Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion in vivo, rabbit (OECD 404) and in vitro (OECD 439): not irritating

Eye irritation in vivo, rabbit (OECD 405) and in vitro (OECD 437 and OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Aug - 19 Aug 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kissleg, Germany
- Age at study initiation: at least 6 weeks old
- Weight at study initiation: at least 1.0 kg
- Housing: controlled environment, individually in labelled cages with perflorated floors
- Diet (e.g. ad libitum): standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits, Altromin, Lage, Germany), approx. 100 g per day. Hay (BMI, Helmond, the netherlands) was provided at least three times a week.
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 +/- 3.0 (actual range: 20.5-21.6)
- Humidity (%): 30-70 (actual range: 44-78)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity.

IN-LIFE DATES: 09 Aug - 19 Aug 2005
Type of coverage:
semiocclusive
Preparation of test site:
other: clipped
Vehicle:
unchanged (no vehicle)
Controls:
other: adjacant areas of untreated skin of each animal were served as controls
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
3 min, 1 and 4 h (1 animal)
4 h ( 2 further animals)
Observation period:
72 h
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 2x3 cm, flank
- Type of wrap if used: metalline patch was mounted on micropore tape , which was wrapped with Coban elastic bandage (Lohmann GmbH, Neuwied, Germany (Metalline) and 3M, St. Paul, Minnesota, USA (Micropore and Coban)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After removal of a dressing, the treated skin was cleaned of residual test substance using water
- Time after start of exposure: 3 min, 1hour and 4 hours (one rabbit); 4 hours (two further rabbits)

SCORING SYSTEM: Erythema and eschar formation:
no erythema: 0; very slight erythema: 1; well-defined erythema: 2; moderate to severe erythema: 3; severe erythema: 4
Oedema formation:
no oedema: 0; very slight oedema: 1; slight oedema: 2; moderate (raised approx. 1 mm) oedema: 3; severe oedema:4
Irritation parameter:
erythema score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Irritation parameter:
edema score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Irritant / corrosive response data:
Four-hour exposure to 0.5 mL of NATU-C resulted in very slight erythema in the treated skin areas of the three rabbits. The skin irritation had resolved within 24 hours after exposure in all animals. No oedema was observed.
There was no evidence of a corrosive effect on the skin.
Other effects:
No staining of the treated skin by the test substance was observed and no test substance remnants were seen.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Base on these results NATU-C is considered to be non-irritating.

Body weights

animal 730, 10 -12 weeks old: prior to application 2274 g; at termination 2399g

animal 739, 7 -9 weeks old: prior to application 1745 g; at termination 1911 g

animal 742, 7 -9 weeks old: prior to application 1757 g; at termination 1639 g

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not irritating
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jul - 24 Aug 2018
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:
adopted 28 Jul 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 06 Jul 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 28 Jun 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: sterilization at 121 °C for 20 min to avoid contaminations of the cell culture





Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDermTM reconstituted three-dimensional human epidermis (EPI-200)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200 SIT kit (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 28646

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 min in the incubator followed by 25 min at room temperature under the sterile flow
- Temperature of post-treatment incubation: 37 ± 1 °C for 42 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with Dulbecco's phosphate buffered saline (DPBS) using a constant stream in about 1.5 cm distance from the tissue surface. This process also occured sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min at 37 ± 1 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm without reference wavelength
- Filter bandpass: ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was 2.216 ± 0.075 and fits to the acceptance criteria of 1.0 - 3.0.
- Barrier function: The barrier function was evaluated by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon treatment with 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.01 h and fits to the acceptance criteria of 4.77 - 8.72 h.
- Contamination: The cells used to produce the EpiDermTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected. Tissue sterility was confirmed by long-term incubation with antibiotic and antimycotic free culture.

