ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate

Endpoint summary

Description of key information

Based on thea available in vitro studies, ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate is considered to be not skin or eye irritating.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August 2019 to 02 August 2019

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: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

FORM AS APPLIED IN THE TEST
The test material was applied as supplied, no formulation was required.

Test system:

human skin model

Remarks:

EPISKIN™(SM)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

100 µL of physiological saline was utilised to ensure good contact with the epidermis.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM) Kit
- Source: SkinEthic, France
- Tissue lot number(s): 19-EKIN-031

TEST FOR DIRECT MTT REDUCTION
- 20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in an incubator with 5 % CO2, in a >95 % humidified atmosphere for 3 hours and any colour change was recorded. After three hours of incubation, yellow colour of the mixture was detected in the test tube indicating the test material did not react with MTT and therefore the use of additional controls was not necessary.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. The test material had colour, therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test mateiral application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

MAIN TEST
PRE-INCUBATION
- The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (at least 18 hours) at 37 °C in an incubator with 5 % CO2 in a > 95 % humidified atmosphere.

APPLICATION
- The Assay Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well.
- 20 mg of test material was applied evenly to each of two test units and each additional control skin units and then 100 µL physiological saline was added to the test material to ensure good contact with the epidermis. 20 mg of the test material covered properly the epidermal surface.
- 50 µL of negative control (Physiological saline (0.9 % (w/v) NaCl solution)) or positive control (glacial acetic acid) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (24.2 - 24.8 °C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After 4 hours incubation time the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove as much as possible of any remaining material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

MTT TEST
- MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37 °C in an incubator with 5 % CO2 for 3 hours, protected from light, in a > 95 % humidified atmosphere.

FORMAZAN EXTRACTION
- At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using the biopsy punch supplied as part of the kit. The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 µL acidified isopropanol (one tube corresponding to one well of the assay plate).
- The capped tubes were thoroughly mixed using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
- A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENTS
- Following the formazan extraction, 2 × 200 µL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. Plate reader linearity range: From 0.0 to 3.0 OD. The mean of 6 wells of acidified isopropanol solution (200 µL/well) was used as blank.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C with MTT

NUMBER OF REPLICATE TISSUES: Two replicates per timepoint. Two negative controls and two positive controls were also run.

DATA EVALUATION
The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).

For 2 disks:
If both disks have mean viability of = 35 % = Non Corrosive
If both disks have mean viability of < 35 % = Corrosive (at the corresponding incubation period)

For more than 2 disks:
If the mean value is = 35 % and the variability is less than 50 % = Non Corrosive
If the mean value is < 35 % and the variability is less than 50 % = Corrosive

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

4 hours

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

113.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- As no colour change (yellow colour) was observed after three hours incubation of the test material in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- Two additional test material-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues were 0.007, Non Specific Colour % was calculated as 1.0 %. No additional calculations were therefore deemed to be necessary.

MAIN STUDY
- The mean OD value for the test material treated skin samples showed 113.4% relative viability compared to the negative control.

QUALITY CRITERIA
- After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper condition.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
- The mean OD value of the two negative control tissues was in the recommended range (0.743 and 0.690, respectively) which satisfied the acceptability criterion of being 0.6 - 1.5.
- The positive control treated tissues showed 0.2 % relative viability when compared to the relevant negative control values, thus demonstrating the proper performance of the assay.
- The variability of the calculated viability values of the test material treated replicates were 14.3 %.
- The variability of the calculated viability values of the negative control treated replicates were 7.5 %.
- High viability results (>>100%) do regularly occur in cases where the test material causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.

Optical Density (OD) and the Calculated Relative Viability % of the Samples in the Corrosivity Test

Substance		Optical Density		Viability (% RV)
		Measured	Blank Corrected
Negative control Physiological saline (0.9 % (w/v) NaCl)	1	0.791	0.743	103.7
	2	0.737	0.690	96.3
	Mean	-	0.717	10.0
Positive control Glacial acetic acid	1	0.051	0.003	0.5
	2	0.047	-0.001	-0.1
	Mean	-	0.001	0.2
Test material	1	0.918	0.871	121.5
	2	0.802	0.754	105.3
	Mean	-	0.813	113.4

Mean blank value was 0.047.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

Historical Control Data: Corrosivity Testing Period 4 Hours

	Negative Control (Physiological Saline Solution)	Positive Control (Glacial Acetic Acid)
Minimum optical density (OD)	0.590	0.000
Maximum optical density (OD)	1.516	0.051
Mean optical density (OD)	0.844	0.010
Standard Deviation (SD)	0.130	0.009
Number of cases	228	208

OD: Optical density (absorbance)

SD: Standard deviation

All optical density (OD) values are background corrected values (measured at 570 ± 30 nm).

