2-[4-(4-Alkoxyphenyl)-(pyridin-2-yl)-heteromonocyclyl]phenol

Endpoint summary

Description of key information

FAT 75637 was found to be non-irritant to eyes and skin.

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:

Study initiation date - 11 January 2021; Experiment start date - 18 January 2021; Experiment completion date - 22 January 2021; Study completion date - 05 March 2021.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item: FAT 75637/B TE
Physical Appearance: Light yellow powder
Purity: 99.4 % all organic constituents; 95.0 % main constituent
Batch No: AT-0063765400
Manufactured Date: 21st April 2020
Expiry Date: May 27th, 2025

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.

One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin kit
EpiSkin is an in vitro reconstructed human epidermis (RHE) from normal human keratinocytes cultured on a collagen matrix at the air liquid interface. This is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Differentiated and stratified epidermis model comprises the main basal, supra basal, spinous and granular layers and a functional stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours ± 1

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

72.03

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Preliminary Tests for Coloring and Direct MTT Reduction Potential of the Test Item: In the coloring potential test, the resultant solution was not colored, hence, it was concluded that the test item does not have the intrinsic ability to become colored when it interacts with water. In the direct MTT reduction potential test, the color of MTT solution did not turn to either blue or purple, hence, it was concluded that the test item did not interact with MTT. Similarly, the water control tested did not show any coloring potential nor did it reduce the MTT.

Skin Irritation Test
The results show the mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues. Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test items. The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 72.03 %. The positive control had a mean cell viability of 4.74 % after 15 minutes exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

Individual Tissue Viability of Epidermis Units (Relative)

	% Individual Viability
	R1	R2	R3	Mean	SD
Positive control	4.58	4.69	4.96	4.74	0.20
Test Item	66.63	74.00	75.46	72.03	4.73

SD: Standard Deviation Negative control mean: 0.9283

R1, R2 & R3: triplicate exposures

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative tissue viability for the test item was 72.03 %. Hence, the test item is predicted to be non-irritant under the experimental conditions described in this report.

Executive summary:

The test item, FAT 75637/B was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application for 15 minutes. This study was conducted according to OECD test guideline 439 in a GLP-certified laboratory. The test item is a solid and was applied directly to the top of the skin tissues at 10 mg/tissue and exposed for 15 minutes. Ten microliters (10 µL) of PBS and 10 µL of 5 % aqueous SDS were used as the negative and positive controls, respectively. After 41-hour post-incubation period, irritation potentials of FAT 75637/B TE were evaluated by assessing the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 minutes treatment with the test item was compared to the negative control tissues. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was 0.9838. The positive control had a mean cell viability of 4.74 % after 15 minutes exposure, indicating that the test system functioned properly. The study indicated that the test item FAT 75637/B is predicted to be non- irritant in this in vitro skin irritation test as the percent viability is 72.03 % using reconstructed human epidermis under the conditions of testing employed.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 25 November 2020; Experiment start date - 25 November 2020; Experiment completion date - 28 November 2020; Study completion date - 14 May 2021

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 (incl. QA statement)

Specific details on test material used for the study:

Test Item: FAT 75637/B TE
Physical Appearance: Light yellow powder
Purity: 99.4 % all organic constituents; 95.0 % main constituent
Batch No: AT-0063765400
Manufactured Date: 21st April 2020
Expiry Date: May 27th, 2025

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

The assessment of skin corrosivity has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.

One of the validated in vitro test methods adopted by the OECD TG 431 makes use of reconstructed human epidermis (RhE). The test is designed to predict and classify the skin corrosivity potential of a test item by assessment of its effect on a reconstituted human epidermis, based on the experience that corrosive substances show cytotoxic effects following short-term exposure of the stratum corneum of the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin kit

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 mg

Duration of treatment / exposure:

3 minutes, 60 minutes and 240 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean 3 minutes

Value:

95.09

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean 60 mins

Value:

97.05

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean 240 minutes

Value:

107.61

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Preliminary Tests for Coloring and Direct MTT Reduction Potential of the Test Item

In the coloring potential test, the resultant solution was not colored, hence, it was concluded that the test item do not have the intrinsic ability to become colored when it interacts with water. Since, the color of MTT solution did not tum to either blue or purple, it was concluded that, the test item did not interact with MTT. Similarly, the water control tested did not show any coloring potential nor did it reduce the MTT.

