Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 701-295-5 | CAS number: -
Endpoint summary
Administrative data
Description of key information
Based on the results observed for the in vitro Skin Irritation and Corrosion Turnkey Testing Strategy (OECD 431; OECD 439) it was concluded that the test substance shows a skin irritation potential Cat. 2 under the test conditions chosen.
Based on the results of the In Vitro Eye Irritation Turnkey Testing Strategy (OECD 437; OECD 492) it was concluded that the test substance does not show an eye irritation potential under the test conditions chosen.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Remarks:
- Turnkey Testing Strategy
- Adequacy of study:
- weight of evidence
- Study period:
- Aug 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Version / remarks:
- adopted 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland-Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: V15 Trienal-Dest. F.4-7+V16 Trienal-Dest.F4-8
- Purity: Around 92 area-% as isomeric mixture determined by GC on an RTX-1701 capillary
- Physical state / color: Liquid / colorless, clear
- Expiry date: May 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room tempreature
- Storage stability: guaranteed by the sponsor
FORM AS APPLIED IN TEST:
- The test substance was applied undiluted
OTHER SPECIFICS:
- Homogeneity: homogeneous by visual inspection
- pH value: Ca. 5 (undiluted test substance, determined in the lab prior to start of the GLP study) - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm 200
- Detailed information: surface 0.6 cm²; cultured in Millicells® ∅ 1 cm
- Date of initiation of testing: 16 Aug 2018
- Evaluation of cell viability: 23 Aug 2018
- Tissue model: EPI-200
- Tissue Lot Number: 28646 (Certificate of Analysis see Appendix)
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 h in the incubator
- Temperature of post-treatment incubation: 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing procedure: The tissues were washed with sterile PBS 3 min or 1h after start of application treatment
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: No reference filter
NUMBER OF REPLICATE TISSUES: 2
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Freezing at –20°C
- N. of replicates : 2
- Method of calculation used: Calculation of mean corrected OD570 KC by subtracting the OD570 KC of the NC from the OD570 KC of the test substance
PREDICTION MODEL / DECISION CRITERIA
- 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
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL undiluted test substance
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL deionized water
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8 N potassium hydroxide solution - Duration of treatment / exposure:
- 3 min and 1h (for treatments with an viability of ≥ 50% after 3 min)
- Number of replicates:
- 2
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL undiluted test substance
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL deionized water
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8 N potassium hydroxide solution
: - Details on study design:
- REMOVAL OF TEST SUBSTANCE
- Washing (if done): with PBS
- Time after start of exposure: 3min or 1h
OBSERVATION TIME POINTS
- 3 min and 1h
SCORING SYSTEM:
- Method of calculation: The individual tissue OD570 is calculated by subtracting the
mean blank value of the respective microtiter plate from the respective individual tissue OD570 value; KC correction was performend to assess the final relative mean viability - Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Exposure period 3min
- Value:
- 104.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Exposure period 1h
- Value:
- 101.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Direct-MTT reduction: yes; KC tissues were applied in parallel and the mean viability was corrected
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: The historical control data demonstrate the reproducibility of results and
robustness of the procedures. - Interpretation of results:
- other: no indication of skin corrosion
- Conclusions:
- No prediction can be made for skin irritation according to GHS criteria based on the results on this in vitro study alone. According to the results of the present study, the substance shows no potential of skin corrosion.
- Executive summary:
The objective was to assess the skin irritation and corrosion potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy:
The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).
In the SCT the potential of the test substance to cause dermal corrosion was assessed by a
single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).
For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each.
Due to the intense smell of the test substance, the plates were sealed with an adhesive protective foil immediately after application.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial
dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal
tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.