 

Interpretation of results:

other: Not skin corrosive

Conclusions:

Following exposure with the test material, the mean relative viability after 4 hours of treatment was 113.4 % compared to the appropriate negative control values of the performed experiments. The observed relative viability value was above the threshold of 35 %, therefore the test material was considered as being non-corrosive.

Executive summary:

The skin corrosion potential of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 431, under GLP conditions.

The in vitro skin corrosivity study was performed in a reconstructed human epidermis model using EPISKIN™(SM), which is designed to predict and classify the corrosivity potential of chemicals by measuring its cytotoxic effect as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue] assay.

During the study, disks of EPISKIN™(SM) were treated with the test material and incubated for 4 hours (in duplicate) at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control). Two additional disks were used to provide in each case an estimate of colour contribution (NSCliving). For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability after 4 hours of exposure is below 35 % of the negative control, the test material is considered to be corrosive to skin.

Following exposure with the test material, the mean relative viability after 4 hours of treatment was 113.4 % compared to the appropriate negative control values of the performed experiments. The observed relative viability value was above the threshold of 35 %, therefore the test material was considered to be non-corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test, the test mateiral can be considered non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2019 to 02 August 2019

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

FORM AS APPLIED IN THE TEST
The test material was applied as supplied, no formulation was required.

Test system:

human skin model

Remarks:

EPISKIN™(SM)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

10 µL distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™(SM) Kit
- Source: SkinEthic, France
- Tissue lot number(s): 19-EKIN-031

TEST FOR DIRECT MTT REDUCTION
- 20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in an incubator with 5 % CO2, in a >95 % humidified atmosphere for 3 hours and any colour change was recorded. After three hours of incubation, yellow colour of the mixture was detected in the test tube indicating the test material did not react with MTT and therefore the use of additional controls was not necessary.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol. The test material had colour, therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test mateiral application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

MAIN TEST (performed under aseptic conditions)
PRE-INCUBATION
- The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (at least 18 hours) at 37 °C in an incubator with 5 % CO2 in a > 95 % humidified atmosphere.

APPLICATION
- The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well.
- An appropriate amount (10 µL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of test material were applied evenly to each of three test units and each additional control skin units. 10 mg of the test material covered properly the epidermal surface.
- 20 µL of negative control (PBS) or positive control (5 % (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature (23.3 - 25.1 °C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- After 15 minutes incubation time the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove as much as possible of any remaining material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (±1 hour) at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere.

MTT TEST
- MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37 °C in an incubator with 5 % CO2 for 3 hours, protected from light, in a > 95 % humidified atmosphere.

FORMAZAN EXTRACTION
- At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using the biopsy punch supplied as part of the kit. The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 µL acidified isopropanol (one tube corresponding to one well of the assay plate).
- The capped tubes were thoroughly mixed using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
- A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENTS
- Following the formazan extraction, 2 × 200 µL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. Plate reader linearity range: from 0.0 to 3.0 OD. The mean of 6 wells of acidified isopropanol solution (200 µL/well) was used as blank.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C

NUMBER OF REPLICATE TISSUES: 3
As the test material was coloured, two additional test material-treated living tissues were used for the non-specific OD evaluation.

DATA EVALUATION
The test material is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes exposure to the test material and 42 hours post incubation is less or equal (=) to 50 % of the mean viability of the negative controls. In case the test material is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (=) to 50 %, the test material is considered to be irritant to skin in accordance with UN GHS Category 2. The test material may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 15 minutes exposure to the test material and 42 hours post incubation is more than (¿) to 50 % of the mean viability of the negative controls.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg

NEGATIVE CONTROL
- Amount(s) applied: 20 µL

POSITIVE CONTROL
- Amount(s) applied: 20 µL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

87

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:

DIRECT MTT REDUCTION
- As no colour change (yellow colour) was observed after three hours incubation of the test material in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- Two additional test material-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.011, Non Specific Colour % (NSCliving%) was calculated as 1.3 %. This is below the threshold of 5 %, therefore correction due to colouring potential was not necessary.

MAIN STUDY
- The mean OD value for the test material treated skin samples showed 87.0 % relative viability compared to the negative control.