CONTROL TISSUES USED IN CASE OF COLOUR INTERFERENCE:
Since the test item mixed with aqua dest. showed colouring detectable by unaided eye assessment and absorbed light in the range of 570 ± 30 nm, the test item was checked for its tissue colouring potential.
- Fresh tissues/killed tissues: Living tissues were treated with 30 µL of the test item (TVT). All steps were performed as in the main study except for the MTT staining (Incubation in medium without MTT).
- No. of replicates: 2 living tissues
- Method of calculation used: The non-specific colour of additional viable tissues (NSCliving) was calculated according to the following formular: NSCliving [%] = [ODTVT / ODNK] x 100
NK: negative control, TVT: additional test item treated living tissue without MTT staining.
If NSCliving is ≤ 5% relative to the negative control of living epidermis, no correction of the result is necessary. If NSCliving is > 5% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formular: TODTT = OTM – ODTVT
TM: test item treated with living tissues
If NSCliving is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues to determine the non-specific reduction of MTT was not performed.


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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 60 min exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 min exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL (47 µL/cm2)

NEGATIVE CONTROL
- Amount applied: 30 µL (47 µL/cm2)

POSITIVE CONTROL
- Amount applied: 30 µL (47 µL/cm2)
- Concentration: 5% (v/v) in deionised water
Duration of treatment / exposure:
35 min at 37 ± 1 °C and 25 min at room temperature
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3 replicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 min exposure
Value:
82.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct MTT reduction: The test substance was not considered to be a MTT reducer. Thus, an additional test with freeze-killed tissues was not performed.
- Colour interference: The substance showed colouring upon mixing with water and absorbed light in the relevant range of 570 ± 30 nm, therefore the non-specific colour of additional viable tissue was determined. Because the non-specific color of additional viable tissues was ≤ 5% (NSCliving: 0.3%) relative to the negative control of living epidermis, no correction of the results was necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 nm of the tissue replicates treated with the negative control is 1.789 and matches the required acceptability criterion.
- Acceptance criteria met for positive control: Mean relative tissue viability is ≤ 20%. Treatment with the positive control for 1 h induced a decrease in relative absorbance to 4.7% as compared to the negative control.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviation of 3 identical replicates is < 18%. The relative standard deviations between the % variability values of the test item, the positive and negative controls are 2.6% at the highest.

Table 1: Results of the test item

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

 

absolute OD570(TODTT)

1.765

1.845

1.763

0.119

0.126

0.125

1.468

1.475

1.530

1.773

1.807

1.778

0.123

0.133

0.134

1.435

1.439

1.538

Mean Absolute OD570

1.789

0.127

1.481

OD570

(blank-corrected)

1.720

1.800

1.718

0.074

0.081

0.081

1.424

1.430

1.485

1.729

1.762

1.734

0.078

0.089

0.089

1.390

1.394

1.493

mean OD570 of the duplicates (blank-corrected)

1.725

1.781

1.726

0.076

0.085

0.085

1.407

1.412

1.489

total mean OD570 of 3 replicate tissues (blank-corrected)

1.744*

0.082

1.436

SD OD570

0.032

0.005

0.046

relative tissue viabilities [%]

98.9

102.1

99.0

4.4

4.9

4.9

80.7

81.0

85.4

mean relative tissue viability [%]

100.0

4.7

82.3

SD tissue viability [%]

1.8

0.3

2.6

CV [% viability]

1.8

6.2

3.2

* Blank-corrected mean OD570 nm of the negative control corresponds to 100% absolute tissue viability.

Table 2: Results of the NSCliving control

NSCliving

TVT

Negative Control

Tissue

1

2

1

2

3

 

Absolute OD570

0.050

0.051

1.765

1.845

1.763

0.051

0.051

1.773

1.807

1.778

 

OD570(Blank Corrected)

0.005

0.006

1.720

1.800

1.718

0.007

0.006

1.729

1.762

1.734

Mean OD570 of the Duplicates (Blank Corrected)

0.006

0.006

1.725

1.781

1.726

Total Mean OD570 of the 2 or 3 Replicate Tissues (Blank Corrected)

0.006

1.744

SD of Mean OD570 of the Duplicates (Blank Corrected)

0.000

0.032

NSCliving[%]

0.3

-

Relative Tissue Viability [%]

-

98.9

102.1

99.0

Mean Relative Tissue Viability [%]

-

100.0

SD of Relative Tissue Viability [%]

-

1.8

CV [% Viabilities]