The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 104.2%, and it was 101.3% after an exposure period of
1 hour. Based on the results of the skin corrosion test no skin corrosion potential can be concluded.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Remarks:
- Turnkey Testing strategy
- Adequacy of study:
- weight of evidence
- Study period:
- 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:
- 28 Jul 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland-Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: V15 Trienal-Dest. F.4-7+V16 Trienal-Dest.F4-8
- Purity: Around 92 area-% as isomeric mixture determined by GC on an RTX-1701 capillary
- Physical state / color: Liquid / colorless, clear
- Storage stability: guaranteed by the sponsor
- Expiry date: May 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room tempreature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Test item preparation: The test substance was applied undiluted
OTHER SPECIFICS:
- Homogeneity: homogeneous by visual inspection
- pH value: Ca. 5 (undiluted test substance, determined in the lab prior to start of the GLP study) - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm 200
- Detailed information: surface 0.6 cm²; cultured in Millicells® ∅ 1 cm
- Date of initiation of testing: 16 Aug 2018
- Evaluation of cell viability: 24 Aug 2018
- Tissue model: EPI-200
- Tissue Lot Number: 28646 (Certificate of Analysis see Appendix)
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall
and for 35 minutes in the incubator
- Temperature of post-treatment incubation: 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing procedure: The tissues were washed with sterile PBS 1h after start of administration
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: No reference filter
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Freezing at –20°C
- N. of replicates : 3
- Method of calculation used: Calculation of mean corrected OD570 KC by subtracting the
OD570 KC of the NC from the OD570 KC of the test substance
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test chemical is identified as requiring classification and labelling according to UN GHS
(Category 2 or Category 1) if the mean percent tissue viability after exposure and posttreatment
incubation is less than or equal (≤) to 50%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL undiluted test substance
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL sterile PBS
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL SDS
- concentraton: 5% - Duration of treatment / exposure:
- 1 h
- Duration of post-treatment incubation (if applicable):
- 42 h
- Number of replicates:
- 3
- Details on study design:
-
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The tissues were washed with sterile PBS
- Time after start of exposure: 1h
OBSERVATION TIME POINTS
- 3h after MTT incubation
SCORING SYSTEM:
- Method of calculation: The mean OD570 for a test group of three tissues treated in the same way is calculated; KC correction was performend to assess the final relative mean viability - Irritation / corrosion parameter:
- % tissue viability
- Value:
- 9.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Direct-MTT reduction: Yes; KC tissues were applied in parallel; the final relative mean viability is given after KC correction
- Interpretation of results:
- other: positive indication of irritation
- Conclusions:
- No predictions can be made for skin irritation according to GHS criteria based on the results of this in vitro study alone.
In the present study, the test substance shows skin irritation potential. - Executive summary:
The objective was to assess the skin irritation and corrosion potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy:
The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).
In the SIT the potential of the test substance to cause dermal irritation was assessed by a
single topical application of 30 μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).
The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation
period. Due to the intense smell of the test substance, the plates were sealed with an adhesive protective foil immediately after application.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial
dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal
tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.
The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction
control KC (freeze-killed control tissues) was introduced. The final relative mean viability of the tissues treated with the test substance determined after
an exposure period of 1 hour with an about 42-hour post-incubation was 9.3%.
Based on the results observed and by applying the evaluation criteria it was concluded that the test substance shows a skin irritation potential in the EpiDerm™ in
vitro skin irritation and corrosion test strategy under the test conditions chosen.
Referenceopen allclose all
Tab. 2: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values,
standard deviations and coefficient of variation
Exposure period: 3 min |
|||||||
Testsubstance identification |
|
|
tissue 1 |
tissue 2 |
mean |
SD |
CV [%] |
NC |
viable tissues |
mean OD570 |
2.071 |
2.098 |
2.084 |
||
viability [% of NC] |
99.4 |
100.6 |
100.0 |
0.9 |
0.9 |
||
KC tissues |
mean OD570 |
0.146 |
0.127 |
0.137 |
|||
viability [% of NC] |
7.0 |
6.1 |
6.5 |
0.6 |
9.8 |
||
18/0072-2 |
viable tissues |
mean OD570 |
2.273 |
2.231 |
2.252 |
||
viability [% of NC] |
109.1 |
107.0 |
108.0 |
1.4 |
1.3 |
||
KC tissues |
mean OD570KC NC corrected |
0.065 |
0.098 |
0.081 |
|||
viability [% of NC] |
3.1 |
4.7 |
3.9 |
1.1 |
29.2 |
||
Final relative mean viability of tissues after KC correction [% of NC]: |
104.2 |
||||||
PC |
viable tissues |
mean OD570 |
0.167 |
0.235 |
0.201 |
||
viability [% of NC] |
8.0 |
11.3 |
9.6 |
2.3 |
23.8 |
||
Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in
parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean
viability 3.9% of NC). Thus, for the test substance, the final relative mean viability is given after KC correction.
