evaluation criteria, it was concluded that the test item does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.

The acceptance criteria for both assays were met and Neova was found to be unable to reduce MTT directly.

 

Eye Irritation

In the chosen key study for eye irritation according to OECD TG 437/492 and GLP (BASF SE 2020; 71V0493/19A064), two assays were considered for an in vitro eye irritation test strategy, i.e. the Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test, since a single in vitro assay is not considered sufficient to cover the full range of an eye irritating potential. However, in the case of testing Neova, the results derived with the EpiOcular alone was sufficient for a final assessment, without further testing.

The potential to cause ocular irritation was assessed by a single topical application of 50μL undiluted Neova to a reconstructed three-dimensional, human cornea model (EpiOcular™), so that the surface of the cornea model was completely covered with the test substance. Two EpiOcular™ tissues were incubated for 30 minutes followed by a 2-hour post-incubation period. In addition to the test substance, 50μL per tissue of a negative control (deionized water) and of a positive control (methyl acetate) were applied to two tissues each. Neova was not able to reduce MTT directly. The relative mean viability of the tissues treated with Neova was 79.0%. The tissues treated with the positive control (methyl acetate) showed a relative mean viability of 22.0% reflecting the expected sensitivity of the tissues. Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that Neova does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

The present data on skin and eye irritation do not fulfill the criteria laid down in regulation (EU) 1272/2008, and therefore, a non-classification is warranted.