 

Skin Corrosion Test

The results show the mean tissue viability obtained after the respective treatment duration with the test item compared to the negative control tissues. Skin corrosion is expressed as the remaining cell viability after exposure to the test items. The relative mean tissue viability obtained after 3, 60 and 240 minutes treatment duration with the test item compared to the negative control tissues was 95.09, 97.05 and 107.61 %, respectively. The positive control had a mean cell viability of 5.32 % after 240 minutes exposure. The absolute mean OD510 of the negative control tissues was within the laboratory historical control data range.

Interpretation of results:

GHS criteria not met

Conclusions:

Since, the tissue viability is ≥35 % after the 240 minute exposure, the test item FAT 75637/B is predicted to be non-corrosive under the experimental conditions described in this report.

Executive summary:

The test item, FAT 75637/B was tested for its possible skin corrosion potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. This study was conducted according to OECD test guideline 431 in a GLP-certified laboratory. The test item is in solid form and was applied directly on the top of the skin tissues at 20 mg/tissue, and exposed for 3, 60 and 240 minutes. 50 μL of Sodium chloride (NaCl) 0.9 % (w/v) was used as the negative control for each time of application (3 min, 60 min and 240 min). Similarly, 50 μL of Glacial Acetic acid was used as the positive control and was tested only at the 240 min exposure time. After the respective post-incubation period, corrosion potential of the test item was determined by assessing the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the respective treatment periods with the test item was compared to the negative control tissues. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was 1.051, 1.160 and 1.059 for the 3-minute, 60-minute and 240-minute exposures, respectively. The positive control had a mean cell viability of 5.32 % after 240-minute exposure, indicating that the test system functioned properly. The study indicated that the test item FAT 75637/B is predicted to be non-corrosive in this In Vitro Skin Corrosion Test using Reconstructed Human Epidermis under the conditions of testing employed.

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:

Study initiation date - 20 November 2020; Experiment start date - 21 November 2020; Experiment end date - 22 November 2020; Study completion date - 22 December 2020.

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)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Item name: FAT 75637/B TE
Batch no.: AT-0063765400
Manufactured date: 21 April 2020
Expiry date: 27 May 2025
Storage condition: Ambient (+15 to +25 °C)
Physical Appearance: Light yellow powder
Solubility: Insoluble in water

Species:

cattle

Details on test animals or tissues and environmental conditions:

The bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 μg/mL) in an ice box.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The positive control (Chlorhexidine) & test item (FAT 75637/B TE) were applied to the corneas as neat (quantity sufficient to cover entire corneal surface area), whereas negative control (Distilled water) was applied 0.75 mL to the each cornea.

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3 corneas/group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Procurement of corneas: The bovine eyes were procured from local abattoir and transported in a jar x containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 μg/mL) in an ice box. On day of experiment 25 numbers were found suitable for experiment.
- Dissection: On day of experiment, post pretest examination, eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container of fresh HBSS (Hank Balanced Salt Solution).
- Mounting: The isolated corneas were mounted in the cornea holder, which separated holder into anterior and posterior chambers. During mounting, it was ensured that the epithelium of the cornea projected into the anterior chamber. The EMEM medium was added in to both the chambers and kept in an incubator (32 °C) for 1 hour.
- Opacity and Permeability: All the selected corneas for the study were tested for their opacity and permeability to calculate IVIS (In vitro irritancy score).

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment 1

Value:

1.765

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
The assay results are found to be accepted because the positive control i.e. Chlorhexidine used in this study has exhibited an IVIS score of 161.704 which fall between two standard deviations of the historical mean i.e 155-172.
Also, the negative control i.e. Distilled water response resulted in opacity and permeability values that are less than established upper limits for negative control opacity and permeability values.

Calculation of IVIS Score:

Treatment group	Mean Opacity value	Constant	Mean Permeability value	IVIS	Classification
Negative control (Distilled water)	0	15	0.0484	0.726	No Category
Chlorhexidine	133	15	1.9136	161.704	Category I
FAT 75637/B	2	15	-0.0157	1.765	No Category

Interpretation of results:

GHS criteria not met

Conclusions:

FAT 75637/B referred as non eye irritant or as agent causing no serious eye damage, with the BCOP test method.

