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EC number: 208-704-1 | CAS number: 538-75-0
- Life Cycle description
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
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- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Toxicological Summary
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- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Endpoint summary
Administrative data
Description of key information
Skin irritation (in-vitro). Key Study. Test method according to OECD Guideline No. 439, GLP study. The test item is non-irritating to the skin under the test conditions.
Skin corrosion (in-vitro). Key Study. Test method according to OECD Guideline No. 430, GLP study. The test item is non-corrosive to the skin under the test conditions.
Eye irritation. Data waiving (study scientifically not necessary / other information available): According to Annex VI of CLP Regulation (EC) Nº 1272/2008, the substance is classified as: Eye Dam 1 H318.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Remarks:
- in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- April 25, 2016- April 30, 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- isolated skin discs
- Source species:
- rat
- Cell type:
- other: dorso lateral skin discs stripped of excess subcutaneous fat
- Cell source:
- other: skin obtained from two female donors
- Source strain:
- Wistar
- Details on animal used as source of test system:
- SOURCE ANIMAL
- Source: skin discs were collected from 2 female rats (outbred) obtained from the conventional husbandry of laboratory animals of the Centre for Experimental Medicine, Medical University in Katowice (reg. No. 0053)
- Sex: female
- Age at study initiation (in days): 21 days old
- Weight at study initiation: not specified
- Housing: both rats were kept in a plastic cage covered with a wire bar lid. Conditions: air temperature: 20-21ºC; relative air humidity: 28-51%; lighting cycle: 12 hours light/12 hours dark; air exhange: about 13 times/hour.
- Diet : ad libitum access to the “Murigran” (batch number 1/16) standard granulated laboratory fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz
- Water: ad libitum drinking water (tap water)
- Acclimation period: 4 days - Justification for test system used:
- OECD Guideline 430 is based on the rat skin transcutaneous electrical resistance (TER) test method, which utilizes skin discs to identify corrosives by their ability to produce a loss of normal stratum corneum integrity and barrier function.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- SKIN DISC PREPARATION
- Procedure used: dorso-lateral skin of each animal (two female Wistar rats of 29 days old) was removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin using a paper towel. Each skin disc was placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface was in contact with the tube. A rubber ‘O’ ring was press-fitted over the end of the tube to hold the skin in place and excess tissue was trimmed away. The rubber ‘O’ ring was then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube was supported by a spring clip inside a receptor chamber containing MgSO4 solution (154 mM). The skin disc should be fully submerged in the MgSO4 solution. As many as 11 skin discs with a diameter of 20-mm each were obtained from a single rat skin. Two of them were used to control the quality of the procedure, whereas the remaining nine were used for the purpose of the experiment.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs should be greater than 10 kΩ
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21-22ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1 (the test item and the control items were removed by washing with a jet of tap water at up to 30°C)
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean TER value obtained for the test item is less than or equal to 5 kΩ and the skin disk is obviously damaged.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test substance is greater than 5 kΩ or if the mean TER value is less than or equal to 5 kΩ and the skin disc is showing no obvious damage. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the test item (ground to a powder) was moistened with 150 µl distilled water and uniformly applied in a sufficient amount to ensure that the whole surface of the epidermis was covered.
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 µl of distilled water
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 µl of 10M hydrochloric acid - Duration of treatment / exposure:
- 24 hours
- Duration of post-treatment incubation (if applicable):
- not apllicable
- Number of replicates:
- three replicates obtained for each rat (two female) were used for the test item and for each control item.
- Irritation / corrosion parameter:
- transcutaneous electrical resistance (in kΩ)
- Run / experiment:
- #1 (mean value)
- Value:
- 18.26
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 14.97 kΩ
- Positive controls validity:
- valid
- Remarks:
- 0.91 kΩ
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- transcutaneous electrical resistance (in kΩ)
- Run / experiment:
- # 2 (mean value)
- Value:
- 15.12
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 16.89 kΩ
- Positive controls validity:
- valid
- Remarks:
- 0.87 kΩ
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: the gross examination showed no changes on the surface of the treated skin discs.
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Yes (study conducted from May to November 2014).
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. TER values within the acceptable range, no changes on surface of the treated skin discs.
- Acceptance criteria met for positive control: yes. TER values within acceptable range and skin discs were perforated.
- Acceptance criteria met for variability between replicate measurements: yes, the difference of TER means were below 5 ± 0,5 kΩ. - Interpretation of results:
- other: non-corrosive
- Remarks:
- EU criteria
- Conclusions:
- The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs. Therefore, the test item is non-corrosive to skin.
