2-(4-amino-2-nitroanilino)ethanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 June 2017 to 19 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Deviation 1
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 no 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.

Deviation 2
The test item was found to have the potential to cause color interference with the MTT endpoint, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

Deviation 3
As the results of the color correction tissues were not used it was therefore unnecessary to use the results of the killed color correction tissues as no double correction for color interference would have occurred.

Deviation 4
The standard deviation for the test item treated group was 18.8% which was marginally higher than the assay acceptance cut-off of 18%. The borderline standard deviation was considered acceptable as each of the three identically treated replicates were clearly negative compared to the negative control.
These deviations were considered to have not affected the integrity or validity of the study.

Deviations:

yes

Remarks:

See remarks above

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-amino-2-nitroanilino)ethanol

EC Number:

220-701-7

EC Name:

2-(4-amino-2-nitroanilino)ethanol

Cas Number:

2871-01-4

Molecular formula:

C8H11N3O3

IUPAC Name:

2-(4-amino-2-nitroanilino)ethanol

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: Batch No. 7215456195
- Expiration date of the lot/batch: 22/05/2018
- Purity test date: 15/12/2016

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material)
The test item was used as supplied

In vitro test system

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

other: Reconstructed Human Epidermis

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
- Tissue batch number(s): 17-EKIN-024
- Production date: not specified
- Shipping date: not specified
- Delivery date: 13 June 2017
- Date of initiation of testing: 8 June 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, 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. Washing steps were performed at the end of exposure and post exposure period.
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time:3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: not specified
- Barrier function: not specified
- Morphology: not specified
- Contamination: not specified
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: triplicates were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- 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 48 ± 1 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. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature
- N. of replicates : three water killed tissues were used
- Method of calculation used: relative mean viability (%) = (mean OD570 of test item / mean OD570 of negative control)x100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: two independant test sequencies were performed : pre test and main test.

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 exposure period is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post exposure is greater than or equal to 50%

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): test item was used as supplied

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- 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

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Test - Negative Control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Test - Positive Control

Value:

15.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main Test

Value:

86.1

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: not specified
- Direct-MTT reduction: An assessment found the test item was potentially able to directly reduce MTT. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that no 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 test item was found to have the potential to cause color interference with the MTT endpoint, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The use of Sodium Dodecyl Sulfate induced positive response to the test system (positive control condition)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.627 and the standard deviation value of the viability was 10.5%. 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 15.6% relative to the negative control treated tissues and the standard deviation value of the viability was 8.2%. 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 18.8%. The test item acceptance criterion was therefore not satisfied. However, the borderline standard deviation was considered acceptable as each of the three identically treated replicates were clearly negative compared to the negative control. This is reported as a deviation
- Range of historical values if different from the ones specified in the test guideline: not specified

Any other information on results incl. tables

Table1 :Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of triplicate tissues	±SDof OD570	Relativeindividualtissueviability(%)	Relativemeanviability(%)	± SD of Relative mean viability (%)
Negative Control Item	0.682	0.627	0.066	108.8	100*	10.5
	0.554			88.4
	0.646			103.0
Positive Control Item	0.092	0.098	0.052	14.7	15.6	8.2
	0.050			8.0
	0.153			24.4
Test Item	0.408	0.540	0.118	65.1	86.1	18.8
	0.636			101.4
	0.577			92.0

 

 

OD= Optical Density

SD=        Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this test system, the registered substance HC Red No.3 did not induced skin irritation 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. Hence, according to CLP criteria, the HC Red No.3 was Not Classified for Skin Irritation.

Executive summary:

The purpose of this GLP compliant study was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours according to the OECD 439 guideline method.

Triplicate tissues 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.  The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes.  At the end of the post exposure incubation period each tissue was taken for MTT-loading in order the quantify the cellular viability.

In this test system, the registered substance HC Red No.3 did not induced skin irritation 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. Hence, according to CLP criteria, the HC Red No.3 was Not Classified for Skin Irritation.