4-(2-acryloyloxy-ethoxy) benzophenone

Endpoint summary

Description of key information

Three in vitro studies were conducted with AEBP to determine its eye and skin irritation/corrosion potential.

The results of the studies were:

Not corrosive when tested according to OECD 431.

Not irritating when tested according to OECD 439.

Not irritating when tested according to OECD 437.

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:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019

Deviations:

no

Remarks:

No deviations ocurred that negatively impacted the integrity of the study.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 30029
- Expiration date of the lot/batch:
- Purity test date: 03 May, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.
- Stability under storage conditions: Stable
- Stability under test conditions: Stable
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): No reactivity observed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, tested as supplied.

FORM AS APPLIED IN THE TEST: The test article was tested as a waxy solid without further manipulation on the surface of the tissues.

Test system:

human skin model

Remarks:

EpiDerm Reconstructed Human Epidermis (RhE)

Source species:

human

Cell type:

other: Reconstructed Human Epidermis - normal human keratinocytes

Cell source:

other: Keratinocyte strain 00267

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Huuman
- Sex: No data
- Age at study initiation (in days): No data

Justification for test system used:

EpiDerm RhE is recommended per OECD 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm RhE
- Tissue batch number(s): Lot 33722, Keratinocyte Strain 00267
- Production date: 9/16/2020
- Shipping date: 14 September, 2020
- Delivery date: 15 September, 2020
- Date of initiation of testing: 16 September, 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 C
- Temperature of post-treatment incubation (if applicable): 37 C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of exposure, tissues were washed with a stream of DPBS which completely removed the control and test articles/substances from the tissue inserts. Tissues were blotted with a sterile cotton swab (inside) and on sterile paper toweling (outside) of the insert, transferred to a fresh plate containing pre-warmed medium returned to the incubator.
- Observable damage in the tissue due to washing: None observed.
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL in DMEM
- Incubation time: 3 hours
- Spectrophotometer: Molecular Devices model i3 plate reader
- Wavelength: 570 nm
- Filter: No data
- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data

NUMBER OF REPLICATE TISSUES: Each group was dosed in triplicate (n=3)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test article/substance was found to be an MTT reductant capable of interference with the MTT assay. Freeze/killed (F/K) tissues were run in parallel with viable tissues as a control as described in the protocol. The F/K tissue control demonstrated that the test article/substance did not interfere with the MTT assay.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
According to OECD TG 439, an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced to or below 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE : None
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL : DPBS
- Amount(s) applied (volume or weight): 30 uL

POSITIVE CONTROL : SDS
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): 5%

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

105

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: None observed
- Direct-MTT reduction: The test article/substance was found to be an MTT reductant capable of interference with the MTT assay. Freeze/killed (F/K) tissues were run in parallel with viable tissues as a control as described in the protocol. The F/K tissue control demonstrated that the test article/substance did not interfere with the MTT assay.
- Colour interference with MTT: None observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The lab that run the study is technically proficient in running the OECD 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean Optical Density (OD) of the negative control (NC) group was ≥ 1.0 and ≤ 2.8.
- Acceptance criteria met for positive control: Yes, the mean viability of the 60 min 5% SDS-treated positive control (PC) group tissues was ≤ 20 % of the NC.
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation (SD) for the test substance groups (n=3) was < 18%.
- Range of historical values if different from the ones specified in the test guideline: Per the guideline.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the study indicating a tissue viability of 105% compared to the negative control, AEBP is not a skin irritant when tested according to OECD 439.