Executive summary:

The evaluation of the ocular irritancy of FAT 75637/B was carried out using the Bovine Corneal Opacity and Permeability assay. This study was conducted according to OECD test guideline 437 in a GLP-certified laboratory. Sufficient quantity to cover entire corneal surface area of the test item (FAT 75637/B) and positive control (Chlorhexidine) was applied to each cornea by means of open chamber method. A volume of 0.75 mL of negative control (Distilled water) were applied on the corneal surface by means of open chamber method. The control and treated corneas were then subjected to the opacity and permeability measurements. In this study, the negative control (Distilled water) was classified as UN GHS No Category (IVIS score 0.726) and the positive control (Chlorhexidine) was classified under Category 1 (IVIS score 161.704). The test item FAT 75637/B exhibited an IVIS score of 1.765 and was classified under UN GHS “No Category”. From the results, it can be concluded as the test item FAT 75637/B referred as non eye irritatant or as agent causing no serious eye damage, with the BCOP test method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In Vitro Skin Irritation EPISKIN test:

The test item, FAT 75637/B was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application for 15 minutes. This study was conducted according to OECD test guideline 439 in a GLP-certified laboratory. The test item is a solid and was applied directly to the top of the skin tissues at 10 mg/tissue and exposed for 15 minutes. Ten microliters (10 µL) of PBS and 10 µL of 5 % aqueous SDS were used as the negative and positive controls, respectively. After 41-hour post-incubation period, irritation potentials of FAT 75637/B were evaluated by assessing the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 minutes treatment with the test item was compared to the negative control tissues. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was 0.9838. The positive control had a mean cell viability of 4.74 % after 15 minutes exposure, indicating that the test system functioned properly. The study indicated that the test item FAT 75637/B is predicted to be non- irritant in this in vitro skin irritation test as the percent viability is 72.03 % using Reconstructed Human Epidermis under the conditions of testing employed.

In Vitro Skin Irritation EPIDERM test:

The test item, FAT 75637/B was tested for its possible skin corrosion potential using a three-dimensional Reconstructed Human Epidermis model, EpiSkin, through topical application. This study was conducted according to OECD test guideline 431 in a GLP certified laboratory. The test item is in solid form and was applied directly on the top of the skin tissues at 20 mg/tissue, and exposed for 3, 60 and 240 minutes. 50 μL of Sodium chloride (NaCl) 0.9 % (w/v) was used as the negative control for each time of application (3 min, 60 min and 240 min). Similarly, 50 μL of Glacial Acetic acid was used as the positive control and was tested only at the 240 min exposure time. After the respective post-incubation period, corrosion potential of the test item was determined by assessing the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the respective treatment periods with the test item was compared to the negative control tissues. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was 1.051, 1.160 and 1.059 for the 3-minute, 60-minute and 240-minute exposures, respectively. The positive control had a mean cell viability of 5.32 % after 240-minute exposure, indicating that the test system functioned properly. The study indicated that the test item FAT 75637/B is predicted to be non-corrosive in this In Vitro Skin Corrosion Test using Reconstructed Human Epidermis under the conditions of testing employed.

In Vitro Eye irritation test

The evaluation of the ocular irritancy of FAT 75637/B was carried out using the Bovine Corneal Opacity and Permeability assay. This study was conducted according to OECD test guideline 437 in a GLP-certified laboratory. Sufficient quantity to cover entire corneal surface area of the test item (FAT 75637/B) and positive control (Chlorhexidine) was applied to each cornea by means of open chamber method. A volume of 0.75 mL of negative control (Distilled water) were applied on the corneal surface by means of open chamber method. The control and treated corneas were then subjected to the opacity and permeability measurements. In this study, the negative control (Distilled water) was classified as UN GHS No Category (IVIS score 0.726) and the positive control (Chlorhexidine) was classified under Category 1 (IVIS score 161.704). The test item FAT 75637/B exhibited an IVIS score of 1.765 and was classified under UN GHS “No Category”. From the results, it can be concluded as the test item FAT 75637/B referred as non eye irritatant or as agent causing no serious eye damage, with the BCOP test method.

Justification for classification or non-classification

Skin irritation / corrosion:

Based on the results of the available studies, FAT 75637 does not warrant classification in accordance with CLP (Regulation EC No.1272/2008).

 

Eye irritation / corrosion:

Based on the results of the available studu, FAT 75637 does not warrant classification in accordance with CLP (Regulation EC No.1272/2008).