Registration Dossier

Administrative data

Description of key information

Skin corrosion: Not corrosive (OECD 431/GLP)

Skin irritation: Not irritating (OECD 439/GLP)

Serious eye damage/eye irritation: Not irritating (OECD 438)

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:
11 May 2020 - 28 August 2020
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)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: sample supplied by sponsor :91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity test date: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was applied in this original form; although it was ground to fine powder in a mortar and pestle.
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes (NHEK)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Model
- Tissue batch number(s): 30865
- Date of initiation of testing:13 May 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C(first 35 min), then 25 minutes at room temperature (23.9-24.7°C)
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiDerm™ units were removed and rinsed thoroughly with DPBS solution(= insert was filled and emptied 20 times in a constant soft stream of DPBS in a glass beaker filled with at least 100 mL DPBS solution) to remove all of the test material from the epidermal surface


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg MTT / ml medium
- Incubation time: 3 hours
- Spectrophotometer: Thermo Fisher Scientific
- Wavelength: 570 nm

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

CONTROL TISSUES USED IN CASE OF MTT DIRECT/COLOUR INTERFERENCE
1. MTT: 25 mg of test item was added to 1 mL MTT medium and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 1 hour protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: other colours
-Test items reacting with MTT: blue or purple
After one hour incubation, red colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

2. Colour: Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
The test item had a light yellow colour, therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritant to skin if the mean cell viability was >50% compared to the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Amount(s) applied (volume or weight with unit):25 µL DPBS
- Lot: RNBH7035 (Sigma-Aldrich)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% (w/v) SDS solution
Duration of treatment / exposure:
35 minutes at 37°C; 25 minutes at room temperature (23.9-24.7°C)
Duration of post-treatment incubation (if applicable):
42 hrs (± 2hrs)
Number of replicates:
3
Vehicle:
other: DPBS
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item
Value:
92.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 92.7±5.9
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: As no colour change was observed after one hour of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Colour interference with MTT:
As the test item was coloured (light yellow), 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 0.003, Non-Specific Colour % (NSCliving%) was calculated as 0.2% compared to negative control (see Table 3). This value was below 5%, therefore additional data calculation was not necessary

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (1.956). Standard deviation of the viability results for negative control samples was 3.2%.
- Acceptance criteria met for positive control: The positive control treated tissues showed 1.9% viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 5.9%. The standard deviation of the viability results for positive control samples was 0.1%.
- Range of historical values if different from the ones specified in the test guideline: OD data of two replicate tissues at the negative control in this study were higher (OD: 2.014 and 1.966) than the maximum OD of the historical control range (OD: 1.941). OD data of one replicate tissue at the positive control in this study was lower (OD: 0.035) than the minimum OD of the historical control range (OD: 0.038). This fact has no impact on the results or integrity of the study since the positive control material showed clear positive result and the acceptable mean percentage viability range for the positive controls is 0-20% according to the OECD No. 439.
Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to CLP.
Conclusions:
In an in vitro skin irritation assay in the human epidermal model EpiDerm, the test item is not a skin irritant.
Executive summary:

In an in vitro skin irritation assay in the human epidermal model EpiDerm (20/071-051B), reconstructed human keratinocytes (moistened with 25µL DPBS) was exposed to 25 mg of the test item (92.4%) for 35 minutes at 37°C and then 25 minutes at room temperature (23.9- 24.7°C). DPBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance via washing, tissues were post-incubated for 42 ± 2hrs. Tissues were then incubated with MTT for 3 hours. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean OD570 for the negative control treated tissues was between ≥0.8 and ≤ 2.8 (1.956). The mean relative tissue viability of the positive control was ≤ 20% (1.9%). Standard deviation of viability of replicate tissues of all dose/negative control/positive control groups was ≤ 18% (5.9%/3.2%/0.1%).

The test item was not directly MTT reducing. The NSCliving % value for two additional test item-treated living tissues was below 5% so additional data calculation was not necessary to consider colour interference. The average viability of tissues treated by the test item was 92.7 ±5.9% of the negative control average value i.e. viability was > 50 %. According to these results, the test item is not irritating.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2020 - 28 August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: sample supplied by sponsor :91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity test date: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was applied in this original form; although it was ground to fine powder in a mortar and pestle.

