2-[(2-nitrophenyl)amino]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:

From 27 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

Guideline:

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

Version / remarks:

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 that 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.
This deviation was considered to have not affected the integrity or validity of the study.

Deviations:

yes

Remarks:

See "Version / remarks" section above

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-nitrophenyl)amino]ethanol

EC Number:

225-555-8

EC Name:

2-[(2-nitrophenyl)amino]ethanol

Cas Number:

4926-55-0

Molecular formula:

C8H10N2O3

IUPAC Name:

2-[(2-nitrophenyl)amino]ethanol

Test material form:

solid

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. 98893196L-1
- Expiration date of the lot/batch: 01 October 2018
- Purity test date: 15.12.2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
at room temperature in the dark
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle:
not applicable, no vehicule was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not appliacle, the test item 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

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen

Cell source:

other: not applicable

Source strain:

other: not applicable

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-021
- Production date: not detailed
- Shipping date:not detailed
- Delivery date: 23 May 2017
- Date of initiation of testing: 22 May 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.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No modification was performed

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: no filter was used
- Filter bandwidth: no filter was used
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
No historical data was mentionned in the report

NUMBER OF REPLICATE TISSUES: triplicates were used per condition

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- water killed tissue
- Procedure used to prepare the killed tissues (if applicable): not detailed
- N. of replicates : triplicates
- Method of calculation used: Relative mean viability (%) = [( mean OD570 of test item) / (mean OD570 of negative control)] x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: two independant tests : Assessments for direct test item reduction of MTT and Color interference ; 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-exposure incubation is equal or 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 incubation is greater than 50% .
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: no different cut off point

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 (corresponding to 26.3 mg/cm2)
- Concentration (if solution): pure (used as supplied)

VEHICLE
No vehicle was used, but 5µL 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): 10µL
- 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 per condition

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:

4.9

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 - Test item

Value:

79.7

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: The solution containing the test item was an orange color therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that 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 positive control showed the profiency of the test sytem to measure the decrease of cellular viability

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.802 and the standard deviation value of the viability was 10.2%. 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 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.8%. 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 15.5%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: not applicable

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	Relative individual tissue viability(%)	Relative mean viability(%)	± SD of Relative mean viability (%)
Negative Control Item^Å	0.721	0.802	0.082	89.9	100*	10.2
	0.800			99.8
	0.885			110.3
Positive Control ItemÅ	0.033	0.039	0.007	4.1	4.9	0.8
	0.039			4.9
	0.046			5.7
Test Item	0.636	0.639	0.124	79.3	79.7	15.5
	0.517			64.5
	0.765			95.4

 

OD= Optical Density

SD=        Standard deviation

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

Å=         Control group shared withEnvigo -Shardlowstudy number QT69YC and BN48LN

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental condition of the study, The test item HC YEllow No 2 did not induce irritant effect to EpiSkin model. Hence, the test item was classified as non-irritant : EU CLP Not classified for Irritation according to CLP criteria.

Executive summary:

The purpose of this GLP-compliant in vitro test was to evaluate the skin irritation potential of the test item HC yellow No 2 using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes according to OECD TG 439 method.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.  The test item was found to have the potential to cause color interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this.  At the end of the exposure period each tissue was rinsed before incubating for 42 hours.  At the end of the post exposure incubation period each tissue was taken for MTT-assay. DPBS was used as negative control and SDS5% w/v was used as positive control. Optical density was measured at 570nm to determine cellular viability.

The relative mean viability of the test item treated tissues was 79.7% after the 15 Minute exposure period and 42 Hours post exposure incubation period.

Under the experimental condition of the study, The test item HC YEllow No 2 did not induce irritant effect to EpiSkin model. Hence, the test item was classified as non-irritant : EU CLP Not classified for Irritation according to CLP criteria