-

1.8

TVT: additional test item treated living tissue without MTTstaining

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
CLP: not irritating
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Mar - 07 Mar 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP- guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS (SPF-quality)
- Source: Broekman Institute, Someren, The Netherlands
- Age at study initiation: approx. 14 weeks
- Weight at study initiation: 2150 - 2537 grams
- Housing: Individually in labelled cages with perforated floors and equipped with an automatic drinking system
- Diet (ad libitum): standard laboratory rabbit diet (LKK-20, pellet diameter 4mm, Hope Farms, Woerden, The Netherlands) approx. 100 gram per day.
- Water (ad libitum): tap-water diluted with decalcified water
- Acclimation period: at least 5 days before start of treatment under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
other: the contralateral flank was similarly prepared (but without test substance) to act as a procedural control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): 100%

Duration of treatment / exposure:
4 h
Observation period:
72 h
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: flank, 2x3 cm
- Type of wrap if used: surgical gauze patch mounted on Micropore tape (3M, St. Paul, USA)
The dressing was wrapped around the abdomen and secured with an elastic bandage (Coban, 3M, St. Paul, USA)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): using a tissue moistened with tap-water and subsequently a dry trissue
- Time after start of exposure: 4 hours

SCORING SYSTEM: according to Draize et al., 1944
Irritation parameter:
erythema score
Basis:
animal: #1 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Remarks on result:
other: brown staining of the treated skin by the test substance
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
edema score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility not applicable
Irritant / corrosive response data:
The observed skin irritation consisted of very slight oedema (score 1) in two animals and very slight erythema (score 1) in all three animals. The skin irritation had resolved within 24 hours after exposure in two animals and within 48 hours in the third animal.
There was no evidence of a corrosive effect on the skin.
Other effects:
Brown staining of the treated skin by the test substance was observed in two animals on day 1.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occured.
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
CLP: not irritating
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:
key study
Study period:
01 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
adopted 09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 28 Jun 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported on ice in Hank's Buffered Salt Solution (HBSS) containing penicillin/streptomycin.
- Time interval prior to initiating testing: On the test day, fresh eyes were collected from the slaughter house. The corneae were prepared immediately after delivery.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for visible defects and defective eyes were discarded.
- Indication of any antibiotics used: penicillin/streptomycin containing HBSS was used for transport.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 0.9% NaCl solution in deionised water
- Lot no: 18095403

POSITIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 100%
- Lot no.: K50117583
Duration of treatment / exposure:
10 ± 1 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
2 h at 32 ± 1 °C
Number of animals or in vitro replicates:
triplicates for each treatment and control groups
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing Hanks’ balanced salt solution (HBSS). A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
After an equilibration period, the initial opacity was determined. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.

TREATMENT METHOD: Closed chamber method
The corneas were mounted in a corneal holder with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. Starting with the posterior chamber, both chambers of the corneal holder were then filled with complete RPMI 1640 medium and the corneas were incubated for 1 h at 32 ± 1 °C. After the equilibration period an initial illuminescence reading the test item, the positive or the negative control was introduced into the anterior chamber (closed chamber method). The corneas were incubated at 32 ± 1 °C and exposed to the appropriate test substance for 10 ± 1 min.

REMOVAL OF TEST SUBSTANCE
After the incubation either the test item or the control substance was removed and the epithelium was washed at least three times with minimum essential medium (MEM) (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI medium (without phenol red).

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH). The light was measured as illuminance (I = luminous flux per area, unit: lux).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Jenway 6405 UV/VIS) at 490 nm (OD 490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was considered as severe irritant/corrosive and labelled Category 1 according to CLP/EPA/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.
Irritation parameter:
in vitro irritation score
Remarks:
mean value of 3 corneae
Run / experiment:
10 min exposure
Value:
1.31
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the test system was demonstrated with 12 substances according to OECD 437.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS value which fell within two standard deviations of the current historical mean.



