(8α,9R,8'''α,9'''R)-1,1'-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(methylene)]bis(6'-methoxycinchonan-1-ium-9-ol) dibromide

Endpoint summary

Description of key information

Not corrosive to skin, in vitro (GLP, OECD TG 431)

Not irritating to skin, in vitro (GLP, OECD TG 439)

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

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)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number(s): Batch No.: 19-EKIN-030
- Production date: 23 July 2019
- Expiry date: 29 July 2019
- Date of initiation of testing: 25 July 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 23.7 to 24.2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washing was done with phosphate buffered saline, volume and number of washes not reported
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL, prepared from 3 mg/mL stock solution
- Incubation time: 3 hours
- Spectrophotometer: not reported
- Wavelength: 570 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

NUMBER OF REPLICATE TISSUES: two

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues: living epidermis units (Manufacturer: SkinEthic, France, Batch No.:19-EKIN-016, Expiry Date: 22 April 2019) on 16 April 2019 were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5% CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the epidermis units were frozen on 18 April 2019
- N. of replicates : two per control
- Method of calculation used:
Blank:
– The mean of the 6 blank (as in 3.8.5) OD values was calculated
Negative control:
– Individual negative control OD values (NCraw) were corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 2 negative control values was also calculated: this corresponds to 100% viability
Positive control:
– Individual positive control OD values (PCraw) were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 2 positive control values was calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control:
Positive Control1 % = (ODPC1 / mean ODNC) ×100
Positive Control2 % = (ODPC2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for positive control was calculated:
Mean PC % = (PC1 % + PC2 % ) / 2
Test item:
– Individual test item OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 2 test item values was calculated
– The % viability for each test item replicate was calculated relative to the mean negative control:
Treated Tissue1 % = (ODTT1 / mean ODNC) ×100
Treated Tissue2 % = (ODTT2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for test item was calculated:
Mean TT % = (TT1 % + TT2 %) / 2
– The variability for 2 disks is calculated as:
(Disk1-Disk2)/((Disk1+Disk2)/2) x 100%
Test items that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability % as follows:
Non-specific MTT reduction calculation (NSMTT%):
NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 50%, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
– The % relative viability (RV%) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSMTT% is > 50% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.
For test items detected as able to stain the tissues the non-specific OD is evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below:
Non-Specific Colour % (NSC living %):
NSC living % = (mean ODCTV / mean ODNC)×100
ODCTV: test item treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode is used.
If NSC living % is > 5 % and ≤ 50 %, then additional correction (TODTT) is undertaken as follows:
TODTT = [ODTT - ODCTV]
ODTT: test item treated viable tissue (incubated with MTT)
ODCTV: test item treated viable tissue (not incubated with MTT)
– The % relative viability (RV%) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.
For test items detected as able to both stain the tissues (3.9.3) and interfere with MTT (3.9.2) may also require a third set of controls before calculation of the “true” viability %.
Non-Specific Colour % with killed tissues (NSCkilled %):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test item treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
In that case the true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
Report Number: 19/181-039B Page 24 of 38
ODTT: test item treated viable tissues (incubated with MTT)
ODKT: test item treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test item treated viable tissues (not incubated with MTT)
ODCTK: test item treated killed tissues (not incubated with MTT)
The % relative viability (RV %) for each test item replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2% = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test item is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The cut-off value of 35% and classification method was validated in an international validation of this kit (Fentem, 1998).
- If both disks have a mean viability of ≥35% = Non Corrosive; If both disks have a mean viability of <35% = Corrosive (at the corresponding incubation period)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg
- 100 μL of physiological saline was added to ensure good contact with the epidermis

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 incubation with MTT solution

Number of replicates:

Two per treatment, negative control, positive control
Two additional with killed epidermis units for testing MTT interaction potential of the test substance
Two additional with killed epidermis units for negative control
Two additional with killed epidermis units for non-specific colour

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of two disks

Value:

111.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After receipt, the two indicators of fitness for the delivered kit (reflecting the storage temperature history and the pH) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay.
The mean OD value of the two negative control tissues was 0.768, which satisfied the acceptability criterion of being 0.6-1.5.
The positive control treated tissues showed 0.4% cell viability demonstrating the proper performance of the assay.
The difference of cell viability between the two test item-treated tissue samples in the MTT assay was 2.3%.
The difference of cell viability between the two negative control treated tissue samples in the MTT assay was 15.1%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.
High viability results (>100%) do regularly occur in cases where the test item causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure of the test model to the substance, the mean cell viability was 111.5% compared to the negative control. This is clearly above the threshold of 35%, and the test substance is considered to be non-corrosive to skin.