Tab. 3: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values,
standard deviations and coefficient of variation
Exposure period: 1 h |
|||||||
Testsubstance identification |
|
|
tissue 1 |
tissue 2 |
mean |
SD |
CV [%] |
NC |
viable tissues |
mean OD570 |
2.043 |
2.084 |
2.063 |
||
viability [% of NC] |
99.0 |
101.0 |
100.0 |
1.4 |
1.4 |
||
KC tissues |
mean OD570 |
0.116 |
0.122 |
0.119 |
|||
viability [% of NC] |
5.6 |
5.9 |
5.8 |
0.2 |
3.6 |
||
18/0072-2 |
viable tissues |
mean OD570 |
2.358 |
2.155 |
2.256 |
||
viability [% of NC] |
114.3 |
104.4 |
109.3 |
7.0 |
6.4 |
||
KC tissues |
mean OD570KC NC corrected |
0.199 |
0.134 |
0.166 |
|||
viability [% of NC] |
9.6 |
6.5 |
8.1 |
2.2 |
27.9 |
||
Final relative mean viability of tissues after KC correction [% of NC]: |
101.3 |
||||||
PC |
viable tissues |
mean OD570 |
0.122 |
0.139 |
0.130 |
||
viability [% of NC] |
5.9 |
6.7 |
6.3 |
0.6 |
9.5 |
||
Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in
parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean
viability 3.9% of NC). Thus, for the test substance, the final relative mean viability is given after KC correction.
Tab. 4: Historic control data of NC and PC of corrosion test
Historic Range of NC |
|
||||
OD570 |
|||||
Exposure Time |
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Jan 2016 - Jul 2018 |
1.751 |
0.217 |
2.184 |
1.318 |
60 minutes |
Jan 2016 - Jul 2018 |
1.723 |
0.225 |
2.172 |
1.273 |
Historic Range of PC |
|
|
|
|
|
OD570 |
|
|
|
|
|
Exposure Time |
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Jan 2016 - Jul 2018 |
0.238 |
0.080 |
0.398 |
0.078 |
60 minutes |
Jan 2016 - Jul 2018 |
0.093 |
0.019 |
0.130 |
0.055 |
Relative Viability (%) |
|
|
|
|
|
Exposure Time |
Period |
Mean % |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Jan 2016 - Jul 2018 |
13.6 |
4.3 |
22.3 |
5.0 |
60 minutes |
Jan 2016 - Jul 2018 |
5.4 |
1.1 |
7.6 |
3.2 |
Tab. 2: Individual and mean OD570 values, individual and mean viability values and standard deviations
Test substance identification |
|
|
tissue 1 |
tissue 2 |
tissue 3 |
mean |
SD |
NC |
viable tissues |
mean OD570 |
2.189 |
2.172 |
2.208 |
2.189 |
|
viability [% of NC] |
100.0 |
99.2 |
100.8 |
100.0 |
0.8 |
||
KC tissues |
mean OD570 |
0.057 |
0.054 |
0.054 |
0.055 |
||
viability [% of NC] |
2.6 |
2.4 |
2.4 |
2.5 |
0.1 |
||
18/0072-2 |
viable tissues |
mean OD570 |
0.191 |
0.136 |
0.374 |
0.234 |
|
viability [% of NC] |
8.7 |
6.2 |
17.1 |
10.7 |
5.7 |
||
KC tissues |
mean OD570KC NC corrected |
0.037 |
0.028 |
0.029 |
0.031 |
||
viability [% of NC] |
1.7 |
1.3 |
1.3 |
1.4 |
0.2 |
||
Final relative mean viability of tissues after KC correction [% of NC]: |
9.3 |
||||||
PC |
viable tissues |
mean OD570 |
0.047 |
0.049 |
0.044 |
0.046 |
|
viability [% of NC] |
2.1 |
2.2 |
2.0 |
2.1 |
0.1 |
||
Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in
parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean
viability 1.4% of NC). Thus, for the test substance, the final relative mean viability is given after KC correction.