Executive summary:

The skin irritation potential of AEBP was evaluated using the reconstructed human epidermal tissue model EpiDerm (EPI-200SIT). The study was conducted according to OECD 439 (2019) in compliance with OECD GLP. The test article (waxy solid) was dosed as received by covering the apical surface of each tissue with 25 mg of test article. Positive (5% SDS) and negative (DPBS) controls were run in parallel. All test article and control exposures were run in triplicate (n=3). Tissues were exposed for 60 minutes at 37 C, washed and incubated for 42 hours at 37 C. Following post-exposure incubation, viability of the tissues was determined using the MTT assay. Tissue viability was expressed as a percent of the negative control. Positive and negative control tissues performed as expected, indicating that the test system was valid. Exposure to AEBP resulted in a mean tissue viability of 105% (4% SD) compared to the negative control. The mean optical density was 2.351 for the treated tissues. Based on the results of the study indicating a tissue viability of 105% compared to the negative control, AEBP is not a skin irritant when tested according to OECD 439.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
3M Company, Lot 30029
- Expiration date of the lot/batch:

- Purity test date:
03 May, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature.
- Stability under storage conditions:
Stable
- Stability under test conditions:
Stable
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):
No reactivity observed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
None, tested as supplied.

FORM AS APPLIED IN THE TEST: The test article was tested as a waxy solid without further manipulation on the surface of the tissues.

Test system:

human skin model

Remarks:

EpiDerm Reconstructed Human Epidermis (RhE)

Source species:

human

Cell type:

other: Reconstructed Human Epidermis - Normal human keratinocytes

Cell source:

other: Keratinocyte strain 00267

Details on animal used as source of test system:

SOURCE ANIMAL
- Source:
Huuman
- Sex:
No data
- Age at study initiation (in days):
No data

Justification for test system used:

EpiDerm RhE is recommended per OECD 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm RhE
- Tissue batch number(s):
Lot 33722, Keratinocyte Strain 00267
- Production date:
9/16/2020
- Shipping date:
14 September, 2020
- Delivery date:
15 September, 2020
- Date of initiation of testing:
16 September, 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
37 C
- Temperature of post-treatment incubation (if applicable):
37 C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
At the end of exposure, tissues were washed with a stream of DPBS which completely removed the control and test articles/substances from the tissue inserts. Tissues were blotted with a sterile cotton swab (inside) and on sterile paper toweling (outside) of the insert, transferred to a fresh plate containing pre-warmed medium returned to the incubator.
- Observable damage in the tissue due to washing:
None observed.
- Modifications to validated SOP:
None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
1 mg/mL in DMEM
- Incubation time:
3 hours
- Spectrophotometer:
Molecular Devices model i3 plate reader
- Wavelength:
570 nm
- Filter:
No data
- Filter bandwidth:
No data
- Linear OD range of spectrophotometer:
No data

NUMBER OF REPLICATE TISSUES:
Each group was dosed in triplicate (n=3)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test article/substance was found to be an MTT reductant capable of interference with the MTT assay. Freeze/killed (F/K) tissues were run in parallel with viable tissues as a control as described in the protocol. The F/K tissue control demonstrated that the test article/substance did not interfere with the MTT assay.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes 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%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and 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(s) applied (volume or weight with unit): 50 mg

VEHICLE : None

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 uL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 uL
- Concentration (if solution): 8N

Duration of treatment / exposure:

3 minutes and 60 minutes.

Duration of post-treatment incubation (if applicable):

None

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 Minute Exposure Mean (%)

Value:

98

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100% Viability

Positive controls validity:

valid

Remarks:

16% Viability

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 Minute Exposure Mean (%)

Value:

101

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100% Viability

Positive controls validity:

valid

Remarks:

6% Viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system:
None observed
- Direct-MTT reduction:
The test article/substance was found to be an MTT reductant capable of interference with the MTT assay. Freeze/killed (F/K) tissues were run in parallel with viable tissues as a control as described in the protocol. The F/K tissue control demonstrated that the test article/substance did not interfere with the MTT assay.
- Colour interference with MTT:
None observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The lab that run the study is technically proficient in running the OECD 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Yes, the mean Optical Density (OD) of the negative control (NC) group was ≥ 0.8.
- Acceptance criteria met for positive control:
Yes, the mean viability of the 60 min 8N KOH treated positive control (PC) group tissues was ≤ 15 % of the NC.
- Acceptance criteria met for variability between replicate measurements:
Yes, the standard deviation (SD) for the test substance groups (n=3) was < 18%.
- Range of historical values if different from the ones specified in the test guideline: Per the guideline.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the study, AEBP was not corrosive in the reconstructed human epidermis (RhE) model when tested according to OECD 431.