QUALITY CRITERIA
- After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper condition.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
- The mean OD value of the three negative control tissues was in the recommended range (0.826) which satisfied the acceptability criterion of being 0.6 - 1.5. Standard deviation of the viability results for negative control samples was 4.3 %.
- The positive control treated tissues showed 8.0 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.2 %.
- The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 14.7 %.
- All these parameters were within acceptable limits and therefore the study was considered to be valid.

Optical Density (OD) and Calculated Relative Viability % of the Samples in the Irritation Test

Substance		Optical Density		Viability (%RV)
		Measured	Blank Corrected
Negative control Phosphate buffered saline	1	0.902	0.855	103.5
	2	0.884	0.836	101.3
	3	0.8834	0.786	95.2
	Mean	-	0.826	100.0
Positive control 5 % (w/v) SDS solution	1	0.103	0.056	6.7
	2	0.114	0.066	8.0
	3	0.124	0.076	9.2
	Mean	-	0.066	8.0
Test material	1	0.626	0.578	70.0
	2	0.827	0.779	94.4
	3	0.844	0.797	96.5
	Mean	-	0.718	87.0

Mean blank value was 0.047.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

SDS: Sodium dodecyl sulphate (5 % (w/v)).

Irritation Testing Historical Control: 20 µL Treatment Volume

	Mean Value (OD)	Standard Deviation (SD)	Minimum Value (OD)	Maximum Value (OD)	Number of Cases
Negative control
Year 2019, absorbance	0.870	0.099	0.617	1.061	80
Positive control
Year 2019, absorbance	0.048	0.031	0.010	0.160	80
Year 2019, viability %	5.6	3.5	1.1	16.3

Negative control: Phosphate buffered saline (PBS)

Positive control: Sodium dodecyl sulphate 5 % (w/v) (SDS)

SD: Standard deviation

OD: Optical density (absorbance)

All OD values (measured at 570) are background corrected values.

Interpretation of results:

GHS criteria not met

Remarks:

Not skin irritating

Conclusions:

Following exposure with the test material, the mean cell viability was 87.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

Executive summary:

The skin irritation potential of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 439, under GLP conditions.

The in vitro skin irritation test was performed in a reconstructed human epidermis model using EPISKIN™(SM), which is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue] assay.

During the study, disks of EPISKIN™(SM) were treated with the test material and incubated for 15 minutes (in triplicate) at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37° C for 42 hours in an incubator with 5 % CO2, in a > 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS treated epidermis were used as negative control and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control). Two additional disks were used to provide in each case an estimate of colour contribution (NSCliving). For each treated tissue, the viability was expressed as a % relative to the negative control. For irritation, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50 % of the negative control, the test mateiral is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 87.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test the test material was considered to be non-irritanting to the skin.

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:

26 July 2019 to 27 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

The solubility of the test material in physiological saline was tested prior to the Experiment I (30 mg test material in 1 mL physiological saline). The test material did not dissolve in physiological saline.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to the Test Facility at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4 - 5 heads per box). The heads were received at the Test Facility and processed within 2 hours of collection in each experiment.
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg

Duration of treatment / exposure:

10 seconds from the end of the application

Number of animals or in vitro replicates:

One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
The 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). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3 - 4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, 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.

EQUILIBRATION AND BASELINE RECORDINGS
If the selected eyes were appropriate for the test, acclimatisation started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatisation and treatment periods.
At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES : One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

NEGATIVE CONTROL USED : 30 µL of physiological saline

POSITIVE CONTROL USED: 30 mg powdered imidazole

TREATMENT
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the test material was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material, taking care not to damage or touch the cornea.
In each experiment a negative control eye was treated with 30 µL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole.
One eye was treated with physiological saline, three eyes with the test material and another three eyes with powdered imidazole in each experiment.

REMOVAL OF TEST MATERIAL
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with at least 20 mL physiological saline solution at ambient temperature in each experiment, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 3 x 20 mL physiological saline was performed at each time point when the test material or positive control material remaining on the cornea was observed in each experiment.

OBSERVATION
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. A Haag-Streit BP 900® slit-lamp microscope was used for the measurements.
Any morphological effects were recorded.

EVALUATION
A test is considered acceptable if the concurrent negative or vehicle/solvent controls and the concurrent positive controls are identified as GHS No Category and GHS Category 1, respectively.

