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Toxicological information

Eye irritation

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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:
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 valid:
yes
Positive controls valid:
yes
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.