Reaction product of propargyl chloride and sodium bisulfite

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:

2016

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:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CA AQ02372

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability

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): 50 µL

Details on study design:

- RhCE tissue construct used, including batch number:
EpiOcular (TM) OCL-200 model, tissue lot number: 23740

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
incubation at 37°C in the incubator for 30 min; for removal of test item tissues were washed with sterile PBS and subsequently incubated for 2 further hours (postincubation)

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
Two tissues were treated with each, the test substance, the PC and the NC. In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control.

- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
Spectrophotometer: SunriseTM Absorbance Reader; measurement using a filter wavelength 570 nm without reference filter

- Description of the method used to quantify MTT formazan
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation.The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Mean tissue viability
(% of negative control)
< 55 Irritant
55 - 65 Borderline
> 65 Non-irritant
The „borderline“-evaluation (60 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
historical data for NC: mean OD = 1.918
historical data for PC: mean OD = 0.575

- Complete supporting information for the specific RhCE tissue construct used
Tissue viability (OD540-570nm): 1.59 ± 0.102
Barrier function: ET500 13.49 min
Sterility:no contamination

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 µL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results.
The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline: Lower acceptance limit ET50 = 12.2 min and Upper acceptance limit: ET50 = 37.5 min

- Acceptable variability between tissue replicates for positive and negative controls
PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
NC: Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5.

- Acceptable variability between tissue replicates for the test chemical
Two tissues were treated under the same conditions. A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Run / experiment:

after 30 min exposure

Value:

83.8

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

27.8%

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met