Corneal swelling was calculated according to the following formulae:

CS at time t = [(CT at time t –CT at t=0) / CT at t=0] x 100

Mean CS at time t = [FECS(at time t) + SECS(at time t) + TECS(at time t)] / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point

Small negative numbers for swelling (0 to -5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

Cornea opacity was calculated according to the following formulae:

¿CO at time t = CO at time t – CO at t=0

Mean ¿COmax = [FECOmax(30min to 240min) + SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
¿CO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:

¿FR at time t = FR at time t – FR at t=0

Mean ¿FR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
¿FR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

Irritation parameter:

percent corneal swelling

Remarks:

(up to 75 min)

Run / experiment:

Experiment I and Experiment II (mean)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Remarks:

(up to 240 min)

Run / experiment:

Experiment I (mean)

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Remarks:

(up to 240 min)

Run / experiment:

Experiment II (mean)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I (mean)

Value:

0.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II (mean)

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I (mean)

Value:

0.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II (mean)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

EYE IRRITATION RESULTS
The test material showed no significant corneal effect in the first experiment. As the test material was solid, the negative results were confirmed by a second experiment. The second experiment confirmed the negative results.
The positive control (Imidazole) was classified as inducing serious eye damage (Category 1) as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
The negative control Physiological saline was not classified for eye irritation or serious eye damage as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

MORPHOLOGICAL EFFECTS
In experiment I the test material was stuck on two cornea surfaces (2/3) after the post-treatment rinse. The cornea surfaces (2/3) were cleared at 180 minutes after the post-treatment rinse.
In experiment II the test material was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 minutes after the post-treatment rinse.
The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse in each experiment.
No other morphological effect was observed in the study.

VALIDITY OF THE TEST
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. The number of eyes used for each treatment group was adequate. These experiments were considered to be valid.

Test Material – Experiment I

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	0.0 %	I
Mean maximum corneal swelling at up to 240 min.	2.2 %	I
Mean maximum corneal opacity change	0.67	I
Mean flouorescein retention change	0.83	I
Other observations	Test material was stuck on two cornea surfaces (2/3) after the post-treatment rinse. The cornea surfaces (2/3) were cleared at 180 minutes after the post-treatment rinse.
Overall ICE class	1 x I 2 x II

 

Test Material – Experiment II

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	0.0 %	I
Mean maximum corneal swelling at up to 240 min.	0.0 %	I
Mean maximum corneal opacity change	0.5	II
Mean flouorescein retention change	0.0	II
Other observations	Test material was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 minutes after the post-treatment rinse.
Overall ICE class	3 x I

 

Positive Control – Experiment I

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	9.6 %	II
Mean maximum corneal swelling at up to 240 min.	25.5 %	III
Mean maximum corneal opacity change	4.0	IV
Mean flouorescein retention change	3.0	IV
Other observations	Imidazole was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE class	1 x III 2 x IV

 

Positive Control – Experiment II

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	10.9 %	II
Mean maximum corneal swelling at up to 240 min.	29.3 %	III
Mean maximum corneal opacity change	4.0	IV
Mean flouorescein retention change	3.0	IV
Other observations	Imidazole was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE class	1 x III 2 x IV

 

Negative Control – Experiment I

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	0.0 %	I
Mean maximum corneal swelling at up to 240 min.	0.0 %	I
Mean maximum corneal opacity change	0.0	I
Mean flouorescein retention change	0.0	I
Other observations	None
Overall ICE class	3 x I

 

Negative Control – Experiment I

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min.	0.0 %	I
Mean maximum corneal swelling at up to 240 min.	0.0 %	I
Mean maximum corneal opacity change	0.0 %	I
Mean flouorescein retention change	0.0 %	I
Other observations	None
Overall ICE class	3 x I

 

Historical Control Data: Negative Control – Physiological Saline

Observation	Min. Value	Max. Value
Mean maximum corneal swelling at up to 75 min.	-3.2 %	3.4 %
Mean maximum corneal swelling at up to 240 min.	-4.8 %	3.4 %
Mean maximum corneal opacity change	0.0	0.5
Mean flouorescein retention change	0.0	0.5
Number of cases	497

 

Historical Control Data: Positive Control – Imidazole

Observation	Min. Value	Max. Value
Mean maximum corneal swelling at up to 75 min.	-6.6 %	25.0 %
Mean maximum corneal swelling at up to 240 min.	-15.9 %	36.7 %
Mean maximum corneal opacity change	3.50	4.00
Mean flouorescein retention change	2.00	3.00
Number of cases	221

Interpretation of results:

GHS criteria not met

Remarks:

Not eye irritating

Conclusions:

Under the conditions of the study, the test material was found to be non-irritant, not requiring classification for eye irritation or serious eye damage.

Executive summary:

The eye irritation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions.