Executive summary:

The skin corrosion potential of AEBP was evaluated using the reconstructed human epidermal tissue model EpiDerm (EPI-200SIT). The study was conducted according to OECD 431 in compliance with OECD GLP. The test article (waxy solid) was dosed as received by covering the apical surface of each tissue with 50 mg of test article. Positive (8N KOH) and negative (distilled water) controls were run in parallel. All test article and control exposures were run in triplicate (n=3). Tissues were exposed for 3 and 60 minutes at 37 C and washed to remove test article or control substances. Following washing, viability of the tissues was determined using the MTT assay. Tissue viability was expressed as a percent of the negative control. Positive and negative control tissues performed as expected, indicating that the test system was valid. Exposure to AEBP resulted in a mean tissue viability of 98% and 101% compared to the negative control following 3- and 60-minute exposures, respectively. Based on the results of the study, AEBP was not corrosive in the reconstructed human epidermis (RhE) model when tested according to OECD 431.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

2017

Deviations:

no

Remarks:

No deviations ocurred that impacted the integrity of the results of the study.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
3M Company, Lot 30029 II
- Expiration date of the lot/batch:
25 July, 2020
- Purity test date:
03 May, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature protected from light
- Stability under storage conditions:
Stable
- Stability under test conditions:
Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test article was dosed neat.

FORM AS APPLIED IN THE TEST: Neat

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Number of animals: No data
- Characteristics of donor animals (e.g. age, sex, weight): No data
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: No data
- indication of any existing defects or lesions in ocular tissue samples: Only eyes with no detectable defects were utilized.
- Indication of any antibiotics used: No data

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 uL

Duration of treatment / exposure:

10 minutes at 32 C

Duration of post- treatment incubation (in vitro):

120 minutes at 32 C

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 C. The corneas were incubated for the minimum of 1 hour at 32 C. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS
Only eyes with no detectable defects were utilized.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Yes, physiological saline

POSITIVE CONTROL USED
Yes, ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 uL, 10 minutes

TREATMENT METHOD
Open chamber

POST-INCUBATION PERIOD:
Yes, 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany).
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

-1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

-0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

-0.1

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: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory that conducted the study is technically proficient in conducting the OECD 437 BCOP assay.

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

Interpretation of results:

GHS criteria not met

Conclusions:

The mean In Vitro Irritancy Score (IVIS) of MTDID 32918 was -0.8. In conclusion, since MTDID 32918 induced an IVIS of <=3, no classification is required for eye irritation or serious eye damage. MTDID 32918 is not irritating to the eye.

Executive summary:

The ocular irritation potential of MTDID 32918 was evaluated in the Bovine Corneal Opacity and Permeability Test. The study was conducted according to OECD 437 in compliance with OECD GLP. Corneas (n=3) were exposed to 0.75mL of MTDID 32918 undiluted, negative control (physiological saline), or positive control (ethanol) for 10 minutes at 32 C. Following the exposure period the corneas were washed and incubated for 120 minutes at 32 C. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). Corneal permeability was evaluated by passage of sodium fluorescein dye through the corneas measured with the aid of microtiter plate reader (OD490). Individual In Vitro Irritancy Scores (IVIS) were -1.5, -0.7, and -0.1. The mean In Vitro Irritancy Score (IVIS) of MTDID 32918 was -0.8. Based on a mean IVIS of -0.8, no classification is required for eye irritation or serious eye damage. MTDID 32918 is not irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on the results of the studies, AEBP is not classified for skin or eye irritation.