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes (NHEK)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Model
- Tissue batch number(s): 30865
- Date of initiation of testing: 14 May, 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (21.9-22.2°C; 3min) and 37°C(1hour);
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with PBS solution to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg MTT / ml medium
- Incubation time: 3 hours
- Spectrophotometer: Thermo Fisher Scientific
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES: 2 per test item, negative and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT/COLOUR INTERFERENCE
1. MTT: 25 mg of test item was added to 1 mL MTT medium and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 1 hour protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: other colours
-Test items reacting with MTT: blue or purple
After one hour incubation, red colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

2. Colour: Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
The test item had a light yellow colour, therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]

- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25mg

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL distilled water
- Lot/batch no. (if required): 73751Y25-2 (B. Braun Pharmaceuticals SA)


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL distilled water


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Potassium hydroxide solution
- Concentration (if solution):8 N
Duration of treatment / exposure:
3 minutes at room temperature (21.9-22.2°C) and 1 hour at 37°C
Duration of post-treatment incubation (if applicable):
Tissues were incubated overnight at room temperature (without shaking) for formazan extraction.
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
109.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hr
Value:
98.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: As no colour change was observed after one hour of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Colour interference with MTT: As the test item was coloured (light yellow), two additional test item-treated living tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) was 0.002 and the NSCliving % value for the test item was calculated as 0.1% in both exposure period compared to negative control (see Table 1 and 2.). This value was below 5%, therefore additional data calculation was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was in the recommended range (1.723 and 1.754).

- Acceptance criteria met for positive control: The two positive control treated tissues showed 23.5% (3 minutes exposure period) and 5.5% (1 hour exposure period) viability demonstrating the proper performance of the assay.

- Acceptance criteria met for variability between replicate measurements:
The difference of cell Coefficient of Variation (CV) between the two test item-treated tissue samples in the MTT assay was 0.5% (3 minutes exposure period) and 4.21% (1 hour exposure period).
The difference of cell Coefficient of Variation (CV) between the two negative control treated tissue samples in the MTT assay was 7.0% (3 minutes exposure period) and 2.36% (1 hour exposure period).

- Range of historical values if different from the ones specified in the test guideline: OD data of one replicate tissue at the positive control in this study at 1 hour exposure period was lower (0.080) than the minimum OD of the historical control range (OD: 0.092). This fact has no impact on the results or integrity of the study since the positive control material showed clear positive result and the acceptable mean percentage viability range for the positive controls is 20-100% according to the OECD No. 431.
Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to CLP.
Conclusions:
In an in vitro skin corrosion assay in the human epidermal model EpiDerm, the test item is non corrosive.
Executive summary:

In an in vitro skin corrosion assay in the human epidermal model EpiDerm ( 20/071-051B), 25mg of test item (92.4%) was applied to the surface of reconstructed human keratinocytes (followed by 25 μL of distilled water) for 3 minutes at room temperature (21.9-22.2°C) and 1 hour at 37°C. Distilled water was used for the negative control and 8 N potassium hydroxide solution was used for the positive control. After removal of the test substance via washing, tissues were then incubated with MTT for 3 hours. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.  

The controls confirmed the validity of the study. The mean OD570 for the negative control treated tissues was between ≥ 0.8 and ≤ 2.8 (1.723 at 3 min and 1.754 at 1h). Mean viability of the tissue replicates were 23.5% (3 minutes) and 5.5% (1 hour) for the positive control (≤15%). The difference of cell Coefficient of Variation (CV) between the two test item-treated tissue samples in the MTT assay was 0.5% (3 minutes exposure period) and 4.21% (1 hour exposure period). The CV between the two negative control treated tissue samples in the MTT assay was 7.0% (3 minutes exposure period) and 2.36% (1 hour exposure period).

The test item was not directly MTT reducing. The NSCliving % value for two additional test item-treated living tissues was below 5% so additional data calculation was not necessary to consider colour interference. The mean cell viability was 109.3±0.7% after 3 minutes treatment and 98.6±6.0% after 1 hour treatment compared to the negative control. These are above the threshold of 50% (3 minutes) and 15% (1 hour). According to these results, the test item is non-corrosive.