Tab.3.: Historic control data of NC and PC of irritation test
Historic Range of NC OD570 |
|
|||
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Jan 2016 - Jul 2018 |
1.840 |
0.182 |
2.205 |
1.476 |
Historic Range of PC OD570 |
|
|
|
|
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Jan 2016 - Jul 2018 |
0.052 |
0.010 |
0.072 |
0.031 |
Relative Viability (%) |
|
|
|
|
Period |
Mean % |
SD |
Mean + 2 SD |
Mean - 2 SD |
Jan 2016 - Jul 2018 |
2.8 |
0.5 |
3.8 |
1.9 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Aug 2018- Sep 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:
- 09 Oct 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: V15 Trienal-Dest. F.4-7+V16 Trienal-Dest.F4-8
- Purity: Around 92 area-% as isomeric mixture determined by GC on an RTX-1701 capillary
- Physical state / color: Liquid / colorless, clear
- Expiry date: May 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room tempreature
- Storage stability: guaranteed by the sponsor
OTHER SPECIFICS:
- Homogeneity: Homogeneous by visual inspection
- pH value: Ca. 5 (undiluted test substance, determined in the lab prior to start of the GLP study) - Species:
- cattle
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Strasse 23, 55232 Alzey, Germany
- Age of donor animals: Minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.) were selected for the experiments - 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): 750 µL undiluted test substance
- Duration of treatment / exposure:
- 10 min
- Duration of post- treatment incubation (in vitro):
- 2 h
- Number of animals or in vitro replicates:
- 3
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS
- Corneas free of defects were selected
- Dissection with a 2 to 3 mm rim of sclera
- Corneas were mounted in cornea holders consisting of 2 chambers (anterior/posterior)
- Equilibration with pre-warmed Eagle’s MEM (without phenol red) at about 32°C for at least 1 h
QUALITY CHECK OF THE ISOLATED CORNEAS
- initial corneal opacity was analyzed; any corneas that showed macroscopic tissue damage or an opacity value < 556 opacity units
were discarded
NUMBER OF REPLICATES : 3 corneas / treatment group
NEGATIVE CONTROL USED
- 750 μL deionized water
POSITIVE CONTROL USED
- PC1: 100% ethanol (750 µL)
- PC 2: 100% dimethylformamide (750 µL)
APPLICATION DOSE AND EXPOSURE TIME
- 750 μL undiluted liquid test substance
- 10 min
POST-INCUBATION PERIOD: yes
- 2 h
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least 3 times
- POST-EXPOSURE INCUBATION: 2h at about 32°C
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer analysis
- Corneal permeability: Passage of sodium fluorescein solution (4 mg/mL) was measured (after incubation for 90 ± 5 min) with the aid of spectrophotometry (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- Mean corneal opacity and permeability values of each treatment group were used for IVIS calculations
- IVIS calculation per treatment group after calculation of IVIS per cornea
DECISION CRITERIA:
- Decision criteria according to the OECD Guideline 437 for evaluation of results - Irritation parameter:
- cornea opacity score
- Value:
- 3.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- other: Permeability score
- Value:
- 0.001
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Value:
- 3.3
- 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:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes - Interpretation of results:
- other: No indication of corrosion
- Conclusions:
- The BCOP test identified the test substance as not corrosive or severe irritant based on a mean IVIS of 3.3.
- Executive summary:
The objective was to assess the eye irritating potential of the test substance. By using the methods
currently available a single in vitro assay is not sufficient to cover the full range of eye irritating
potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The
Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.
The potential of the test substance to cause ocular irritation or serious damage to the eyes was
assessed by a single topical application of 750 μL undiluted test substance to the epithelial
surface of isolated bovine corneas.Three corneas were treated with the test substance for 10 minutes followed by a 2-hour postincubation
period. In addition to the test substance, a negative control (NC; deionized water) and two positive
controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each.
