Registration Dossier

Administrative data

Description of key information

In-Vitro Skin Irritation:

Following exposure with CAS 577978-76-8, the mean cell viability was 88.1% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

In-Vitro Skin Corrosion:

Following exposure with CAS 577978-76-8, the mean cell viability was 96.2% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

In-Vitro Eye Irritation:

Based on the performed in vitro eye irritation assay (one run) in isolated chicken eyes with CAS 577978-76-8, the test item is non-irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2019 to 29 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report
Test system:
human skin model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Cell source:
other: EPISKINTM(SM) (Manufacturer: SkinEthic, France,
Source strain:
not specified
Details on animal used as source of test system:
Not applicable for In-Vitro tests
Justification for test system used:
The EPISKINTM(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)
Details on test system:
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-026, Expiry Date: 01 July 2019) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity and irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM(SM) Test Kits used in the present study)
Amount/concentration applied:
In case of the irritation testing, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of test item were applied evenly to each of three test units and each additional control skin units.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
In this assay, two replicates per time point were used for test item (in case of corrosivity part of the test) and three replicates per time point were used for test item (in case of irritation part of the test). Two negative controls and two positive controls were also run in corrosivity testing and three negative controls and three positive controls were also run in irritation testing. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation in both cases.
Species:
other: Not applicable
Strain:
other: Not applicable
Details on test animals and environmental conditions:
Not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Corrosion testing
Value:
96.2
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:
Corrosivity testing:
Following exposure with CAS 577978-76-8, the mean cell viability was 96.2% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

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

Colour control for corrosivity testing:

Two additional test item-treated living tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.006, Non Specific Colour % was calculated as 0.7% . This value was below 5%, therefore additional data calculation was not necessary.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues (Corrosivity test)

Additional control

Optical Density (OD)

NSC%

(living)

 

Measured

Blank corrected

Treated with

CAS 577978-76-8

1

*0.049

*0.002

 

0.7

2

0.057

0.010

 

Mean

-

0.006

Notes:

1. Mean blank value was 0.047.

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

3. *One sample was used for counting because another sample was excluded from the OD means calculation (negative value)

Corrosivity testing:

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in the table below. The OD values for the test item treated skin samples showed 96.2% relative viability compared to the negative control.

Optical Density (OD) and the calculated relative viability % of the samples (Corrosivity test)

Substance

Optical Density (OD)

Viability

(% RV)

 

Measured

Blank corrected

Negative Control:

Physiological saline

(0.9% (w/v) NaCl)

1

0.851

0.804

91.5

2

1.000

0953

108.5

Mean

-

0.879

100.0

Positive Control:

Glacial acetic acid

1

0.053

0.006

0.7

2

0.059

0.012

1.4

Mean

-

0.009

1.0

Test Item

CAS 577978-76-8

1

0.915

0.868

98.7

2

0.870

0.823

93.7

Mean

-

0.846

96.2

Notes:

1. Mean blank value was 0.047.

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

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro EPISKINTM (SM) model test with CAS 577978-76-8, the results indicate that the test item is not corrosive and not irritant to the skin,
Executive summary:

An in vitro skin corrosivity of CAS 577978-76-8 was performed in a reconstructed human epidermis model. EPISKINTM(SM) 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; CAS number 298-93-1) assay. The corrosivity potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines [1, 2].

Disks of EPISKINTM(SM) were treated with the powdered test item and incubated for 4 hours (two units for corrosivity testing) at room temperature. Exposure of the test item 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) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin.

Following exposure with CAS 577978-76-8, the mean cell viability was 96.2% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

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

In conclusion, in this in vitro EPISKINTM (SM) model test with CAS 577978-76-8, the results indicate that the test item is not corrosive to the skin, UN GHS Classification: No Category.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2019 to 29 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report
Test system:
human skin model
Source species:
human
Cell type:
other: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
Cell source:
other: EPISKINTM(SM) (Manufacturer: SkinEthic, France,
Source strain:
not specified
Details on animal used as source of test system:
Not applicable for In-Vitro tests
Justification for test system used:
The EPISKINTM(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)
Details on test system:
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-026, Expiry Date: 01 July 2019) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity and irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM(SM) Test Kits used in the present study)
Amount/concentration applied:
In case of the irritation testing, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of test item were applied evenly to each of three test units and each additional control skin units.
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
In this assay, two replicates per time point were used for test item (in case of corrosivity part of the test) and three replicates per time point were used for test item (in case of irritation part of the test). Two negative controls and two positive controls were also run in corrosivity testing and three negative controls and three positive controls were also run in irritation testing. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation in both cases.
Species:
other: Not applicable
Strain:
other: Not applicable
Details on test animals and environmental conditions:
Not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Irritation testing
Value:
88.1
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:
Irritation testing:
Following exposure with CAS 577978-76-8, the mean cell viability was 88.1% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

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

Colour control for irritation testing:

Two additional test item-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 0.013, Non Specific Colour % (NSCliving%) was calculated as 1.8% (see Table 2). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues (Irritation test)

Additional control

Optical Density (OD)

NSC%

(living)

 

Measured

Blank corrected

Treated with

CAS 577978-76-8

1

0.053

0.006

 

1.8

2

0.067

0.020

 

Mean

-

0.013

Notes:

1. Mean blank value was 0.047.

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

Irritation testing:

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented. The mean OD value for the test item treated skin samples showed 88.1% relative viability compared to the negative control.

