Registration Dossier

Administrative data

Description of key information

Skin:

The results obtained from this in vitro skin irritation test, using the EPISKIN model according to OECD guideline 439, indicated that the test item reveals no skin irritation potential. T

Eye:

In an eye irritation assay (isolated chicken eye) according to OECD guideline 438, no prediction could be made.

In an in vitro eye irritation test, using the EpiOcular™ model according to OECD guideline 492, the test item did not show eye irritating properties.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 31 - October 10, 2016
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)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM)
- Tissue batch number: 16-EKIN-037

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
see "Any other information on Materials and Methods"

NUMBER OF REPLICATE TISSUES:
3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls,

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.
- Method of calculation used:
Data calculation for normal test items
Blank
– The mean of the 6 blank OD values is calculated:
Negative control
– Individual negative control OD values are corrected with the mean blank OD: :
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 3 negative control values are calculated: this corresponds to 100% viability
Positive control
– Individual positive control OD values are corrected with the mean blank OD: :
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control values are calculated
– The % viability for each positive control replicate is 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 viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test item
– Individual test item OD values are corrected with the mean blank OD: :
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 3 test item values are calculated
– The % viability for each test item replicate is 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 viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3

Data calculation for MTT-interacting items
Test items that interfere with MTT can produce non-specific reduction of the MTT. It is necessary to evaluate the OD due to non-specific reduction and to subtract it before calculations of viability %.
– Non-specific MTT reduction calculation (NSMTT):
NSMTT = [(ODKT - ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD (mean)
ODKT: test item treated killed tissues OD (mean)
ODNC: negative control OD (mean)
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.
– True MTT metabolic conversion (TODTT) is undertaken if NSMTT is < 50%:
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 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / 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

Data calculation for dyes and chemicals able to colour the tissue
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 %.
– Non Specific Colour % (NSC %):
NSC % = (mean ODCT / mean ODNC) × 100
ODCT: test item treated tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
– True MTT metabolic conversion (TODTT) is undertaken if NSC % is > 5 % and ≤ 50 %.
TODTT = [ODTV – mean ODCT]
ODTV: test item-treated tissues (incubated with MTT)
ODCT: test item-treated 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 / ODNC] × 100
% RV 2= [TODTT2 / ODNC] × 100
% RV 3 = [TODTT3 / 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
– If NSC % is ≤ 5 % then the normal calculation mode is used.
– If NSC % 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.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating or corrosive to skin if the viability after exposure is less than or equal to 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after exposure is greater than 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg of the test item was applied evenly to the epidermal surface.

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 1x PBS

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq. solution
Duration of treatment / exposure:
15 minutes (± 0.5 min) at room temperature.
Duration of post-treatment incubation (if applicable):
42 hours (± 1h) at 37 °C in an incubator with 5 % CO2, ≥95 % humidified atmosphere.
Number of replicates:
In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-3
Value:
>= 115 - <= 144
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean viability 128%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: As the test item has an intrinsic colour (light red), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary.
The non-specific MTT reduction (NSMTT) was determined (0%)*, the correction of viability percentage was not necessary
- Colour interference with MTT: Yes, as the test item has an intrinsic colour (light red), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.012. The Non Specific Colour % (NSC %) was calculated as 1.4 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 2: OD values and viability percentages of the controls

Substance

Optical Density (OD)

Viability (%)

Negative Control:
1x PBS

1

1.018

117

2

0.823

95

3

0.765

88

mean

0.869

100

standard deviation (SD)

15.22

Positive Control:
SDS (5 % aq.)

1

0.167

19

2

0.168

19

3

0.072

8

mean

0.136

16

standard deviation (SD)

6.34

Table 3: OD values and viability percentages of the test item

Test Item

Optical Density (OD)

Viability (%)

1

1.000

115

2

1.073

124

3

1.253

144

mean

1.109

128

standard deviation (SD)

14.93

 Table 4: OD values of additional controls for MTT-interacting test item

Additional controls

Optical Density (OD)

Negative control killed tissues:
1x PBS

1

0.065

2

0.078

3

0.079

mean

0.074

Test item treated killed tissues:

1

0.048

2

0.053

3

0.045

mean

0.048

 

Table 5: OD values and NSC % of additional control:

Additional colour control

Optical Density (OD)

Non Specific Colour % (NSC %)

(test item treated tissueswithout MTT incubation)

1

0.012

1.4

2

0.013

mean

0.012

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model according to OECD guideline 439, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Executive summary:

In an in vitro skin irritation test (EpiSkin SM) according to OECD guideline 492, the skin irritating potential of the test item was assessed.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. SDS (5 % aq.) and 1× PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (light red), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated for 42 hours in an incubator at 37°C with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (light red). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not significantly reduce cell viability in comparison to the negative control (mean viability: 128 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. 

The results obtained from this in vitro skin irritation assay, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test is considered to be not irritating to skin and is therefore not classified (UN GHS No Category).

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 August - 22 September, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
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)
Version / remarks:
26 July, 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.4 ºC to 20.3 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4 - 5 heads/box).
- indication of any existing defects or lesions in ocular tissue samples: 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 fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with 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.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 mg, The test item was applied in its original form, no formulation was required. The test item and the positive control were finely ground before application.