Corneal opacity was quantitatively measured as the amount of light transmitted through the
cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye
that passes across the full thickness of the cornea. Both measurements were used to calculate
an In Vitro Irritancy Score of the test substance.
The mean IVIS of 3.3 of the test substance treated corneas did not indicate a corrosive or severe
eye irritation potential.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Aug 2018 - Oct 2018
- Reliability:
- 1 (reliable without restriction)
- 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:
- 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: V15 Trienal-Dest. F.4-7+V16 Trienal-Dest.F4-8
- Purity: Around 92 area-% as isomeric mixture determined by GC on an RTX-1701 capillary
- Physical state / color: Liquid / colorless, clear
- Storage stability: guaranteed by the sponsor
- Expiry date: May 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room tempreature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Test item preparation: The test substance was applied undiluted
OTHER SPECIFICS:
- Homogeneity: homogeneous by visual inspection
- pH value: Ca. 5 (undiluted test substance, determined in the lab prior to start of the GLP study) - Species:
- human
- Details on test animals or tissues and environmental conditions:
- Tissue model: OCL-200
Tissue Lot Number: 27065 (test run 1) and 27073 (test run 2)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Characteristics: Surface 0.6 cm², cultured in Millicells® ∅ 1 cm - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- other: MTT reduction control (KC)
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL undiluted liquid test substance
- Duration of treatment / exposure:
- 30 min
- Duration of post- treatment incubation (in vitro):
- 2 h
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- - RhCE tissue construct:
: OCL-200; 27065 (test run 1) and 27073 (test run 2)
- Tissue supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Doses of test chemical and control substances used : undiluted test substance
- Duration and temperature of exposure: 30 min; 37°C
- Duration of the post-exposure incubation period: 2 h; 37°C
- Indication of controls used for direct MTT-reducers: Killed control tissues were analysed in parallel
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan : After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and
the tissues were incubated in the incubator for 3 h; formazan was extracted by overnight incubation/ for at least 2 h on a plate shaker in isopropanol at room temperature
- Complete supporting information for the specific RhCE tissue construct used: The supplier demonstrates that each batch of the model
used meets the defined production release criteria.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : A chemical is considered as "non-irritant”
(no UN GHS Category) if the mean relative tissue viability with a test material is greater than 60% (according to OECD guideline 492). A “borderline“ evaluation (60 ± 5%) was determined statistically using historic BASF data and
hence considers the variance of the test method. This evaluation is an amendment to the
evaluation provided in OECD Guideline 492.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Historical data demonstrate the reproducibility of results and
robustness of the procedures
- Reference to historical data of the RhCE tissue construct
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
- Positive and negative control means and acceptance ranges based on historical data
- Acceptable variability between tissue replicates for positive and negative controls - Irritation parameter:
- other: mean tissue viability [% of NC]
- Run / experiment:
- 1st test run
- Value:
- 67.9
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- other: mean tissue viability [% of NC]
- Run / experiment:
- 2nd test run
- Value:
- 82.4
- 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:
- - After the 1st test run the final mean relative tissue viability of the test substance lies slightly
above the borderline range for evaluation. In addition, high OD570 values of the NC tissues
were noted, thus, another test run was performed to verify the results.
- Acceptance criteria met for negative control for the 1st and 2nd test run:
The negative control OD values exceed the upper acceptance limit of 2.5 for both test runs. According to a statement provided by the RhCE model developer/supplier, the upper limit has gradually increased. Thus, a request to the OECD to change the upper limit in the OECD TG 492 for the EpiOcular™ EIT model from 2.5 to 2.8 was submitted. Taking this update into account the negative control OD values of the current study are acceptable and do not influence the result of the study. - Interpretation of results:
- other: No indication of irritation
- Conclusions:
- Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. In combination with the BCOP assay, which identified the test substance as not corrosive or severe irritant, the test substance is not classified for eye irritation.
- Executive summary:
The objective was to assess the eye irritating potential of the test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating
potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.
The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).
Two test runs were performed. Two EpiOcular™ tissues per test run were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial
dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal
tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.
