2-[(2-nitrophenyl)amino]ethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 November 2016 to 12 May 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

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: 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 Lowenstein, Lot No. 47
- Expiration date of the lot/batch: 13 July 2019
- Purity test date: 14 July 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle used

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment, the test item was used as supplied
- Final dilution of a dissolved solid, stock liquid or gel: no dilution performed
- Final preparation of a solid: no preparation performed

Test animals / tissue source

Species:

human

Strain:

other: human-derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.
The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
Approximately 50 mg of the test item (83.3 mg/cm² according to guideline) were tested topically on duplicate EpiOcular™ tissues

VEHICLE
No vehicle was used

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

Duplicate

Details on study design:

- Details of the test procedure used

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 6 hours.After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.. The tissues were incubated for approximately 18 hours standard culture conditions (post-treatment incubation).

- RhCE tissue construct used, including batch number
EpiOcularTM (MatTek Corporation) Lot Number : 23755 & 23779

- Doses of test chemical and control substances used: 50µL of control substances and 50 mg of the test article were used

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
6 hours for incubation time at 37°C / 18 hours of post exposure at 37°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable):
50μL of deionised water in 1 mL of 1.0 mg/mL MTT solution

- Number of tissue replicates used per test chemical and controls (positive control, negative control,
NSMTT, NSCliving and NSCkilled, if applicable) : duplicates were used

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software
Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

- Description of the method used to quantify MTT formazan
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions. Inserts were removed from the 24-well plate after 180 minutes; then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so
that no isopropanol is flowing into the insert. The plates were and were extracted overnight at 2-8 °C in the dark and shaken for 2 hours at room temperature (main experiment) afterwards or immediately extracted (shaken for 2 hours at room temperature; additional viable and freeze-killed tissues), respectively. The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s). The absorbance at 570 nm (OD570) of each well was m
easured with a plate reader.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
See attached-Table 1

- Complete supporting information for the specific RhCE tissue construct used
Yes, certificate of analysis was provided in the report

- Reference to historical data of the RhCE tissue construct:
Yes, See attached-Table 1

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
- Positive and negative control means and acceptance ranges based on historical data : Yes
- Acceptable variability between tissue replicates for positive and negative controls: Yes
- Acceptable variability between tissue replicates for the test chemical: Yes

Concerning acceptance criteria:
• The mean negative control OD (blank corrected) is > 0.8 and < 2.5 (1.471 and 1.530).
• The mean relative viability of the positive control is below 50% of the negative control viability (28.0%).
• The difference of viability between the two relating tissues of a single item is < 20% (0.7% and 1.2%) in the same run (for positive and negative control tissues and tissues of single test items). This applies also to the additional viable tissues (without MTT addition), which were calculated as percent values related to the viability of the relating negative control.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 28.0%, thus the validity of the test system is ensured. The acceptance criteria were met

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Viability (Percent)

Run / experiment:

Test Article

Value:

2.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Viability (Percent)

Run / experiment:

Negative Control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: Viability (Percent)

Run / experiment:

Positive Control

Value:

28

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not showed blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 2.5% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye. Since the test item proved to be clearly eye irritant, correction of the viability using the determined correction factors derived from the additional tests was not necessary.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 28.0%, thus the validity of the test system is ensured.
The acceptance criteria were met.
Relevant irritating effects were observed following 6 hours incubation with HC Yellow No. 2 (B041). The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 2.5% compared with the value of the negative control (threshold for irritancy: ≤ 60%).

Applicant's summary and conclusion

Interpretation of results:

other: Eye Irritating Potential

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, HC Yellow No. 2 (B041) possesses an eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of HC Yellow No.2 by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test, but it changed colour in the presence of water (orange) and isopropanol (orange) in the colour interference pre-test. Therefore, an additional test with freeze-killed tissues did not have to be performed, but an additional test with viable tissues (without MTT addition) was necessary to determine a correction factor for calculating the true viability in the main experiment.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

Irritating effects were observed following incubation with HC Yellow No. 2 (B041). Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased to 2.5% (threshold for irritancy: < 60%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, HC Yellow No. 2 (B041) possesses an eye irritating potential.