Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
The prediction model used for evaluating the results was amended by the implementation of "borderline" criteria indicating inconclusive test results. The "borderline" criteria are based on historic BASF data and consider the variance of the test method.
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 5222A20170420
- Purity test date: 20 Apr 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was applied undiluted.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was applied undiluted.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Tissue model: EPI-200.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM model
- Tissue batch number(s): 25830

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature or incubator at 37°C
- Temperature of post-treatment incubation (if applicable): 37°C ± 1°C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter:without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570 of the NC between 0.8 and 2.8
- Barrier function: Lower acceptance limit: ET50 = 4.0 hours. Upper acceptance limit: ET50 = 8.7 hours.

NUMBER OF REPLICATE TISSUES: 2 per exposure time and test group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze-killed control tissues
- Procedure used to prepare the killed tissues (if applicable): freezing
- N. of replicates : 2
- Method of calculation used: quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean tissue viability after 3 minutes of exposure is less than 45%, or if the viability after 3 minutes exposure is greater than or equal to 45 % and the viability after 1 hour exposure is less than 10%.
- The test substance is considered to be borderline corrosive (inconclusive) to skin if the mean tissue viability after 3 minutes of exposure is 45-55%, or if the viability after 3 minutes exposure is greater than or equal to 55 % and the viability after 1 hour exposure is 10-20%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 55% and the viability after 1 hour exposure is greater than or equal to 20%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: The “borderline“ evaluation (50 ± 5%, 25 ± 5% and 15 ± 5%) was statistically determined by 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 431.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted, purity 99.57%

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
Duration of treatment / exposure:
3 minutes at room temperature or 1 hour in the incubator
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1, mean of 2 replicate tissues / 3 min exposure period
Value:
102
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1, mean of 2 replicate tissues / 1 hour exposure time
Value:
52.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

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:Exposure period 3 min: Individual and mean OD570values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification   tissue 1 tissue 2 mean SD CV [%]
NC mean OD570 1.637 1.613 1.625    
viability
[% of NC]
100.8 99.2 100 1.1 1.1
test
substance
mean OD570 1.589 1.727 1.658    
viability
[% of NC]
97.8 106.3 102.0 6.0 5.9
PC mean OD570 0.136 0.159 0.147    
viability
[% of NC]
8.3 9.8 9.0 1.0 11.1

NC: Negative control

PC: Positive control

Table 2:Exposure period 1 h: Individual and mean OD570values, individual and mean viability values, standard deviations and coefficient of variation

Test substance identification   tissue 1 tissue 2 mean SD CV [%]
NC mean OD570 1.661 1.743 1.702    
viability
[% of NC]
97.6 2.4 100    
test
substance
mean OD570 1.042 0.742 0.892    
viability
[% of NC]
61.2 43.6 52.4 12.4 23.7
PC mean OD570 0.061 0.069 0.065    
viability
[% of NC]
3.6 4 3.8 0.3 8.2

NC: Negative control

PC: Positive control

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was identified as non corrosive to the skin in the EpiDerm™ in vitro skin corrosion test.
Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of 50 µL of the 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.

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 following results were obtained in the EpiDerm™ skin corrosion test:

The test substance is not able to directly reduce MTT.

The mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 102.0% and it was 52.4% after an exposure period of 1 hour.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test material shows no skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 5222A20170420
- Purity test date: 20 Apr 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was applied undiluted.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was applied undiluted.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Tissue model: EPI-200.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit, EPI-200
- Tissue batch number(s): 25830

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 1 hour
- Temperature of post-treatment incubation (if applicable): 37°C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 24 ± 2 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD570 of the NC between 0.8 and 2.8
- Barrier function: Lower acceptance limit: ET50 = 4.0 hours. Upper acceptance limit: ET50 = 8.7 hours.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze-killed control tissues
- Procedure used to prepare the killed tissues (if applicable): freezing
- N. of replicates : 3
- Method of calculation used: quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the mean tissue viability after exposure is less than 45%.
- The test substance is considered to be borderline irritant (inconclusive) to skin if the mean tissue viability after exposure is 45-55%.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than or equal to 55%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: The “borderline“ evaluation (50 ± 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted, purity 99.57%

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% (w/v)
Duration of treatment / exposure:
25 minutes at room temperature and for 35 minutes in the incubator at 37°C
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1, mean of 3 replicate tissues
Value:
3.1
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

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

Any other information on results incl. tables

Table 1: Results of the Skin Irritation Test.Individual and mean OD570values, individual and mean viability values, standard deviations and coefficient of variation.

Test substance identification   tissue 1 tissue 2 tissue 3 mean SD CV [%]
NC mean OD570 1.806 1.678 1.523 1.669    
viability
[% of NC]
108.2 100.5 91.2 100 8.5 8.5
test
substance
mean OD570 0.054 0.050 0.051 0.051    
viability
[% of NC]
3.2 3 3.1 3.1 0.1 3.9
PC mean OD570 0.046 0.041 0.041 0.043    
viability
[% of NC]
2.8 2.4 2.5 2.5 0.2 7.2

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test material was identified as irritant to the skin in the EpiDerm™ in vitro skin irritation test.
Executive summary:

The potential of the test substance to cause dermal irritation was assessed by a single topical application of 30 µL of the 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.

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 following results were obtained in the EpiDerm™ skin irritation test:

The test substance is not able to directly reduce MTT.

The mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubationwas 3.1%.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test material shows a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.