Duration of treatment / exposure:
10 s
Duration of post- treatment incubation (in vitro):
30 min
Number of animals or in vitro replicates:
3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
See "Details on test animals or tissues and environmental conditions"

EQUILIBRATION AND BASELINE RECORDINGS
appropriate eyey for the test,were acclimated for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods

NUMBER OF REPLICATES
3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.

NEGATIVE CONTROL USED
Sodium chloride, NaCl (9 g/L saline)

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
NaCl: 30 µL/eye
Imidazole: 30 mg/eye

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Removal of test item: The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum. The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 minutes of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.


METHODS FOR MEASURED ENDPOINTS:
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance .
- Corneal opacity:
- Damage to epithelium based on fluorescein retention:
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm.
- Macroscopic morphological damage to the surface: The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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.


SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA:
The decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The negative controls showed the expected results. The experiment was considered to be valid.
Positive controls validity:
valid
Remarks:
The positive controls showed the expected results. The experiment was considered to be valid.
Remarks on result:
no indication of irritation
Remarks:
The overall ICE class was 1xI, 2xII; thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made"

Table 5: Summary of the test item results 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

3%

I

Mean maximum corneal swelling at up to 240 min

4%

I

Mean maximum corneal opacity

1.0

II

Mean fluorescein retention

1.0

II

Other Observations

None

Overall ICE Class1

1xI, 2xII

 

Table 6: Summary of the positive control results 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

25%

III

Mean maximum corneal swelling at up to 240 min

33%

IV

Mean maximum corneal opacity

4.0

IV

Mean fluorescein retention

3.0

IV

Other Observations

Cornea opacity score 4 was observed in two eyes at
30 minutes after the post-treatment rinse.

Overall ICE Class1

3xIV

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

Table 7: Summary of the negative control results 

Observation

Value

ICE Class1

Mean maximum corneal swelling at up to 75 min

2%

I

Mean maximum corneal swelling at up to 240 min

3%

I

Mean maximum corneal opacity

0.5

I

Mean fluorescein retention

0.0

I

Other Observations

None

Overall ICE Class1

3xI

The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Interpretation of results:
other: in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.
Conclusions:
In an eye irritation assay (isolated chicken eye) according to OECD guideline 438, no prediction could be made.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The study was performed according to the OECD guideline 438. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

 The test item and Imidazole (positive control) was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 µL saline solution (9 g/L). After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with ca. 20 mL saline solution at ambient temperature. Positive and negative controls showed the expected results. The experiment was considered to be valid. In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with Leuco Sulfur Red 14, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xI, 2xII.

According to the overall in vitro evaluation criteria classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 28 - June 2, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model (10 February 2017).
Version / remarks:
2017 February 10
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: Keratinocyte strain 4F1188
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
see " Any other information on materials ad methods"
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg (in its original form on top of 0.6 cm² tissue; approx. 83.3 mg/cm²)


Duration of treatment / exposure:
6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90 ± 10 % humidified atmosphere).
Duration of post- treatment incubation (in vitro):
18 hours ± 15 minutes at standard culture conditions.
Number of animals or in vitro replicates:
In this assay 2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.
Details on study design:
- Details of the test procedure used : The tissues were equilibrated to room temperature for about 15 minutes, while the Assay Medium was pre-warmed to 37±1°C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37 °C in an incubator with 5 ± 1 % CO2, 90 ± 10 % humidified atmosphere for one hour in the Assay Medium, than the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours). During the pre-treatement the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Approximately 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium. After rinsing, the tissues were transferred to and immersed in 5 mL RT Assay Medium in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation (Post-Soak) at RT; after it, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37 °C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation).

- RhCE tissue construct used, including lot number : EpiOcular™ (OCL-200-EIT), Lot No 23779

- Doses of test chemical and control substances used : 50 mg of the grounded test item, 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 h exposure at 37 °C, 25 min post-soak period at room temperature, 18 h post-treatment incubation at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: 2 killed treated tissues and 2 killed negative control tissues were used for the MTT evaluation in one run. Additional controls for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item treated tissues were used for the non-specific OD evaluation. In addition to the normal procedure, two killed treated tissues were used for avoiding a possible double correction for colour interference. The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90 ± 10 % humidified atmosphere). After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with minimum of 100 mL Ca++Mg++ Free-DPBS per test item. Each test item utilizes a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the tissues was dipped into the first beaker of DPBS and was swirled in a circular motion in the liquid for approximately 2 seconds and after was lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The tissue was rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

- Number of tissue replicates used per test chemical and controls: In this assay 2 replicates per test item and 2 replicates negative controls, 2 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 2 killed treated tissues and 2 killed negative control tissues are used for the MTT evaluation.