Executive summary:

The skin corrosion potential of the substance was studied under GLP to OECD TG 431, in an in vitro study using a reconstructed human epidermis model, EpiSkin. After receipt, the two indicators of fitness for the delivered test model kit (reflecting the storage temperature history and the pH of the agar medium) were checked. Based on the observed colours, the epidermis units were in suitable condition for use in the assay. All cell units were maintained under proper conditions before their use. On the day prior to the test, the epidermis units were placed in the appropriate number of wells in an assay plate, which were filled with pre-warmed maintenance medium, and incubated overnight at 37 °C in an incubator with 5% carbon dioxide in a >95% humidified atmosphere. On the day of the experiments, two epidermis disks were treated with 20 mg of the powdered test substance for a period of 4 hours at room temperature. Exposure was terminated by rinsing the units with phosphate buffered saline solution. The cell viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution (at a concentration of 0.5 mg/mL). The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically at 570 nm. The two negative control epidermis units were treated with physiological saline (0.9% w/v NaCl solution in purified water). The two positive control epidermis units were treated with glacial acetic acid. Two additional disks were used to provide an estimate of colour distribution from the test substance. The possible interaction of the test substance with MTT was examined using two killed epidermis units treated with the substance and two additional killed epidermis units treated with physiological saline. To avoid a possible double correction for colour interference, a third set of two control epidermis units for non-specific colounr in killed tissues were used. For each treated tissue, the viability was expressed as a percentage relative to the negative control. The experiment met all the validity criteria, e.g. with regards to cell tissue suitability, negative and positive controls. Following exposure to the test substance over a period of four hours, the mean cell viability in the treated epidermis units was 111.5% compared to the negative control. This is above the threshold of 35%, and the test substance was therefore considered to be not corrosive to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 EpiSkinTM model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 19-EKIN-031
- Production date: 30 July 2019
- Shipping date: not reported
- Expiry date: 05 August 2019
- Date of initiation of testing: 31 July 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 23.3 to 25.1 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: test substance on the skin disks was removed by washing with phosphate buffered physiological saline, the volume and number of washing steps was not reported
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: not reported
- Wavelength: 570 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

NUMBER OF REPLICATE TISSUES: three for test substance

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : negative and positive control, further two tissues for testing the non-specific optical density
- Method of calculation used:
Blank:
– The mean of the six blank OD values was calculated
Negative control:
– Individual negative control OD values (NCraw) were corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The mean corrected OD of the 3 negative control samples was calculated: this value corresponds to 100% viability
Positive control:
– Individual positive control OD values (PCraw) were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The mean corrected OD of the 3 positive control samples was calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual relative viability % for positive control was calculated:
Mean PC % = (%PC1 +%PC2 +%PC3) / 3
Test item:
– Individual test item OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The mean corrected OD of the 3 test item samples was calculated
– The % viability for each test item replicate was calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual relative viability % for test item was calculated:
Mean TT % = (%TT1 +%TT2 +%TT3) / 3

Test items that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability% as detailed below:
Non-specific MTT reduction calculation (NSMTT%):
NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test item is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
If NSMTT% is > 30% relative to the negative control, then additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

For test items detected as able to stain the tissues the non-specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % as detailed below:
Non-Specific Colour % with viable tissues (NSCliving %):
NSC living % = (mean ODCTV / mean ODNC) ×100
ODCTV: test substance treated viable tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode is used (see 3.9.1).
If NSCliving % is > 5% and ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows.
TODTT = [ODTT – mean ODCTV]
ODTT: test substance treated viable tissue (incubated with MTT)
ODCTV: test substance treated viable tissue (not incubated with MTT)
The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test item is calculated:
Mean Relative Viability% = (% RV 1 +% RV 2 +% RV 3) / 3
If NSC living % is > 30 % relative to the negative control, additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.

