Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: 130022 P040
- pH-value: ca. 6 (undiluted test substance, moistened with de-ionized water)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test substance was applied moistened with de-ionized water.

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

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature or 1 hour in the incubator at 37°C (corrosion test); 25 minutes at room temperature overall and 35 minutes in the incubator at 37°C (irritation test)
- Temperature of post-treatment incubation (if applicable): 37°C (irritation test)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL assay medium
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: For every treatment, except the killed controls, 2 tissues (corrosion test) or 3 tissues (irritation test) were treated in parallel.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (sub-category 1A) if the mean tissue viability after 3 min exposure is < 50%
- The test substance is considered to be corrosive to skin (sub-category 1B and 1C) if the mean tissue viability after 3 min exposure is ≥ 50% and after 1 h exposure < 15%
- The test substance is considered to be non-corrosive to skin if the mean tissue viability after 3 min exposure is ≥ 50% and after 1 h exposure ≥ 15%

- The test substance is considered to be irritant to skin if the mean tissue viability after exposure is ≤ 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure is > 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: MTT-reduction control: De-ionized water or test substance (corrosion test); sterile PBS or test substance (irritation test)
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL de-ionized water (corrosion test) or sterile PBS (irritation test) was applied first. Thereafter, a bulk volume of 25 μL (about 20 mg) of the solid test material was applied with a sharp spoon and homogeneously distributed with the fluid.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)
Duration of treatment / exposure:
3 minutes at room temperature or 1 hour in the incubator at 37°C (corrosion test); 25 minutes at room temperature overall and 35 minutes in the incubator at 37°C (irritation test)
Duration of post-treatment incubation (if applicable):
approx. 42 hours (irritation test)
Number of replicates:
For every treatment, except the killed controls, 2 tissues (corrosion test) or 3 tissues (irritation test) were treated in parallel.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicate tissues
Run / experiment:
corrosion test (3 min-exposure)
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive (in combination with the result of the 1h-exposure)
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicate values
Run / experiment:
corrosion test (1h-exposure)
Value:
102
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non-corrosive (in combination with the result of the 3 min-exposure)
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 replicate values
Run / experiment:
irritation test
Value:
96
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:
- Direct-MTT reduction: yes (This ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing (performed with corrosion test, only).)
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes (demonstrated by historical control values of negative and positive controls, gathered over an appropriate time period)

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: Corrosion test - Individual and mean OD570values, individual and mean viability values

 

 

Exposure: 3 min

Exposure: 1 hour

Test

substance

 

tissue 1

tissue 2

KC

mean

tissue 1

tissue 2

KC

mean

 

NC

mean OD570

1.927

2.168

0.113

2.047

1.799

1.762

0.108

1.780

viability

[% of NC]

94.1

105.9

-

100

101.0

99.0

-

100

Test substance

mean OD570

1.948

2.094

0.123

2.021

1.797

1.839

0.123

1.818

viability

[% of NC]

95.2

102.3

-

99

101.0

103.3

-

102

 

PC

mean OD570

0.441

0.435

-

0.438

0.130

0.125

-

0.127

viability

[% of NC]

21.5

21.2

-

21

7.3

7.0

-

7

  NC: negative control

  PC: positive control

  KC: MTT-reduction control

 

Due to the ability of the test substance to reduce MTT directly, a KC was applied in parallel. However, the result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability

calculation.

Table 2: Irritation test - Individual and mean OD570values, individual and mean viability values

 

Test

substance

 

tissue 1

tissue 2

tissue 3

mean

SD

 

NC

mean OD570

2.533

2.719

2.389

2.547

viability

[% of NC]

99.4

106.8

93.8

100

6.51

Test substance

mean OD570

2.619

2.388

2.360

2.456

viability

[% of NC]

102.8

93.8

92.7

96

5.59

 

PC

mean OD570

0.075

0.070

0.061

0.068

viability

[% of NC]

2.9

2.7

2.4

3

0.29

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.
Executive summary:

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 25 μL bulk volume (about 20 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™).

For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiDerm™ skin corrosion/irritation test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing (performed with corrosion test, only).

Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 99%, and it was 102% after an exposure period of 1 hour.

Irritation test:

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 96%.