- Executive summary:
A transcutaneous electrical resistance test (TER) was performed on the test item according to OECD TG 430 (GLP study). Skin discs used in the experiment were obtained from two 29-day-old female Wistar rats. The test item (ground to a powder) was uniformly applied to the epidermal surface of these skin discs placed inside a tube. Positive (10M hydrochloric acid) and negative (distilled water) controls were conducted concurrently. Three skin discs obtained from each animal were used for the test item and three for each control item. The skin discs were incubated for 24 hours and kept at 21-22°C. Then, substances were removed by washing with a jet of tap water. Prior to measuring the electrical resistance, the surface tension of the skin discs was reduced by adding a sufficient volume of 70% ethanol. After removing the ethanol the tissue was hydrated by the addition of 3 ml of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin discs (in kΩ) by placing the databridge electrodes on either side of the skin disc. After the measurements, the skin discs were subjected to a gross examination in order to reveal possible damage. The mean TER results for the skin discs treated with the test item were 18.26 kΩ (animal no. 1) and 15.12 kΩ (animal no. 2), higher than the 5 kΩ cut-off value. Positive and negative control values felt within the acceptable ranges for the method. Gross examinations of the skin discs treated with the test item did not reveal any changes. On the basis of these conditions, the test item was considered to be non-corrosive to skin.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Remarks:
- in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- September 14, 2016 - September 16, 2016
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: Adult human-derived epidermal keratinocytes
- Source strain:
- other: adult human
- Details on animal used as source of test system:
- not applicable
- Justification for test system used:
- The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM model
- Tissue batch number(s): 16-EKIN-037
- Expiration date: September 19, 2016
- Validation: After receipt, the two indicators of the delivered kit were checked (for pH and temperature) in order to assess if they were in good order.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 26.4-27.2°C (room temperature)
- Temperature of post-treatment incubation (if applicable): 37ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: first they were rinsed thoroughly with PBS to remove all remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- Observable damage in the tissue due to washing: no.
- Modifications to validated SOP: no.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 hours and 10 minutes
- Spectrophotometer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018.
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD values with negative controls should be within historical range: 0.573- 1.362
- Barrier function: IC 50 determination: specification 1.5 mg/ml <=IC 50 <= 3 mg/ml. Value obtained: 1.9 mg/ml
- Morphology: well- differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: on blood of the same donors it was verified the absence of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs. On epidermal cells of the same donors it was verified the absence of bacteria, fungus and mycoplasma.
- Reproducibility: historical control data. Negative control: Mean optical density (OD): 0.802, SD 0.157, range 0.573- 1.362, n= 111. Positive control: Mean optical density (OD): 0.094, SD 0.048, range 0.032-0.354, n= 102
NUMBER OF REPLICATE TISSUES: three replicates for the test item, negative and positives controls. Two additional test item-treated tissues were used for the non-specific OD evaluation (the test item was coloured).
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- No interference found, no additional controls were necessary.
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance was considered to be irritant to skin (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation was less or equal (≤) to 50% of the negative control
- The test substance was considered to be non-irritant to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation was greater than 50% of the negative control. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg of the test substance.
VEHICLE
- no vehicle used
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl of PBS. Supplied by Sigma-Aldrich Co.; Batch number: BCBQ2925V; Expiry date: January 2020.
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl of a 5% SDS solution. Supplied by Sigma-Aldrich Co.; Batch number: MKBV0024V; Expiry date: 19 August 2017. - Duration of treatment / exposure:
- 15 minutes (± 0.5 min) at room temperature (26.4-27.2°C)
- Duration of post-treatment incubation (if applicable):
- 42 hours (± 1h) at 37ºC in an incubator with 5% CO2, in a >95% humidified atmosphere
- Number of replicates:
- three replicates for test item, negative and positive controls,
two replicates for the non-specific OD evaluation. - Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean
- Value:
- 95
- 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:
- - OTHER EFFECTS:
- Visible damage on test system: no.
- Direct-MTT reduction: no.
- Colour interference with MTT: The optical density (measured at 570 nm) of tissues were 0.016, Non-specific Colour % was calculated as 2.3%. This value was below 5%, therefore additional data calculation was not necessary.
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. Proficiency verification with 10 reference chemicals. The 5 non-irritant chemicals all gave a clearly non-irritant response and the 5 irritant chemicals all gave a clearly irritant response. (CiToxLAB Hungary Ltd., study code: 10/292-043B)
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD value was in the recommended range (0.694 - range: 0.6 to 1.5). Standard deviation of the viability results for negative control samples was 2.4 (shoul be <=18)
- Acceptance criteria met for positive control: 3.7% viabilty (within the range: 0-40%). Standard deviation of the viability results for positive control samples was 0.5 (should be <=18)
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.9 (shoul be <=18) - Interpretation of results:
- GHS criteria not met
- Remarks:
- based on GHS/CLP classification
- Conclusions:
- The test item was considered to be non-irritant to skin under the test conditions.
- Executive summary:
The skin irritation potential of the test substance was determined according to the OECD Guideline No. 439 (GLP study). This in vitro test was performed in a reconstructed human epidermis model: EPISKIN (SM). Three disks of EPISKIN were treated with the test item (20 mg), PBS as negative control and SDS as positive control and incubated for 15 minutes at room temperature. Two additional disks were used to provide an estimate of colour from the test item. Exposure to the substances was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. For each treated tissue, the viability was expressed as a % relative compared to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation was less than or equal to 50% of the negative control, the test item is considered to be irritant to skin. The mean cell viability for the test substance was determined to be 95% as compared to the negative control. All validity criteria were met, so the results indicated that the substance was not irritating to the skin.