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:
07 May 2020 - 26 August 2020
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)
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: test material provided by sponsor: 91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity test date: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25℃, ≤70% relative humidity)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was applied in its original form (although it was ground to a fine powder in a mortar and pestle).
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: H-9600 Sárvár, Rábasömjéniutca 129., Hungary)

- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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.`

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30mg

VEHICLE: unchanged (no vehicle)
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
30, 75, 120, 180 and 240 minutes after the post-treatment rinse
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
3 for positve control group
1 for negative control group
3 for test group
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). 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 a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. 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.

EQUILIBRATION AND BASELINE RECORDINGS
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 significant corneal thickness changes (-1.6% was observed on two eyes in Experiment I and -1.6% was noted on one eye or 1.6% was noted on eye in Experiment II) and no corneal thickness changes were observed in the other eyes in the Experiments. 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.

NUMBER OF REPLICATES :
3 for positve control group
1 for negative control group
3 for test group

NEGATIVE CONTROL USED
physiological saline [B. Braun Pharmaceuticals SA (194948162)]

POSITIVE CONTROL USED
powdered Imidazole [Acros Organics (A0386897)]

APPLICATION DOSE AND EXPOSURE TIME
30mg for 10 seconds

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, Additional gentle rinsing with 3*20 mL saline was performed at each time point when the positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
Haag-Streit BP 900® slit lamp microscope was used for the measurements. Corneal thickness and corneal opacity were measured at all time points. For opacity determination, the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. The fluorescein retention determination the settings of the slit lamp microscope was the same as for opacity assessment, but the green light filter was used.

- Macroscopic morphological damage to the surface: In each experiment 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 effects were observed in each experiment in any group.

SCORING SYSTEM:
- Mean corneal swelling (%) Corneal swelling is determined from corneal thickness measurements made with an optical pachymeter on a slit lamp microscope. It is expressed as a percentage and is calculated from corneal thickness measurements according to the guideline formulae.
- Mean maximum opacity score : See below.
- Mean fluorescein retention score at 30 minutes post-treatment : See below.

DECISION CRITERIA: As per OECD 438 criteria.
Irritation parameter:
percent corneal swelling
Remarks:
up to 75min
Run / experiment:
Experiment I
Value:
1.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
up to 240 min
Run / experiment:
Experiment I
Value:
-7.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class II
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment I
Value:
0.33
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment I
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
up to 75min
Run / experiment:
Experiment II
Value:
-1.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment II
Value:
-4.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment II
Value:
0.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment II
Value:
0.33
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The mean corneal swelling at 240 min was -7.6%. As no severe loosening of the epithelium and no other morphological effects were observed, the mean maximum corneal swelling is considered to be slight and therefore classified as ICE class: II. In each experiment 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 effects were observed in each experiment in any group.

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.
Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro eye irritation test in isolated chicken eyes, the test item is not an eye irritant.
Executive summary:

In an in vitro eye irritation test in isolated chicken eyes (ICET) assay (20/071-038CS), isolated chicken eyes were exposed to the test item (92.4%) for 10 seconds. Physiological saline (0.9% (w/v) NaCl) was used for the negative control and powdered Imidazole was used for the positive control. 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. 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. Any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

In each experiment 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 effects were observed in the study in any group.

In Experiment I, slight corneal swelling change (mean = -7.6%) was observed during the four-hour observation period on test item treated eyes (As no severe loosening of the epithelium and no other morphological effects were observed, the mean maximum corneal swelling is considered to be slight and therefore classified as ICE class: II.). Minimal cornea opacity change (mean = 0.33) was observed. No fluorescein retention change (mean = 0.0) was noted. No other morphological effects were observed.  The Overall ICE Class was 2xI 1xII. In experiment II, minimal corneal swelling change (mean = -4.3%) was observed during the four-hour observation period on test item treated eyes. Minimal cornea opacity change (mean = 0.50) was observed. Minimal fluorescein retention change (mean = 0.33) was noted. No other morphological effects were observed. The Overall ICE Class was 3xI.  Therefore, based on these results, the test item was a non-irritant.