The following results were obtained in the EpiOcular™ eye irritation assay:
The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.
In the 1st test run the final relative mean viability of the tissues treated with the test substance compared to the negative control was 67.9%. Another test run was performed to verify the
result, because the OD570 value of the negative control tissues lies out of the acceptance range and the mean viability value of the test substance lies slightly above the borderline
range for evaluation. In the 2nd test run the final relative mean viability of the tissues treated with the test substance compared to the negative control was 82.4% and therefore well above the value for eye
irritation. The mean viability value of the negative control lies out of the acceptance range again. However, as meanwhile the acceptance range for the negative control was updated by the
developer/supplier and the results of the 2nd test run verified the results of the 1st test run, the study is considered to be valid.
Based on the results of the EpiOcular Tests and by applying the evaluation criteria, it was concluded that the test substance does not show an eye irritation potential
in the in vitro eye irritation test strategy under the test conditions chosen.
Referenceopen allclose all
Tab. 2: Opacity score of the test substance, the NC and the PC
Test substance identification |
Cornea-No. |
Initial opacity |
Final opacity |
Opacity Change |
Corrected Opacity Change |
Mean |
SD |
|
13 |
6.0 |
15.4 |
9.4 |
5.0 |
|
|
18/0072-2 |
14 |
4.7 |
9.4 |
4.7 |
0.3 |
3.2 |
2.5 |
|
15 |
2.9 |
11.4 |
8.5 |
4.2 |
|
|
|
1 |
4.6 |
8.5 |
3.9 |
NA |
|
|
NC |
2 |
4.1 |
7.9 |
3.7 |
NA |
4.3 |
0.9 |
|
3 |
5.2 |
10.6 |
5.4 |
NA |
|
|
|
4 |
4.5 |
32.7 |
28.2 |
23.9 |
|
|
PC1 |
5 |
4.4 |
34.6 |
30.1 |
25.8 |
24.8 |
1.0 |
|
6 |
4.8 |
33.9 |
29.1 |
24.8 |
|
|
|
7 |
5.6 |
110.7 |
105.0 |
100.7 |
|
|
PC2 |
8 |
4.8 |
102.4 |
97.6 |
93.3 |
102.1 |
9.6 |
|
9 |
6.3 |
123.0 |
116.7 |
112.3 |
|
|
Tab. 3: Permeability score of the test substance, the NC and the PC
Test substance identification |
Cornea- No. |
Mean OD490 |
Dilution Factor |
Mean Corrected OD490 * |
Mean |
SD |
|
13 |
0.002 |
1 |
0.000 |
|
|
18/0072-2 |
14 |
0.005 |
1 |
0.003 |
0.010 |
0.015 |
|
15 |
0.029 |
1 |
0.027 |
|
|
|
1 |
0.000 |
1 |
NA |
|
|
NC |
2 |
0.000 |
1 |
NA |
0.001 |
0.001 |
|
3 |
0.002 |
1 |
NA |
|
|
|
4 |
0.842 |
1 |
0.841 |
|
|
PC1 |
5 |
0.534 |
1 |
0.533 |
0.751 |
0.190 |
|
6 |
0.881 |
1 |
0.880 |
|
|
|
7 |
0.407 |
1 |
0.406 |
|
|
PC2 |
8 |
0.483 |
1 |
0.482 |
0.389 |
0.102 |
|
9 |
0.280 |
1 |
0.279 |
|
|
* Negative values are set to zero for further calculation
Tab. 4: In Vitro Irritancy score (IVIS) of the test substance, the NC and the PC
Test substance identification |
Cornea No. |
Opacity per cornea |
Permeability per cornea |
per cornea |
IVIS per group |
|
mean |
SD |
|||||
|
13 |
5.0 |
0.000 |
5.0 |
|
|
18/0072-2 |
14 |
0.3 |
0.003 |
0.4 |
3.3 |
2.6 |
|
15 |
4.2 |
0.027 |
4.6 |
|
|
|
1 |
3.9 |
0.001 |
3.9 |
|
|
NC |
2 |
3.7 |
0.001 |
3.8 |
4.4 |
0.9 |
|
3 |
5.4 |
0.002 |
5.4 |
|
|
|
4 |
23.9 |
0.841 |
36.5 |
|
|
PC1 |
5 |
25.8 |
0.533 |
33.8 |
36.1 |
2.1 |
|
6 |
24.8 |
0.880 |
38.0 |
|
|
|
7 |
100.7 |
0.406 |
106.8 |
|
|
PC2 |
8 |
93.3 |
0.482 |
100.5 |
107.9 |
8.1 |
|
9 |
112.3 |
0.279 |
116.5 |
|
|
HISTORICAL CONTROL DATA
Tab. 5: Historic range of NC (protocol for liquids and surfactants)
Historic period: Jan 2015 - Jul 2018 (no. of studies performed: 22)
Opacity |
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
5.1 |
1.8 |
8.8 |
1.4 |
Permeability (OD490) |
Mean OD |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
0.004 |
0.003 |
0.009 |
-0.002 |
Tab.6: Historic range of PC1 (100% ethanol)
Historic period: Jan 2015 - Jul 2018 (no. of studies performed: 21)