Table 1: Results on opacity

Cornea No. Test Item Initial Opacity Final Opacity Change of Corrected Opacity Value Opacity Value
1 Negative 1.92 2.24 0.32  
2 1.96 2.13 0.18  
3 1.96 2.24 0.29  
MV 1.94 2.21 0.26  
4 Positive 2.86 26.48 23.62 23.35
5 2.9 29.68 26.78 26.52
6 3.42 31.81 28.39 28.13
MV 3.06 29.32 26.26 26
7 Test Item 1.85 2.79 0.94 0.68
8 1.85 2.1 0.25 -0.01
9 2.39 5.66 3.27 3.01
MV 2.03 3.51 1.49 1.22

MV= mean value

Table 2: Results on permeability

Cornea No. Test Item OD490 Corrected OD490 Value
1 Negative 0.004
2 0.019
3 0.015
MV 0.013
4 Positive 1.262 1.249
5 1.585 1.572
6 1.289 1.276
MV 1.379 1.366
7 Test Item 0.018 0.005
8 0.027 0.014
9 0.01 -0.003
MV 0.018 0.006

MV= mean value

Table 3: Results in vitro irritation score (IVIS)

Cornea No. Test Item Corrected Opacity Corrected
OD490 Value
IVIS
1 Negative 0.32 0.004  
2 0.18 0.019  
3 0.29 0.015  
MV 0.26 0.013 0.45
4 Positive 23.35 1.249  
5 26.52 1.572  
6 28.13 1.276  
MV 26 1.366 46.49
7 Test Item 0.68 0.005  
8 -0.01 0.014  
9 3.01 -0.003  
MV 1.22 0.006 1.31

MV= mean value

Table 4: Historical mean In Vitro Irritation Score of the positive control

  IVIS Positive Control - Ethanol 100 %
Mean Value (MV)  48.41
Standard Deviation (SD) 9.25
MV- 2xSD  29.91
MV+2xSD 66.91
Number of Replicates providing Historical Mean: 56

Positive controls are updated after every single experiment or at least every 3 months.

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) 1272/2008.
Conclusions:
CLP: not classified
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jul - 06 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
adopted 25 Jun 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: sterilization at 121 °C for 20 min to avoid contaminations of the cell culture.
Species:
human
Strain:
other: EpiOcularTM, reconstructed three-dimensional human corneal epithelium
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL (83.3 µL / cm²)

NEGATIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: RNBG3520

POSITIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: S6943111
Duration of treatment / exposure:
30 ± 2 min at 37 ± 2 °C
Duration of post- treatment incubation (in vitro):
120 ± 15 min at 37 ± 2 °C
Number of animals or in vitro replicates:
2 tissues for each treatment and control group
Details on study design:
DETAILS OF THE TEST PROCEDURE USED
The EpiOcular™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

RHCE TISSUE CONSTRUCT
- Model used: OCL-200-EIT (MatTek Corporation, Bratislava, Slovakia)
- Tissue Batch number: 27062 (main experiment), 27066 (viable tissue controls), 27027 (killed tissue control negative control) and 27019 (killed tissue control test item treated)

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiOcular™ tissues was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was in the accepted range of 1.0 - 3.0.
- Barrier function: The barrier function was evaluated by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon treatment with 100 µL of 0.3% Triton X-100. The ET-50 value fits to the acceptance criteria of 12.2 – 37.5 min.
- Contamination: The cells used to produce the EpiOcularTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected. Tissue sterility was confirmed by long-term incubation with antibiotic and antimycotic free culture.

DURATION AND TEMPERATURE
- EXPOSURE: 30 ± 2 min at 37 ± 2 °C
- POST-EXPOSURE IMMERSION: 12 ± 2 min at room temperature
- POST-EXPOSURE INCUBATION: 120 ± 15 min at 37 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS: The tissues were extensively rinsed with Dulbecco's phosphate buffered saline (DPBS).

INDICATION OF CONTROLS USED FOR DIRECT MTT-REDUCERS AND/OR COLOURING TEST CHEMICALS
The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour turned blue/purple, the test substance is presumed to have reduced the MTT. Therefore, an additional test using freeze-killed tissues was performed. A second pre-test revealed that the test item was also colour interfering upon mixing with water. Therefore coloured tissue controls were performed using two additional viable tissues. Moreover, since the test item showed non-specific colouring of living tissues, a third control for non-specific tissue colour in killed tissues was performed to avoid a possible double-correction for colour interference. The true tissue viability was then calculated as the percent living tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
True Tissue Viability = [%] mean Tissue viability - NSMTT - NSCliving + NSCkilled
NSMTT: non-specific reduction of MTT
NSCliving: non-specific colour of additional viable tissues
NSCkilled: non-specific colour of additional killed tissues
- No. of replicates: 2 tissues (killed/viable) for each test item and controls for determination of NSMTT, NSCliving and NSCkilled.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min at 37 ± 2 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter band pass: ± 30nm

EVALUATION CRITERIA
The test substance was considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.

