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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 February 2017 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Deviation 1
General Study Plan Number 1107 version 07 was withdrawn prior to the start of the study therefore General Study Plan Number 1107 version 09 was followed.

Deviation 2
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

These deviations were considered to have not affected the integrity or validity of the study
Deviations:
yes
Remarks:
See "Version / remarks" above
GLP compliance:
yes (incl. certificate)

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
- Source and lot/batch No.of test material: supplied by the Sponsor, batch no. 7215603732
- Expiration date of the lot/batch: 31 January 2018 (retest date)
- Purity test date: 21.08.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable, the test substance was used as supplied
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: highly differentiated and stratified epidermis
Cell source:
other: not appicable
Source strain:
other: adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen
Justification for test system used:
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM reconstructed human epidermis model
- Tissue batch number(s): 17-EKIN-050
- Production date: not detailed
- Shipping date: not detailed
- Delivery date: 12 December 2017
- Date of initiation of testing: 12 December 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 °C, 5% CO2 in air

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item
- Observable damage in the tissue due to washing: no observable damage
- Modifications to validated SOP: no modification of SOP

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: no filter
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not detailed

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No reference to historical data was mentionned

NUMBER OF REPLICATE TISSUES: triplicate were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Water- killed tissues
- Procedure used to prepare the killed tissues (if applicable): Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months
- N. of replicates : triplicate
- Method of calculation used: True viability = Viability of treated tissue - direct MTT interferance from test item = OD treated viable tissue - (mean OD treated killed tissue - mean OD untreated killed tissue) OD = opical density

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: two independant sequences : pretest and main test (including MTT assay)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation period is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if ithe viability after 15 minutes exposure and 42 hours post incubation period is higher than 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not different cutpoint
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)
- Concentration (if solution): not applicable, used as supplied

VEHICLE
No vehicle was sued, however, 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of DPBS
- Concentration (if solution): pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of SDS
- Concentration (if solution): 5% w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
triplicates were used per condition

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - Test item
Value:
86.1
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - Negative control
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test - Positive control
Value:
6.8
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.
- Colour interference with MTT: The solution containing the test item was a pale grey color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control condition showed the ability of the test system to quantify decrease of tissue viability

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.910 and the standard deviation value of the viability was 3.0%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.8% relative to the negative control treated tissues and the standard deviation value of the viability was 2.8%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.3%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: Not specified

Any other information on results incl. tables

Table & : Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD570of tissues

Mean OD570of triplicate tissues

± SD of OD570

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.880

0.910

0.028

96.7

100*

3.0

0.916

100.6

0.935

102.7

Positive Control Item

0.042

0.062

0.026

4.6

6.8

2.8

0.053

5.8

0.091

10.0

Test Item

0.772

0.784

0.057

84.8

86.1

6.3

0.734

80.6

0.846

92.9

 

 

 

 


SD=        Standard deviation

*=         The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
other: Not skin irritant
Conclusions:
Under the experimental condition of the study, the registered substance was applied on EpiSkinTM model and the relative mean viability of treated tissues was 86.1% after the 15 Minute exposure period and 42 Hours post exposure incubation period. Hence, according to CLP criteria, the test item 2-Amino-4-Hydroxyethylaminoanisole Sulfate was not classified for skin irritation.
Executive summary:

The purpose of this GLP-compliant in vitro test was to evaluate the skin irritation potential of the test item 2-Amino-4-Hydroxyethylaminoanisole Sulfate using the EPISKINTM reconstructed human epidermis model.

Triplicate tissues of EPISKINTM model were treated with the test item for an exposure period of 15 minutes.  At the end of the exposure period each tissue was rinsed before incubating for 42 hours. DPBS was used as negative control and SDS as positive control condition. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes.  At the end of the post exposure incubation period each tissue was taken for MTT-loading.  

The relative mean viability of the test item treated tissues was 86.1% after the 15 Minute exposure period and 42 Hours post exposure incubation period.

Under the experimental condition of the study, the registered substance did not indeuced adverse effect on EpiSkinTM model. Hence, according to CLP criteria, the test item 2-Amino-4-Hydroxyethylaminoanisole Sulfate was not classified for skin irritation.