- Description of the method used to quantify MTT formazan : After the post-incubation the EpiOcular™ units were transferred into the MTT ready to use solution filled 24-well plate (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 10 min) at 37 ± 1 °C in an incubator with 5 ± 1 % CO2 protected from light, 90 ± 10 % humidified atmosphere. Inserts were removed from the 24-well plate after 3 hours ± 10 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm. To extract the MTT, the plates were placed on an orbital plate shaker and shaken (150 rpm) for approximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative, positive, and colorant controls were treated identically.
Following the formazan extraction, 200 µL sample(s) from each tube (preferably 2×200 µL if possible) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank (8×200 µL).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
According to the United Nations Globally Harmonized System (UN GHS) of Classification and Labelling of Chemicals (6th revised edition; 2015) and as implemented in the European Commission Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (EU CLP), the irritancy potential of test items according to this OECD test guidance is used to distinguish between irritant or corrosive (Category 2 or Category 1) and non-irritant (No Category) substances.
In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control. However, this test method (OECD 492) cannot resolve between UN GHS Categories 1 and 2, further testing with other test methods will be required to decide on its final classification.
Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing with the help of other test methods is required.
The prediction model (PM) is described below:
Mean tissue viability % is ≤ 60 % --> Category 2 or Category 1
Mean tissue viability % is > 60 % --> No Category



Irritation parameter:
in vitro irritation score
Run / experiment:
1-2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: tissue viability mean %
Run / experiment:
1-2
Value:
78
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: test item viability results were above 60 % when compared to the viability values obtained from the negative control.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

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

Table 3: OD values and viability percentages of the controls

Controls

Optical Density (OD)

Viability (%)

Δ%

Negative Control:
Sterile deionized water

1

1.740

104

7.5

2

1.614

96

mean

1.677

100

 

Positive Control:
Methyl acetate

1

0.509

30

8.6

2

0.364

22

mean

0.437

26

 

 

 Table 4: OD values and viability percentages of the test item

Test Item

Optical Density (OD)

Viability (%)

Δ%

Tissue

1

1.435

86

15.6

2

1.173

70

mean

1.304

78

 

  

Table 5: OD values of additional controls for MTT-interacting test item

Additional controls

Optical Density (OD)

Negative control killed tissues:
Sterile deionized water

1

0.042

2

0.193

mean

0.118

Test item treated killed tissues:

1

0.060

2

0.070

mean

0.065

 

Table 6: OD values and NSC % of additional control

Additional colour control

Optical Density (OD)

Non Specific Colour %(NSCliving %)

Δ%

Tissue

1

0.008

0.55

0.1

2

0.010

mean

0.009

 

 Remark: Δ% The difference of viability between the two relating tissues

 

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro eye irritation test, using the EpiOcular™ model according to OECD guideline 492, the test item did not show skin irritating properties. Thus, the test item is considered as non-irritant to eye (UN GHS No Category).
Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro according to OECD guideline 492.

Before treatment the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90 ± 10 % humidified atmosphere). Exposure of test material was determinated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. 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, 90 ± 10 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes. The test item has an intrinsic colour (light red), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. The test item is a possible MTT-reducer and has an intrinsic colour (light red). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% compared to the negative control. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The test item did not induce significantly reduced cell viability in comparison to the negative control (mean viability: 78%). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be not irritating to eye according to the UN GHS classification.

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

Additional information

Skin irritation:

In an in vitro skin irritation test (EpiSkin SM) according to OECD guideline 492, the skin irritating potential of the test item was assessed.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (light red), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated for 42 hours in an incubator at 37 °C with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.

The test item is a possible MTT-reducer and has an intrinsic colour (light red). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item did not significantly reduce cell viability in comparison to the negative control (mean viability: 128 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. 

The results obtained from this in vitro skin irritation assay, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test is considered to be not irritating to skin and is therefore not classified (UN GHS No Category).

Eye irritation:

The objective of the present studies was to determine a possible eye irritating/eye damaging potential of the test substance. A single in vitro assay may not always be sufficient, using the currently available methods, to cover the full range of eye irritating potential. Consequently, in accordance with the IATA for the assessment of eye irritation/eye damage two in vitro assays were performed: The isolated chicken eye test (ICE Test) according to OECD guideline 438 and the EpiOcularEye Irritation Test according to OECD guideline 492.

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The study was performed according to the OECD guideline 438. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points.

 The test item and Imidazole (positive control) was ground before use in the study. The test item and positive control applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 µL saline solution (9 g/L). After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with ca. 20 mL saline solution at ambient temperature. Positive and negative controls showed the expected results. The experiment was considered to be valid. In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with Leuco Sulfur Red 14, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xI, 2xII.

According to the overall in vitro evaluation criteria classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

In a trailing study, following the top down approach, the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro according to OECD guideline 492 was determined.
Before treatment the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90 ± 10 % humidified atmosphere). Exposure of test material was determinated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incuba
tion). Fresh Assay Medium was used during the Post-Soak and Post-incubation. 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, 90 ± 10 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes. The test item has an intrinsic colour (light red), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. The test item is a possible MTT-reducer and has an intrinsic colour (light red). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% compared to the negative control. Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The test item did not induce significantly reduced cell viability in comparison to the negative control (mean viability: 78%). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be not irritating to eye according to the UN GHS classification.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin irritation/corrosion and eye irritation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.