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Administrative data

Description of key information

In an in vitro skin corrosion assay according to OECD Guideline 431, the test item showed a skin corrosive potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2017-08-09 to 2017-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2016-07-29
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”
Version / remarks:
2008-05-31
Deviations:
no
Qualifier:
according to
Guideline:
other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test”
Version / remarks:
December 2011 / February 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch No.: D1608206

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM
- Tissue batch numbers: 17-EKIN-032; used for first experiment (4 h exposure) / 17-EKIN-035; used for additional experiment (4 h, 1 h and 3 min exposure)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature

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

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

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: distilled water
- No of replicates: 2 killed treated tissues and 2 killed negative control tissues were used for the MTT evaluation

NUMBER OF INDEPENDENT TEST SEQUENCES / 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 viability after 3 minutes exposure is less than 35 %, or if the viability after 3 minutes exposure is greater than or equal to 35 % and the viability after 1 hour exposure is less than 35% or the mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure
- The test substance is considered to be non-corrosive to skin if the mean tissue viability after 4 h exposure is greater than or equal to 35 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
3 min, 1 h or 4 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
90
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h
Value:
51
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
4 h
Value:
13
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined to be (0%)*. in first experiment and was determined to be 15.095 % at 4 hours exposure, 23.915 % at 1 hour exposure and 6.248 % at 3 minutes exposure in the additional experiment. As the NSMTT were below 50 % the true MTT metabolic conversion in all occasions and the correction of viability percentages were undertaken.
* The calculated NSMTT was -4.151%. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).

- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is yellowish and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

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

Table 1: Result of the first experiment

Test Substance

Optical Density (OD)

Viability (%)

Δ%

Negative Control

1

0.962

107

13

2

0.845

93

mean

0.903

100

 

Positive Control

1

0.060

7

4

2

0.023

2

mean

0.041

5

 

Test Item

1

0.250

28

5

2

0.203

22

mean

0.226

25

 

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

Table 2: Result of the second experiment

Test Substance

Optical Density (OD)

TODTT

Viability (%)

Relative Viability (%)

Δ%

Negative Control, 4 h

1

1.160

-

99

-

3

2

1.194

-

101

-

mean

1.177

-

100

-

 

Negative Control, 1 h

1

0.9.23

-

96

-

8

2

1.003

-

104

-

mean

0.963

-

100

-

 

Negative Control, 3 min

1

0.982

-

96

-

8

2

1.061

-

104

-

mean

1.021

-

100

-

 

Positive Control, 4 h

1

0.031

-

3

-

0

2

0.035

-

3

-

mean

0.033

-

3

-

 

Test Item, 4 h

1

0.320

0.143

27

12

2

2

0.347

0.169

29

14

mean

0.334

0.156

28

13

 

Test Item, 1 h

1

0.677

0.447

70

46

9

2

0.762

0.531

79

55

mean

0.720

0.489

75

51

 

Test Item, 3 min

1

1.029

0.965

101

95

9

2

0.938

0.875

92

86

mean

0.984

0.920

96

90

 

TODTT: true MTT metabolic conversion

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

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Remarks:
Optional Sub- categories 1B and 1C
Conclusions:
In an in vitro skin corrosion assay according to OECD Guideline 431, the test item showed a skin corrosive potential.
Executive summary:

The skin corrosive potential of the test item was determined in an in vitro assay according to OECD Guideline 431. 50 µL of the test item, negative (9 g/L NaCl in water) or positive (glacial acetic acid) control were apllied upon reconstituted human epidermis (EPISKIN model). Dublicates of EPISKIN were treated with test item and incubated for 4 hours at room temperature (first experiment). Furthermore, disks of EPISKIN (two units / exposure time) were treated with test item and incubated for 4 hours, 1 hour and 3 min at room temperature (second experiment). Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The test item showed significantly reduced cell viability (below 35 %) in comparison to the negative control after 4 hours of exposure in both experiments. In the first experiment the average test item treated tissue viability was 25 % and in the second experiment it was (corrected value) 13 % at 4 hours of exposure. In the second experiment the test item treated tissue viabilities (corrected value) were above 35 % of the mean negative control value after 1 hour and 3 min of exposure. The average test item treated tissue viabilities (corrected value) were 51 % at 1 hour and 90 % at 3 minutes of exposure.

In conclusion, in this in vitro skin corrosion test in EPISKIN model the results indicate that the test item is corrosive to skin after 4 hours exposure and not corrosive after 1 hour and 3 min exposure. According to the UN GHS classification systems, the test item has been categorized as “Corrosive: Optional Sub- categories 1B and 1C”.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)

Additional information

Skin:

The skin corrosive potential of the test item was determined in an in vitro assay according to OECD Guideline 431. 50 µL of the test item, negative (9 g/L NaCl in water) or positive (glacial acetic acid) control were apllied upon reconstituted human epidermis (EPISKIN model). Dublicates of EPISKIN were treated with test item and incubated for 4 hours at room temperature (first experiment). Furthermore, disks of EPISKIN (two units / exposure time) were treated with test item and incubated for 4 hours, 1 hour and 3 min at room temperature (second experiment). Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The test item showed significantly reduced cell viability (below 35 %) in comparison to the negative control after 4 hours of exposure in both experiments. In the first experiment the average test item treated tissue viability was 25 % and in the second experiment it was (corrected value) 13 % at 4 hours of exposure. In the second experiment the test item treated tissue viabilities (corrected value) were above 35 % of the mean negative control value after 1 hour and 3 min of exposure. The average test item treated tissue viabilities (corrected value) were 51 % at 1 hour and 90 % at 3 minutes of exposure.

In conclusion, in this in vitro skin corrosion test in EPISKIN model the results indicate that the test item is corrosive to skin after 4 hours exposure and not corrosive after 1 hour and 3 min exposure. According to the UN GHS classification systems, the test item has been categorized as “Corrosive: Optional Sub- categories 1B and 1C”.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Skin corrosive properties were documented. As a result the substance should be classified as skin corrosive (UN GHS Category 1B and 1C, H314) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.