For test substances detected as able to stain the tissues and interfere with MTT, a third set of controls is also required before calculation of the “true” viability %.
Non-Specific Colour % with killed tissues (NSCkilled %):
NSCkilled % = (mean ODCTK / mean ODNC) ×100
ODCTK: test substance treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
In that case the true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: test substance treated viable tissues (incubated with MTT)
ODKT: test substance treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test substance treated viable tissues (not incubated with MTT)
ODCTK: test substance treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test substance is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin (Cat. 2) if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls (as the substance was found to be not corrosive to skin)
- The test substance is considered to be non-corrosive to skin if the mean relative viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours post incubation is more than (>) 50% of the mean viability of the negative controls

Control samples:

yes, concurrent negative control

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):

42 hours

Number of replicates:

Three per treatment, negative and positive control, two further for for testing the non-specific optical density

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three individual tissues

Value:

96.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After receipt, the two indicators included in the package of the delivered kits (reflecting the storage temperature history and the pH of the agar medium) were checked. Based on the observed colours, the epidermis units were in a suitable condition for use in the assay.
The mean OD value of the three negative control tissues was in the recommended range (0.826). 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 item-treated tissue samples in the MTT assay was 6.4%.
The mean OD value of the blank samples (acidified isopropanol) was 0.047.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean relative cell viability of three individual tissues treated with the test substance for 15 minutes and then incubated for 42 hours was 96.1% compared to the negative control. This is above the threshold of 50% and the substance was considered as not irritating to skin.

Executive summary:

The skin irritation potential of the substance was tested under GLP to OECD TG 439, in an in vitro study using a reconstructed human epidermis model, EpiSkin. After receipt, the two indicators included in the package of the delivered test model kits (reflecting the storage temperature history and the pH of the agar medium) were checked. Based on the observed colours, the epidermis units were in a suitable condition for use in the assay. Maintenance medium was pre-warmed to 37 °C and 2 mL each of this medium was filled into the appropriate number of wells in an assay plate. The epidermis units were placed in the wells, ensuring contact of the epidermis with the medium, and incubated overnight at 37 °C in an incubator with 5% carbon dioxide in >95% relative humidity. On the day of the experiment, three disks of the epidermis model were treated with 10 μL distilled water and then 10 mg of the powder test substance, which was sufficient to cover the full epidermal surface. A volume of 20 μL of phosphate buffered physiological saline or 5% sodium dodecyl sulphate solution were added to each skin unit of the negative and positive control and were spread gently to cover evenly the surface, without damaging the epidermis. The treated epidermis units were incubated at room temperature for 15 minutes. All units were then rinsed with phosphate buffered physiological saline to remove all material, placed in plate wells filled with fresh maintenance medium and incubated for 42 hours at 37 °C under the conditions of the pre-conditioning. After the incubation, the units were transferred into wells filled with the MTT working solution and incubated for 3 hours under the same conditions as previously. The two living epidermis used for the colour control were incubated with assay medium instead. A formazan extraction was performed after the incubation with MTT. The cell viability was then determined by measuring the optical density of each plate at 570 nm. The validity criteria for the usability of the test models, the optical density of the negative and positive controls and the standard deviation of the three tissues treated with the test substance were met and the study was considered as valid. The mean cell viability of the three individual epidermis units treated with the substance was 96.1% compared to the negative controls. The substance was considered to be not irritating to 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