TEST ACCEPTANCE CRITERIA
- Negative control: The mean absolute OD 570 nm is between 0.8 and 2.5.
- Positive control: The mean relative tissue viability is < 50% of the negative control viability.
- Relative tissue viability difference of replicate tissues is < 20%.

REFERENCE TO HISTORICAL DATA OF THE RHCE TISSUE CONSTRUCT: Please refer to Table 1
Irritation parameter:
other: % tissue viability mean value of 2 tissues
Run / experiment:
30 min exposure
Value:
93.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
tissue viability was corrected for non-specific reduction of MTT and non-specific colour interference
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 (1.961).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was <50% compared to the negative control (31.1%).
- Relative tissue viability difference of replicate tissues was < 20% (values between 1.5 and 9.7).

Table 2: Results of the test item and controls

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570 values

1.950

1.957

0.553

0.737

1.796

1.894

1.944

1.995

0.543

0.732

1.819

1.931

OD570 values (blank-corrected)

1.905

1.911

0.508

0.692

1.751

1.849

1.899

1.950

0.498

0.687

1.774

1.886

mean of the duplicates

1.902

1.931

0.503

0.689

1.762

1.868

mean OD

1.916*

0.596

1.815

TODTT- NSMTT

-

-

1.791

TODTTNSMTT and NSCliving

-

-

1.786

tissue viability [%]

99.2

100.8

26.3

36.0

92.0

97.5

relative tissue viability difference [%]

1.5

9.7

5.5

mean tissue viability [%]

100.0

31.1

94.7

mean tissue viability [%]

- NSMTT corrected

-

-

93.4

mean tissue viability [%]

- NSMTT and NSCliving corrected

 

-

 

-

 

93.1

True Tissue Viability

-

-

93.4

* Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

OD: optical density

NSMTT: non-specific reduction of MTT

NSC living: non-specific color of additional viable tissues

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not irritating
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jul - 06 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
adopted 25 Jun 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 24 Jan 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: sterilization at 121 °C for 20 min to avoid contaminations of the cell culture
Species:
human
Strain:
other: EpiOcularTM, reconstructed three-dimensional human corneal epithelium
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL (83.3 µL / cm²)

NEGATIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: RNBG3520

POSITIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: S6943111
Duration of treatment / exposure:
30 ± 2 min at 37 ± 2 °C
Duration of post- treatment incubation (in vitro):
120 ± 15 min at 37 ± 2 °C
Number of animals or in vitro replicates:
2 tissues for each treatment and control group
Details on study design:
DETAILS OF THE TEST PROCEDURE USED
The EpiOcular™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

RHCE TISSUE CONSTRUCT
- Model used: OCL-200-EIT (MatTek Corporation, Bratislava, Slovakia)
- Tissue Batch number: 27062 (main experiment), 27066 (viable tissue controls), 27027 (killed tissue controls)

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiOcular™ tissues was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was in the accepted range of 1.0 - 3.0.
- Barrier function: The barrier function was evaluated by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon treatment with 100 µL of 0.3% Triton X-100. The ET-50 value fits to the acceptance criteria of 12.2 – 37.5 min.
- Contamination: The cells used to produce the EpiOcularTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected. Tissue sterility was confirmed by long-term incubation with antibiotic and antimycotic free culture.

DURATION AND TEMPERATURE
- EXPOSURE: 30 ± 2 min at 37 ± 2 °C
- POST-EXPOSURE IMMERSION: 12 ± 2 min at room temperature
- POST-EXPOSURE INCUBATION: 120 ± 15 min at 37 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS: The tissues were extensively rinsed with Dulbecco's phosphate buffered saline (DPBS).