Opacity |
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
24.4 |
4.2 |
32.8 |
16 |
Permeability (OD490) |
Mean OD |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
0.821 |
0.17 |
1.162 |
0.481 |
In Vitro Irritation Score (IVIS) |
Mean OD |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
36.7 |
5.1 |
47.0 |
26.5 |
Tab. 7: Historic range of PC2 (100% dimethylformamide)
Historic period: May 2015 - Jul 2018 (no. of studies performed: 18)
Opacity |
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
92.3 |
8.0 |
108.3 |
76.4 |
Permeability (OD490) |
Mean OD |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
0.577 |
0.184 |
0.944 |
0.209 |
In Vitro Irritation Score (IVIS) |
Mean OD |
SD |
Mean + 2 SD |
Mean – 2 SD |
|
101.0 |
8.2 |
117.4 |
84.5 |
Tab. 2: 1st test run: Individual and mean OD570 values, individual and mean viability values and intertissue variability
Test substance identification |
|
|
tissue 1 |
tissue 2 |
mean |
Inter-tissue variability[%] |
NC |
viable tissues |
mean OD570 |
2.596 |
2.598 |
2.597 |
|
viability [% of NC] |
100.0 |
100.0 |
100.0 |
0.1 |
||
KC tissues |
mean OD570 |
0.044 |
0.041 |
0.042 |
||
viability [% of NC] |
1.7 |
1.6 |
1.6 |
0.1 |
||
18/0072-2 |
viable tissues |
mean OD570 |
1.717 |
1.945 |
1.831 |
|
viability [% of NC] |
66.1 |
74.9 |
70.5 |
8.8 |
||
KC tissues |
mean OD570 KC NC corrected |
0.080 |
0.058 |
0.069 |
||
viability [% of NC] |
3.1 |
2.2 |
2.6 |
0.8 |
||
Final relative mean viability of tissues after KC correction [% of NC]: |
67.9 |
|||||
PC |
viable tissues |
mean OD570 |
0.772 |
0.769 |
0.771 |
|
viability [% of NC] |
29.7 |
29.6 |
29.7 |
0.1 |
||
Tab. 3: 2nd test run: Individual and mean OD570 values, individual and mean viability values and intertissue variability
Test substance identification |
|
|
tissue 1 |
tissue 2 |
mean |
Inter-tissue variability [%] |
NC |
viable tissues |
mean OD570 |
2.699 |
2.728 |
2.713 |
|
viability [% of NC] |
99.5 |
100.5 |
100.0 |
1.1 |
||
KC tissues |
mean OD570 |
0.040 |
0.041 |
0.040 |
||
viability [% of NC] |
1.5 |
1.5 |
1.5 |
0.1 |
||
18/0072-2 |
viable tissues |
mean OD570 |
2.201 |
2.351 |
2.276 |
|
viability [% of NC] |
81.1 |
86.6 |
83.9 |
5.5 |
||
KC tissues |
mean OD570 KC NC corrected |
0.041 |
0.037 |
0.039 |
||
viability [% of NC] |
1.5 |
1.4 |
1.4 |
0.1 |
||
Final relative mean viability of tissues after KC correction [% of NC]: |
82.4 |
|||||
PC |
viable tissues |
mean OD570 |
0.762 |
0.628 |
0.695 |
|
viability [% of NC] |
28.1 |
23.1 |
25.6 |
5.0 |
||
HISTORIC CONTROL DATA
Tab. 4: Historic range of NC
OD570
Protocol |
Period * |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Protocol for liquids |
Feb 2016 - Aug 2018 |
1.885 |
0.415 |
2.715 |
1.055 |
Protocol for solids |
Feb 2016 - Aug 2018 |
1.787 |
0.343 |
2.473 |
1.100 |
* current results not included
Tab. 5: Historic range of PC
OD570
Protocol |
Period* |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Protocol for liquids |
Feb 2016 - Aug 2018 |
0.542 |
0.170 |
0.882 |
0.202 |
Protocol for solids |
Feb 2016 - Aug 2018 |
0.325 |
0.109 |
0.544 |
0.106 |
* current results not included
Relative viability [%]
Protocol |
Period* |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Protocol for liquids |
Feb 2016 - Aug 2018 |
28.9 |
7.5 |
43.8 |
14.0 |
Protocol for solids |
Feb 2016 - Aug 2018 |
18.2 |
5.3 |
28.7 |
7.6 |
* current results not included
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
SKIN IRRITATION/ CORROSION
The objective was to assess the skin irritation and corrosion potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).
In the SCT the potential of the test substance to cause dermal corrosion was assessed by a
single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).