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

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni u 129., Hungary
Chicken heads were collected from a commercial abattoir after chickens (approximately 7 weeks old) had been slaughtered for human consumption. Heads were collected by a slaughter house technician. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a sealed plastic box (4-5 heads/box). The heads were immediately transported to the test laboratory at ambient temperature. The heads were processed within 2 hours of collection.
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, to avoid 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 still 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.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting 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 retainer. The cornea was positioned vertically with the eye in the correct relative position (same position as in the chicken head), taking care to avoid putting too much pressure on the eye by the retainer. Due to 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 retainer holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes (approximately nine to twelve) were selected, after being placed in the superfusion apparatus they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to clearly see 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 with an optical pachymeter on a slit-lamp microscope, which was set at a 0.095 mm slit-width. Any eye with cornea thickness deviating by 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 was started and 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied as supplied (although it was ground to fine powder). An amount of 30 mg test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The negative and positive control eyes as well as all 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.

Number of animals or in vitro replicates:

One eye for negative control (physiological saline), three eyes for treatment with test substance, three eyes for positive control (imidazole)

Details on study design:

The time of application was observed, 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 (B. Braun Pharmaceuticals SA, Lot number 90352Y05-2) at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item if possible. The eye was returned to the chamber after rinsing. The time while the eye was out of the chamber was limited to the minimum required.
Additional gentle rinsing with 20 mL saline (3 or 4 times) was performed at each time point when the test item or the positive control material was observed to remain on the cornea.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. A slit-lamp microscope was used for the measurements and was set at a 0.095 mm slit-width.

Irritation parameter:

corneal swelling 

Run / experiment:

Mean of three eyes

Value:

3.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A mean maximum corneal swelling of 3.2% was noted at up to 75 minutes and 240 minutes after application.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean of three eyes

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean of three eyes

Value:

0.83

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Test substance was stuck on all corneal surfaces after the post-treatment rinse. All surfaces were clear at 75 minutes after the post-treatment rinse. Positive control substance was stuck on the cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. No other morphological effects were observed.
The positive control substance imidazole was classified as severely irritating, based on the observations on corneal swelling, corneal opacity change and fluorescein retention change. The negative control physiological saline (Sasol solution, NaCl 0.9% w/v) was classified as non-irritating.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eye based on the results of the in vitro eye irritation study.

Executive summary:

An in vitro eye irritation study was performed under GLP on isolated chicken eyes to OECD TG 438 (June 2018). Fresh isolated chicken eyes were obtained from a commercial abattoir and used within 2 hours of receipt. Three eyes were used for the treatment with the test substance, alongside one negative (physiological saline) and three positive (imidazole) control eyes. After the preparation of the chicken eyes and the zero reference (baseline) measurements, each eye in the treatment group was held in a horizontal position and 30 mg of powdered, neat test substance applied onto the centre of the cornea such that the entire surface of the cornea was covered. After ten seconds, the surface was rinsed with physiological saline. In the experiment the positive control eyes were treated in a similar way with 30 mg of powdered imidazole. The negative control eye was treated with 30 μL of physiological saline. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects were evaluated over a four hour observation period. No significant corneal swelling (mean 3.2%) and no significant cornea opacity change (severity 0.5) was observed on any of the three eyes treated with the substance. Slight fluorescein retention change (severity 1.0 on two eyes and severity 0.5 on one eye) was noted on the three eyes. Based on these findings, the test substance was considered as non-irritant. The results obtained with negative and positive controls demonstrated the sensitivity of the assay and that the study was valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of the substance to be corrosive or irritating to the skin was studied under GLP with the EpiSkin model using reconstructed human epidermis in valid in vitro studies performed to OECD 431 and 439. The cell viability in the assays with the test substance was clearly above the respective threshold values of the tests, showing that the substance was not corrosive or irritating to human skin under the conditions of the test.

The potential of the substance to be corrosive or irritating to the eyes was studied under GLP in a valid isolated chicken test performed to OECD TG 438. The observed corneal swelling, corneal opacity and fluorescein retention change in the eyes treated with the test substance indicated that the substance was not irritating to the eyes under the conditions of the test.

Justification for classification or non-classification

On the basis of the information obtained in reliable and valid in vitro studies, the substance is not classified for skin irritation or eye irritation in accordance with Regulation (EC) No. 1272/2008.