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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

There is one in vitro skin corrosion study available and one in vitro skin irritation study available (combined OECD 431/439 test).

Skin corrosion:

In an in vitro skin corrosion assay in the human epidermal modelEpiDerm (OECD 431/GLP),25mg of test item (92.4%) was applied to the surface of reconstructed human keratinocytes (followed by25 μL of distilled water) for 3 minutes at room temperature (21.9-22.2°C) and 1 hour at 37°C.Distilled waterwas used for the negative control and8 N potassium hydroxide solutionwas used for the positive control. After removal of the test substance via washing, tissues were then incubated with MTT for 3 hours. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.  

The controls confirmed the validity of the study. The mean OD570 for the negative control treated tissues was between ≥ 0.8 and ≤ 2.8 (1.723 at 3 min and 1.754 at 1h).Mean viability of the tissue replicates were23.5% (3 minutes)and 5.5% (1 hour) for the positive control (≤15%).The difference of cell Coefficient of Variation (CV) between the two test item-treated tissue samples in the MTT assay was 0.5% (3 minutes exposure period) and 4.21% (1 hour exposure period). The CV between the two negative control treated tissue samples in the MTT assay was 7.0% (3 minutes exposure period) and 2.36% (1 hour exposure period). The test item was not directly MTT reducing. The NSCliving % value for two additional test item-treated living tissues was below 5% so additional data calculation was not necessary to consider colour interference.The mean cell viability was 109.3±0.7% after 3 minutes treatment and 98.6±6.0% after 1 hour treatment compared to the negative control. These are above the threshold of 50% (3 minutes) and 15% (1 hour). According to these results, the test item is non-corrosive.

Skin irritation:

In an in vitro skin irritation assay in the human epidermal model EpiDerm (OECD 439/GLP), reconstructed human keratinocytes (moistened with 25µL DPBS) was exposed to 25 mg of the test item (92.4%) for 35 minutes at 37°C and then 25 minutes at room temperature (23.9- 24.7°C). DPBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance via washing, tissues were post-incubated for 42 ± 2hrs. Tissues were then incubated with MTT for 3 hours. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. The controls confirmed the validity of the study. The mean OD570 for the negative control treated tissues was between ≥0.8 and ≤ 2.8 (1.956). The mean relative tissue viability of the positive control was ≤ 20% (1.9%). Standard deviation of viability of replicate tissues of all dose/negative control/positive control groups was ≤ 18% (5.9%/3.2%/0.1%). The test item was not directly MTT reducing. The NSCliving % value for two additional test item-treated living tissues was below 5% so additional data calculation was not necessary to consider colour interference. The average viability of tissues treated by the test item was 92.7 ±5.9% of the negative control average value i.e. viability was > 50 %. According to these results, the test item is not irritating.

Serious eye damage/eye irritation

There is one in vitro eye irritation study available.

In an in vitro eye irritation test in isolated chicken eyes (ICET) assay (OECD 438/GLP), isolated chicken eyes were exposed to the test item (92.4%) for 10 seconds. Physiological saline (0.9% (w/v) NaCl) was used for the negative control and powdered Imidazole was used for the positive control. 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. 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. Any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

In each experiment 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 effects were observed in the study in any group. In Experiment I, slight corneal swelling change (mean = -7.6%) was observed during the four-hour observation period on test item treated eyes (As no severe loosening of the epithelium and no other morphological effects were observed, the mean maximum corneal swelling is considered to be slight and therefore classified as ICE class: II.). Minimal cornea opacity change (mean = 0.33) was observed. No fluorescein retention change (mean = 0.0) was noted. No other morphological effects were observed.  The Overall ICE Class was 2xI 1xII. In experiment II, minimal corneal swelling change (mean = -4.3%) was observed during the four-hour observation period on test item treated eyes. Minimal cornea opacity change (mean = 0.50) was observed. Minimal fluorescein retention change (mean = 0.33) was noted. No other morphological effects were observed. The Overall ICE Class was 3xI.  Therefore, based on these results, the test item was a non-irritant.


Justification for classification or non-classification

Based on the available information in the dossier, the test item is not classified for skin irritation/corrosion or serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.