For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. Due to the intense smell of the test substance, the plates were sealed with an adhesive protective foil immediately after application. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 104.2%, and it was 101.3% after an exposure period of
1 hour. Based on the results of the skin corrosion test no skin corrosion potential can be concluded.
In the SIT the potential of the test substance to cause dermal irritation was assessed by a single topical application of 30 μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. Due to the intense smell of the test substance, the plates were sealed with an adhesive protective foil immediately after application. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.
The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 9.3%.
Based on the results observed and by applying the evaluation criteria it was concluded that the test substance shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
EYE IRRITATION
Two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.
BCOP Test
The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hour postincubation period. In addition to the test substance, a negative control (NC; deionized water) and two positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.
The mean IVIS (3.3) of the test substance treated corneas did not indicate a corrosive or severe eye irritation potential.
EpiOcular
The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).
Two test runs were performed. Two EpiOcular™ tissues per test run were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular™ eye irritation assay: The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction
control (freeze-killed control tissues (KC)) was introduced. In the 1st test run the final relative mean viability of the tissues treated with the test substance compared to the negative control was 67.9%. Another test run was performed to verify the result, because the OD570 value of the negative control tissues lies out of the acceptance range and the mean viability value of the test substance lies slightly above the borderline range for evaluation. In the 2nd test run the final relative mean viability of the tissues treated with the test substance compared to the negative control was 82.4% and therefore well above the value for eye irritation. The mean viability value of the negative control lies out of the acceptance range again. However, as meanwhile the acceptance range for the negative control was updated by the developer/supplier and the results of the 2nd test run verified the results of the 1st test run, the study is considered to be valid.
Based on the results of the BCOP and EpiOcular, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.
Justification for classification or non-classification
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. A GLP-compliant OECD 431 and OECD 439 study is available for the skin irritation test strategy. A GLP-compliant OECD 437 (BCOP test) and OECD 492 study (EpiOcular) is available for the eye irritation test strategy.
In the in vitro EpiDerm™ skin irritation test (OECD 431 and 439), the substance showed no skin corrosion potential, however by applying the evaluation criteria it was concluded that the test substance showed a skin irritation potential. As a result, the test the substance is considered to be classified for skin irritation Cat. 2 under Regulation (EC) No. 1272/2008.
In both studies of the in vitro eye irritation test strategy, the scores for the test item treated tissues were below the thresholds for classification as an irritant. As a result, the substance is not considered to be classified for eye irritation under Regulation (EC) No. 1272/2008
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