INDICATION OF CONTROLS USED FOR DIRECT MTT-REDUCERS AND/OR COLOURING TEST CHEMICALS
The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour turned black, the test substance is presumed to have reduced the MTT. Therefore, an additional test using freeze-killed tissues was performed. A second pre-test revealed that the test item was also colour interfering upon mixing with water. Therefore coloured tissue controls were performed using two additional viable tissues. Moreover, since the test item showed non-specific colouring of living tissues, a third control for non-specific tissue colour in killed tissues was performed to avoid a possible double-correction for colour interference. The true tissue viability was then calculated as the percent living tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
True Tissue Viability = [%] mean Tissue viability - NSMTT - NSCliving + NSCkilled
NSMTT: non-specific reduction of MTT
NSCliving: non-specific colour of additional viable tissues
NSCkilled: non-specific colour of additional killed tissues
- No. of replicates: 2 tissues (killed/viable) for each test item and controls for determination of NSMTT, NSCliving and NSCkilled.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min at 37 ± 2 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter band pass: ± 30nm

EVALUATION CRITERIA
The test substance was considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.

TEST ACCEPTANCE CRITERIA
- Negative control: The mean absolute OD 570 nm is between 0.8 and 2.5.
- Positive control: The mean relative tissue viability is < 50% of the negative control viability.
- Relative tissue viability difference of replicate tissues is < 20%.

REFERENCE TO HISTORICAL DATA OF THE RHCE TISSUE CONSTRUCT: Please refer to Table 1
Irritation parameter:
other: % tissue viability mean value of 2 tissues
Run / experiment:
30 min exposure
Value:
94.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
tissue viability was corrected for non-specific reduction of MTT and non-specific colour interference
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 (1.961).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was <50% compared to the negative control (31.1%).
- Relative tissue viability difference of replicate tissues was < 20% (values between 1.5 and 9.7).

Table 2: Results of the test item and controls

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570 values

1.950

1.957

0.553

0.737

1.890

1.820

1.944

1.995

0.543

0.732

1.893

1.875

OD570 values (blank-corrected)

1.905

1.911

0.508

0.692

1.845

1.775

1.899

1.950

0.498

0.687

1.848

1.830

mean of the duplicates

1.902

1.931

0.503

0.689

1.847

1.803

mean OD

1.916*

0.596

1.825

TODTT- NSMTT

-

-

1.809

TODTTNSMTT and NSCliving

-

-

1.804

tissue viability [%]

99.2

100.8

26.3

36.0

96.4

94.1

relative tissue viability difference [%]

1.5

9.7

2.3

mean tissue viability [%]

100.0

31.1

95.2

mean tissue viability [%]

- NSMTT corrected

-

-

94.3

mean tissue viability [%]

- NSMTT and NSClivingcorrected

 

-

 

-

 

94.0

True Tissue Viability

-

-

94.2

* Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

OD: optical density

NSMTT: non-specific reduction of MTT

NSC living: non-specific color of additional viable tissues

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not irritating
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jul - 05 Aug 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan, Belton, Leics, England and Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: at least 6 weeks old
- Weight at study initiation: body weights were at least 1.0 kg.
- Housing: labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany,
dimensions 40 x 32 x 23 cm).
- Diet (e.g. ad libitum): Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100
grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least three times a week.
- Water (e.g. ad libitum): tap water.
- Acclimatization period: at least 5 days before start of treatment under laboratory conditions.
A health inspection was performed prior to commencement of treatment, to ensure that the animals were in a good state of health.
Special attention was paid to the eyes, which were free from any abnormality.
Results of analysis for diet (nutrients and contaminants), hay and water were assessed and did not reveal any findings that were
considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.
Animal specifications (sex, age and body weight) are specified in the attached table.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 – 20.0
- Humidity (%): 50 - 80
Temporary deviations from the maximum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN LIFE DATES: 19 Jul - 05 Aug 2010
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 0.1 mL.
Duration of treatment / exposure:
Single instillation on Day 1.
Observation period (in vivo):
1, 24, 48 and 72 h post-instillation
Number of animals or in vitro replicates:
3
Details on study design:
STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 2 weeks later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Each animal was treated by instillation of 0.1 mL of the test substance in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.

Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing (if done): No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation.
- Necropsy: No necropsy was performed according to protocol.
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours \\\and 7, 14 and 21 days after instillation of the test
substance.
The irritation scores and a description of all other (local) effects were recorded.The irritation was assessed according to the following
numerical scoring system. At each observation, the highest scores given were recorded:

CORNEAL IRRITATION
Opacity: degree of density (area most dense taken for reading)
0: No ulceration or opacity (may include slight dulling of normal luster)
1: Scattered or diffuse areas of opacity, details of iris clearly visible
2: Easily discernible translucent area, details of iris slightly obscured
3: Nacreous area, no details of iris visible, size of pupil barely discernible
4: Opaque cornea, iris not discernible through the opacity
Area of cornea involved:
0: No ulceration or opacity
1: One quarter or less but not zero
2: Greater than one quarter, but less than half
3: Greater than half, but less than three quarters
4: Greater than three quarters, up to whole area

IRIS
0: Normal
1: Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination thereof,
iris still reacting to light (sluggish reaction is positive)
2: No reaction to light, hemorrhage, gross destruction (any or all of these)
CONJUNCTIVAL IRRITATION
Redness (refers to palpebrae and sclera, excluding cornea and iris):
0: Blood vessels normal
1: Some blood vessels definitely hyperaemic (injected)
2: Diffuse, crimson color, individual vessels not easily discernible
3: Diffuse beefy red
Chemosis (refers to lids and/or nictitating membranes):
0: No swelling
1: Any swelling above normal (includes nictitating membranes)
2: Obvious swelling with partial eversion of lids
3: Swelling with lids about half closed
4: Swelling with lids more than half closed
Discharge:
0: No discharge (may include small amounts observed in inner canthus of normal animals)
1: Any amount different from normal and/or lacrimation
2: Discharge with moistening of the lids and hairs just adjacent to lids
3: Discharge with moistening of the lids and hairs (considerable area around the eye)

Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic
examination lamp.
In cases of equivocal results when comparing the treated and untreated eyes, the illustrated guide from the Consumer Product Safety
Commission, Washington, D.C. 20207 was used for additional control purposes.
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Instillation of 0.1 mL of Vinasses into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The latter two findings were only present at 1 hour after instillation in two animals. The irritation completely resolved within 48 hours in all animals.

No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Coloration / Remnants

Brown staining of the fur on the head and paws, caused by the test substance, was noted during the observation period.

Toxicity / Mortality

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual eye irritation scores are specified in the attached table.

Interpretation of results:
not irritating
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Two in vivo studies and one in vitro study on skin irritation are available for Vinasses, residue of fermentation.

In vivo

Vinasses, residue of fermentation were tested for acute dermal irritation/corrosion in two in vivo studies according to OECD guideline 404 and complying with GLP (Beerens-Heijnen, 2005; Pels Rijcken, 1992). In each study, 3 albino rabbits were exposed to 0.5 mL of the undiluted test material, applied onto the clipped or shaved skin for 4 h using a semi-occlusive dressing. The treated skin was observed for reactions after patch removal and evaluations were made at 1, 24, 48 and 72 h post-application.

In one study, very slight erythema was observed at the treated skin areas of all 3 animals at 1 h post-application. This effect was fully reversible within 24 hours post-application in all animals. No oedema was observed (Beerens-Heijnen, 2005).

In the second study, the observed skin reaction consisted of very slight oedema in 2 animals and very slight erythema in all 3 animals at 1 h post-application. These effects were fully reversible within 24 h post-application in 2 animals and within 48 h in the third animal (Pels Rijcken, 1992).

There was no evidence of an irritating/corrosive effect of the test materials on the skin and no other signs of intoxication were seen. The mean erythema and edema scores over 24, 48 and 72 h were equal to 0 for all 3 animals in both studies.