The in vitro study was performed in isolated chicken eyes. In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes were examined in each experiment, furthermore three positive control treated eyes and one negative control treated eye were examined in each experiment.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. The number of eyes used for each treatment group was adequate. Thus, the experiments were considered to be valid.

Experiment I: No significant corneal swelling (mean = 5 %) was observed during the four-hour observation period on all test material treated eyes. Slight corneal opacity change (severity 0.5 on two test material treated eyes and severity 1.0 on one test material treated eye) was noted on test material treated eyes. Slight fluorescein retention change (severity 0.5 on one test material treated eye and severity 1.0 on two test material treated eyes) was noted on test material treated eyes. Test material was stuck on two cornea surfaces (2/3) after the post-treatment rinse. The cornea surfaces (2/3) were cleared at 180 minutes after the post-treatment rinse.

No other corneal effect was observed.

Experiment II: No corneal swelling was observed during the four-hour observation period on all test material treated eyes. No significant corneal opacity change (severity 0.5 on all test material treated eyes) was noted on test material treated eyes. No fluorescein retention change was observed on all test material treated eyes. Test material was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 minutes after the post-treatment rinse. No other corneal effect was observed.

Based on these in vitro eye irritation results in the isolated chicken eyes tests, the test material was concluded to be non-irritant, not requiring classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosion study (2019)

The skin corrosion potential of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 431, under GLP conditions.

The in vitro skin corrosivity study was performed in a reconstructed human epidermis model using EPISKIN™(SM), which is designed to predict and classify the corrosivity potential of chemicals by measuring its cytotoxic effect as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue] assay.

During the study, disks of EPISKIN™(SM) were treated with the test material and incubated for 4 hours (in duplicate) at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control). Two additional disks were used to provide in each case an estimate of colour contribution (NSCliving). For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability after 4 hours of exposure is below 35 % of the negative control, the test material is considered to be corrosive to skin.

Following exposure with the test material, the mean relative viability after 4 hours of treatment was 113.4 % compared to the appropriate negative control values of the performed experiments. The observed relative viability value was above the threshold of 35 %, therefore the test material was considered to be non-corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test, the test mateiral can be considered non-corrosive to the skin.

In vitro skin irritation study (2019)

The skin irritation potential of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 439, under GLP conditions.

The in vitro skin irritation test was performed in a reconstructed human epidermis model using EPISKIN™(SM), which is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue] assay.

During the study, disks of EPISKIN™(SM) were treated with the test material and incubated for 15 minutes (in triplicate) at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2, in a > 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 protected from light, in a > 95 % humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS treated epidermis were used as negative control and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control). Two additional disks were used to provide in each case an estimate of colour contribution (NSCliving). For each treated tissue, the viability was expressed as a % relative to the negative control. For irritation, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50 % of the negative control, the test mateiral is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 87.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test the test material was considered to be non-irritanting to the skin.

 

In vitro eye irritation/corrosion study (2019)

The eye irritation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions.

The in vitro study was performed in isolated chicken eyes. In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes were examined in each experiment, furthermore three positive control treated eyes and one negative control treated eye were examined in each experiment.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. The number of eyes used for each treatment group was adequate. Thus, the experiments were considered to be valid.

Experiment I: No significant corneal swelling (mean = 5 %) was observed during the four-hour observation period on all test material treated eyes. Slight corneal opacity change (severity 0.5 on two test material treated eyes and severity 1.0 on one test material treated eye) was noted on test material treated eyes. Slight fluorescein retention change (severity 0.5 on one test material treated eye and severity 1.0 on two test material treated eyes) was noted on test material treated eyes. Test material was stuck on two cornea surfaces (2/3) after the post-treatment rinse. The cornea surfaces (2/3) were cleared at 180 minutes after the post-treatment rinse.

No other corneal effect was observed.

Experiment II: No corneal swelling was observed during the four-hour observation period on all test material treated eyes. No significant corneal opacity change (severity 0.5 on all test material treated eyes) was noted on test material treated eyes. No fluorescein retention change was observed on all test material treated eyes. Test material was stuck on all cornea surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 180 minutes after the post-treatment rinse. No other corneal effect was observed.

Based on these in vitro eye irritation results in the isolated chicken eyes tests, the test material was concluded to be a non-irritant, not requiring classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Based on the available data , no classification of ethyl (3-benzoyl-2,4,6-trimethylbenzoyl)(phenyl)phosphinate is required for skin or eye irritation according to the Regulation (EC) No 1272/2008.