Optical Density (OD) and the calculated relative viability % of the samples (Irritation test)

Substance

Optical Density (OD)

Viability

(% RV)

 

Measured

Blank corrected

Negative Control:

Phosphate buffered saline

1

0.755

0.708

94.5

2

0.822

0.775

103.4

3

0.813

0.766

102.1

Mean

-

0.750

100.0

Positive Control:

5% (w/v) SDS solution

1

0.098

0.051

6.8

2

0.124

0.077

10.2

3

0.127

0.080

10.7

Mean

-

0.070

9.3

Test Item

CAS 577978-76-8

1

0.718

0.671

89.5

2

0.834

0.687

91.7

3

0.670

0.623

83.1

Mean

-

0.661

88.1

  Notes:

1. Mean blank value was 0.047.

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

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro EPISKINTM (SM) model test with CAS 577978-76-8, the results indicate that the test item is not corrosive and not irritant to the skin,
Executive summary:

An in vitro skin irritation test of CAS 577978-76-8 was performed in a reconstructed human epidermis model. EPISKINTM(SM) 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; CAS number 298-93-1) assay. The irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines.

Disks of EPISKINTM(SM) were treated with the powdered test item and incubated for 15 minutes (three units for irritation testing) at room temperature. Exposure of the test item 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/w) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) for the irritation testing. Two additional disks were used to provide in each case an estimate of colour contribution (NSCliving) from the test item. 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 item is considered to be irritant to skin.

Following exposure with CAS 577978-76-8, the mean cell viability was 88.1% compared to the negative control. This is above the threshold of 50%, therefore the test item 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 EPISKINTM (SM) model test with CAS 577978-76-8, the results indicate that the test item is not irritant to the skin, UN GHS Classification: No Category.

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:
13 June 2019 to 13 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
SELECTION AND PREPARATION OF EYES FOR THE TEST

Eyes selection
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.

Preparation of 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.

Eyes examination and acclimatization time
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. If the selected eyes were appropriate for the test, acclimatization 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 acclimatization and treatment periods.
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 Charles River Laboratories Hungary Kft. 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 Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.

Identification
The eyes were identified by chamber number, marked on the door of the chamber.

THE BASELINE ASSESSMENTS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. 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 any eye. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg of the powdered test item was applied onto the entire surface of the cornea
Duration of treatment / exposure:
10 seconds from the end of the application
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
3 eyes per test group.
Details on study design:
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. 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
The 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 powdered test item and another three eyes with powdered Imidazole.

Test item removal
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 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 3x20 mL saline was performed at each time point when the test item and positive control material remaining on the cornea was observed.
Note: Physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 90352Y05-2, Expiry date: December 2021) was used for rinsing.

OBSERVATION
Observation and assessment of corneal effects
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. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
Irritation parameter:
percent corneal swelling
Run / experiment:
swelling at up to 75 min
Value:
4.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Run / experiment:
swelling at up to 240 min
Value:
4.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Value:
0.17
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:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 75 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.
No other morphological effect was observed in the study.

TEST ITEM

Observation

 

Value

 

ICE Class

 

Mean maximum corneal swelling at up to 75 min

4.9%

I

Mean maximum corneal swelling at up to 240 min

4.9%

I

Mean maximum corneal opacity

0.50

I

Mean fluorescein retention

0.17

I

Other Observations

 

Test item was stuck on all cornea surface after the post-treatment rinse. The cornea surfaces were cleared at 75 minutes after the post-treatment rinse.

Overall ICE Class

3xI

 

Based on thisin vitroeye irritation study in isolated chicken eyes with CAS 577978-76-8, the test item is non-irritant. At the Sponsor request, the second run (tor confirm the negative outcome according to the OECD No 438 guideline) was not performed.

POSITIVE CONTROL

Observation

 

Value

 

ICE Class

 

Mean maximum corneal swelling at up to 75 min

8.6%

II

Mean maximum corneal swelling at up to 240 min

28.3%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

 

Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Overall ICE Class

1xIII 2xIV

 

The positive control Imidazole was classified as severely irritating, UN GHS Classification: Category 1.

NEGATIVE CONTROL

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

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the performed in vitro eye irritation assay (one run) in isolated chicken eyes with CAS 577978-76-8, the test item is non-irritant.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (25 June 2018).

After the zero reference measurements, the eye was held in horizontal position and 30 mg powdered test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds exposure time, 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). Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

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 this experiment. Thus, the study was considered to be valid.

No significant swelling (mean swelling ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on any eyes. No significant fluorescein retention change (severity 0.5 on one eye and no fluorescein retention change on two eyes) was noted. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 75 minutes after the post-treatment rinse.

At the Sponsor request the second run (to confirm the negative outcome according to the OECD No. 438 guideline) was not performed.

Based on the performed in vitro eye irritation assay (one run) in isolated chicken eyes with CAS 577978-76-8, the test item is non-irritant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The results indicate that the test item is not corrosive and not irritant to the skin, UN GHS Classification: No Category.