In vitro

The skin irritation potential of the test substance was determined in vitro by using the skin irritation test with reconstructed human epidermis according to OECD Guideline 439 and in compliance with GLP (The Ethanol REACH Association, 2018). Pre-tests revealed that the test item was non-MTT-reducing but colour-interfering with water. However, the non-specific colour of additional viable tissues was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

In the main study 30 μL each of the undiluted test substance, the negative control (DPBS) and the positive control (5% SDS) were applied topically to the surface of the EpiDermTM tissues for 60 min (35 min at 37 °C, 25 min at room temperature). After the exposure and 42 h post-incubation period, cytotoxicity was evaluated by MTT reduction.

Compared to the relative absorbance value of the negative control, the mean relative absorbance value of the test item was reduced to 82.3% after 60 min of exposure. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is considered as non-irritant to skin. The controls confirmed the validity of the study.

Conclusion skin irritation:

Based on the results of two in vivo studies in rabbits and one in vitro study using the EpiDermTM standard model, Vinasses, residue of fermentation has no skin irritating potential.

Eye irritation

One in vivo study and three in vitro tests on eye irritation are available for Vinasses, residue of fermentation.

In vivo

Vinasses, residue of fermentation were tested for eye irritation/corrosion in albino rabbits in a study performed according to the OECD guideline 405 and complying with GLP (van Otterdijk, 2010b). The test material (0.1 mL) was applied into the conjunctival sac of one eye, the other eye serving as control. The eyes were examined and scored 1, 24, 48 and 72 h after application. Initially, one animal was tested only, in which no corrosive effects were observed. The negative response was confirmed using two additional animals. Irritation of the conjunctivae, which consisted of redness, chemosis and discharge, was observed at 1 h post- application. The latter two findings were only present in two animals. These effects were fully reversible within 48 h post-instillation in all animals. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 h post-instillation revealed no corneal epithelial damage. The average cornea, iris, and chemosis scores over 24, 48, and 72 h were all 0 for all animals. The mean conjunctivae score over 24, 48 and 72 h was 0.3 for each of the 3 animals.

In vitro

The eye irritating potential of Vinasses, residue of fermentation were investigated in three in vitro studies, one bovine corneal opacity and permeability (BCOP) test and two assays with reconstructed human cornea-line epithelium (RhCE).

The BCOP test was conducted according to OECD guideline 437 and in compliance with GLP (The Ethanol REACH association, 2018). After an initial reading of the basal corneal opacity, 750 µL of undiluted test substance, negative control (physiological saline) and positive control (ethanol) was applied to the epithelial surface of each three corneas. After 10 min of incubation the substances were rinsed off and the corneas were post-incubated for 2 h at 32 ± 1 °C. Afterwards a second opacity reading was performed. In addition, permeability of the corneas were determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation for 90 min at 32 ± 1 °C. The negative control and the positive control confirmed the validity of the test system and were in the range of the historic controls. Relative to the negative control, the calculated mean IVIS of the test substance was 1.31 and thus the test substance is not considered to be irritant to the eye.

Two further in vitro test for eye irritation were performed for two batches of Vinasses, residue of fermentation, by using the EpiOcularTM model according to OECD guideline 492 and in compliance with GLP (The Ethanol REACH association, 2018).

Each 30 μL of the undiluted test item, the negative control (distilled water) or the positive control (methyl acetate) were applied topically to the EpiOcularTM tissue for an incubation period of 30 min. After the exposure period and removal of the test substances, the tissues were immersed for 12 min post-exposure and post-incubated for 120 min. Afterwards, cytotoxic effects were determined via MTT reduction assay. Absorbance values of the negative control (OD ≥ 0.8 and ≤ 2.8) and the positive control (tissue viability < 50%) confirmed the validity of the test system.

In pre-tests the substance turned out to reduce MTT and to colour-interfere with water. The amount of colour interference and MTT-reduction was quantified using additional killed and living control tissues and the results were quantitatively corrected. Compared to the mean tissue viability of the negative control, the two batches of the test item revealed a tissue viability of 94.2% and 93.4%, respectively. Therefore, Vinasses, residue of fermentation are not considered irritating to the eyes.

Conclusion eye irritation:

Based on the results of one in vivo and three in vitro studies it can be assumed that the test substance Vinasses, residue of fermentation have no eye irritating potential.

 

Respiratory tract:

This information is not available.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.