Rifamycin, 3-amino-1,4-dideoxy-1,4-dihydro-4-imino-1-oxo-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-06-26 to 2017-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

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)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Rifamycin, 3-amino-1,4-dideoxy-1,4-dihydro-4-imino-1-oxo-

Cas Number:

62041-01-4

Molecular formula:

C37 H47 N3 O11

IUPAC Name:

Rifamycin, 3-amino-1,4-dideoxy-1,4-dihydro-4-imino-1-oxo-

Specific details on test material used for the study:

Batch: 16012R87A
Purity: not specified

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 51.3 to 61.2 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL sterile Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Methyl Acetate (positive control) or Triton X (0.3%)

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcularTM (OCL-200-EIT MatTek Corporation, Lot: 23487 and 23491)

- Doses of test chemical and control substances used: 51.3 to 61.2 mg test item, 50 µL Milli-Q water (negative control) and 50 µL Methyl Acetate or Triton X (0.3%) (positive control).

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure period: 6 hours ± 15 minutes at 37.0 ± 1.0°C, post-exposure immersion: 25 ± 2 minute at room temperature, post-exposure incubation: 18 hours ± 15 minutes at 37°C.

- Description of the Interference of the Test Item with the MTT Endpoint
Test for Color Interference by the Test Item: the test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of the test item or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mLMilli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, approximately 50 mg of test item or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Centrifugation was not considered necessary. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement. Since the test item showed color interference. A functional test with living skin tissues was performed to show that the test item did not bind to the tissue and resulted in a false MTT reduction signal.
Test for Reduction of MTT by the Test Item: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 50 mg of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 µL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

- Number of tissue replicates used per test chemical and controls: 2

-ACCEPTABILITY CRITERIA, the in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.
d) The non-specific color of the test item should be ≤50% relative to the negative control OD.

-INTERPRETATION for ACCEPTABILITY CRITERIA
The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: relative mean tissue viability%

Run / experiment:

first test

Value:

58

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues

Results 2

Irritation parameter:

other: relative mean tissue viability%

Run / experiment:

second test

Value:

78

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues.

Other effects / acceptance of results:

OTHER EFFECTS:
-Visible damage on test system:
- Colour interference with MTT: the test item did not interfere with the MTT endpoint.
- Color interference in aqueous conditions: the test item induced color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 6 hours ± 15 minutes exposure is 1.9%. The positive control had a mean cell viability after 6 hours ± 15 minutes exposure is 29% in the second test.

Applicant's summary and conclusion

Interpretation of results:

other: non-irritant

Conclusions:

The test item is non-irritant in the EpiOcularTM test under the experimental conditions described in this report.

Executive summary:

The test item was topically applied on the Reconstructed Human EpiOcularTM Model to evaluate the potential eye hazard. The study procedures described in this report were based on OECD 492 (2015) guideline.

 

The test item (at least 50 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.

 

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

 

The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color of the test item was 0.53% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

 

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 58%. Since the individual viabilities were spread over 2 categories (individual values 55 and 61 respectively), the test was inconclusive and a repeat experiment was performed.

 

The non-specific color of the test item was 0.39% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.

 

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 78% in the second test (individual values 82 and 75%, respectively). Since the mean relative tissue viability for the test item was above 60% after 6 hours ± 15 minutes treatment the test item is considered to be non-irritant in this test.

 

Overall since 3 out of 4 individual viabilities over 2 independent performed tests were above 60% the test item is considered to be non-irritant.

 

In conclusion, the test item is non-irritant in the EpiOcularTM test under the experimental conditions described in this report.