Referenceopen allclose all
Table 1. Results of the control transcutaneous electrical resistance test (TER): skin discs accepted if TER value > 10 (kΩ)
Animal number |
Skin disc number |
TER value (kΩ) |
1 |
1 |
14.31 |
2 |
13.03 |
|
2 |
1 |
15.01 |
2 |
14.17 |
Table 2. Results of the transcutaneous electrical resistance test (TER)
Animal number |
Tested substance |
Skin disc number |
TER value (kΩ) |
Mean TER value ± SD (kΩ) |
Control ranges (kΩ) |
1 |
Positive control – 10M HCl |
1 |
0.91 |
0.91± 0.01 |
0.5 -1.0 |
2 |
0.92 |
||||
3 |
0.90 |
||||
Negative control – Distilled water |
1 |
14.78 |
14.97 ± 0.20 |
10 -25 |
|
2 |
14.95 |
||||
3 |
15.17 |
||||
Test item |
1 |
18.23 |
18.26 ± 0.32 |
|
|
2 |
17.96 |
||||
3 |
18.59 |
||||
2 |
Positive control – 10M HCl |
1 |
0.88 |
0.87± 0.01 |
0.5 -1.0 |
2 |
0.87 |
||||
3 |
0.86 |
||||
Negative control – Distilled water |
1 |
17.35 |
16.89 ± 0.52 |
10 -25 |
|
2 |
17.01 |
||||
3 |
16.32 |
||||
Test item |
1 |
15.36 |
15.12 ± 0.21 |
||
2 |
14.98 |
||||
3 |
15.01 |
Table 3. Gross changes on the surface of the treated skin discs.
Animal number |
Tested substance |
Skin disc number |
Gross changes |
1 |
Positive control – 10M HCl |
1 |
Perforation |
2 |
Perforation |
||
3 |
Perforation |
||
Negative control – Distilled water |
1 |
No changes |
|
2 |
No changes |
||
3 |
No changes |
||
Test item |
1 |
No changes |
|
2 |
No changes |
||
3 |
No changes |
||
2 |
Positive control – 10M HCl |
1 |
Perforation |
2 |
Perforation |
||
3 |
Perforation |
||
Negative control – Distilled water |
1 |
No changes |
|
2 |
No changes |
||
3 |
No changes |
||
Test item |
1 |
No changes |
|
2 |
No changes |
||
3 |
No changes |
Table 1. Optical Density (OD) and the calculated Non-Specific Colour %
Additional control |
Optical density (OD) |
NSC % |
||
|
Measured |
Blank corrected |
||
Treated with DCC |
1 |
0.059 |
0.013 |
2.3 |
2 |
0.066 |
0.019 |
||
Mean |
- |
0.016 |
Notes: Mean blank value was 0.046
Table 2. Optical Density (OD) and the calculated relative viabillity (%) of the samples
Substance |
Optical density (OD) |
Viability (% RV) |
||
|
Measured |
Blank corrected |
||
Negative Control: PBS |
1 |
0.754 |
0.707 |
102.0 |
2 |
0.722 |
0.675 |
97.4 |
|
3 |
0.745 |
0.698 |
100.7 |
|
Mean |
- |
0.694 |
100.0 |
|
Positive Control: SDS |
1 |
0.074 |
0.028 |
4.0 |
2 |
0.073 |
0.027 |
3.9 |
|
3 |
0.068 |
0.022 |
3.1 |
|
Mean |
- |
0.026 |
3.7 |
|
Test item: DCC |
1 |
0.707 |
0.660 |
95.2 |
2 |
0.732 |
0.686 |
98.9 |
|
3 |
0.678 |
0.631 |
91.0 |
|
Mean |
- |
0.659 |
95.0 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- the study need not be conducted because the substance is classified as irritating to eyes with risk of serious damage to eyes
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
No further testing on the substance is deemed necessary since there is valid data available to allow classification of the substance according to the rules laid down in Regulation (EC) no. 1272/2008 (CLP).
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irreversible damage)
Additional information
Skin irritation (in-vitro). Key Study. Test method according to OECD Guideline No. 439, GLP study. The test item was applied to EPISKIN discs (Reconstructed Human Epidermis Model Test) in order to determine the viability of the tissues after the exposure to the test item. The test item was found to be non-irritating to the skin under the test conditions.
Skin corrosion (in-vitro/ex vivo). Key Study. Test method according to OECD Guideline No. 430, GLP study. Rat skin discs were treated with the test item and the electrical resistance thereof measured (Transcutaneous Electrical Resistance Test - TER). The test item was found to be non-corrosive to the skin under the test conditions.
Eye irritation. Data waiving: the substance is classified under CLP regulation as Eye Dam 1 H318.
Justification for classification or non-classification
Based on the available data, the substance is not classified as a skin irritant or skin corrosive, but is classified as an eye irritant according to CLP Regulation no. 1272/2008. The substance is classified as Eye Dam 1 H318.
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.
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