2-((4-methyl-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

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-((4-methyl-2-nitrophenyl)amino)ethanol

EC Number:

408-090-7

EC Name:

2-((4-methyl-2-nitrophenyl)amino)ethanol

Cas Number:

100418-33-5

Molecular formula:

C9H12N2O3

IUPAC Name:

2-[(4-methyl-2-nitrophenyl)amino]ethan-1-ol

Test material form:

solid: crystalline

Details on test material:

orange powder
Batch # : 7215587794

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg of the test item (83.3 mg/cm² according to guideline)

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Remarks:

% of negative control

Run / experiment:

6 hours

Value:

3.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Results after treatment for 6 hours withHydroxyethyl-2-Nitro-p-Toluidine (B075)and the controls

Dose Group	Ab-sorbance Well 1 (Tissue 1/2)	Ab-sorbance Well 2 (Tissue 1/2)	Mean Absor-bance (Tissue 1/2)	Mean Absorbance Tissue 1 and 2 minus Mean Blank	Mean Absorbance of 2 Wells	Rel. Absorbance [%] Tissue 1 and 2*	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Absorbance [% of Negative Control]*
	0.038	0.038	0.038	0.000
Negative Control	1.742	1.751	1.747	1.709	1.703	100.3	0.7	100.0
	1.767	1.703	1.735	1.697		99.7
Positive Control	0.340	0.363	0.351	0.313	0.303	18.4	1.2	17.8
	0.332	0.331	0.332	0.293		17.2
Test Item	0.096	0.094	0.095	0.056	0.059	3.3	0.3	3.5
	0.100	0.101	0.100	0.062		3.6
Blank	0.038	0.038	0.038	0.000
Negative Control Add. Viable Tissue	0.041	0.040	0.040	0.003	0.003	0.1	0.0	0.2
	0.041	0.041	0.041	0.003		0.2
Test Item Add. Viable Tissue	0.039	0.039	0.039	0.001	0.001	0.1	0.0	0.0
	0.039	0.039	0.039	0.001		0.0

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 3.5% (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.

Concerning acceptance criteria:

·        The mean negative control OD (blank corrected) is > 0.8 and < 2.5(1.703 and 1.697).

·        The mean relative viability of the positive control is below 50% of the negative control viability (17.8%).

The difference of viability between the two relating tissues of a single item is < 20% (values between 0.0% and 1.2%) in the same run (for positive and negative control tissues and tissues of single test items).This applies also to theadditional viable tissues (without MTT addition), which were calculated as percent values related to the viability of the relating negative control.

Applicant's summary and conclusion

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Hydroxyethyl-2-Nitro-p-Toluidine (B075) possesses an eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of Hydroxyethyl-2-Nitro-p-Toluidine (B075) 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 (reddish) 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.After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 0.8 and < 2.5 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% compared with thenegative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with Hydroxyethyl-2-Nitro-p-Toluidine (B075). Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased to 3.5% (threshold for irritancy: < 60%). Since the test item was classified as irritant in the main experiment due to a viability being lower than the cut-off of 60%, it was not necessary to correct the viability value in the main experiment with the determined correction factor. In conclusion, it can be stated that in this study and under the experimental conditions reported, Hydroxyethyl-2-Nitro-p-Toluidine (B075) possesses an eye irritating potential.