Dipotassium oxalate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Nov - 16 Dec 2016

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:

adopted in 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium oxalate hydrate

Cas Number:

6009-70-7

Molecular formula:

C2H8N2O4.H2O

IUPAC Name:

Diammonium oxalate hydrate

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

in duplicates for each treatment and control group

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23757
- Viability: The quality of the final tissue was assessed by undertaking an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 μL of 0.3% Triton X-100. The ET-50 value was determined to be 20.43 min.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a preexperiment. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT. An additional test with freeze-killed tissues did not have to be performed.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength: 570 nm
- Evaluation criteria: The test substance is considered to be not irritating to eye if the test substance-treated tissue viability is >60%.
- Acceptance criteria: The results are acceptable if the negative control OD is >0.8 and <2.5, if the mean relative viability of the positive control is below 50% of the negative control viability and if the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items).
- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Run / experiment:

6 h exposure

Value:

3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER OBSERVATIONS:
The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in water or isopropanol did not lead to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD values between 1.361 and 1.417 was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 18.2%, thus the validity of the test system is ensured.
The difference of viability between the two relating tissues of a single substance is < 20% (values between 1.3% to 4.6%) in the same run (for positive and negative control tissues and tissues of single test substance).

Any other information on results incl. tables

Table 1: Results after treatmen for 6 h with the test substance and the controls

Dose Group	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Mean Absorbance (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2 minus Mean Blank	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Difference of the Rel. Absorbances [%] Tissue 1 and 2	Viability [% of Negative Control]**
Blank	0.038	0.038	0.038	0.000
Negative Control	1.384	1.361	1.373	1.335	1.355	98.5	3.0	100.0
	1.417	1.410	1.414	1.376		101.5
Positive Control	0.249	0.258	0.253	0.215	0.247	15.9	4.6	18.2
	0.318	0.314	0.316	0.278		20.5
Test substance	0.072	0.072	0.072	0.034	0.042	2.5	1.3	3.1
	0.089	0.089	0.089	0.051		3.8

Table 2 Historical Control Data

Positive Control		Negative Control [OD570]
Mean Viability	32.47%	Mean Absorption	1.49
Rel. Standard Deviation	10.29%	Rel. Standard Deviation	0.24
Range of Viabilities	15.90% - 42.30%	Range of Absorbance	1.24 - 2.05
Mean Absorption	0.48
Rel. Standard Deviation	0.14
Range of Absorbance	0.22- 0.64

Applicant's summary and conclusion

Interpretation of results:

other: the results of this study as a stand-alone study are not suitable for classification according to CLP/EU GHS criteria; the results may be used for classification purposes in a weight of evidence approach

Conclusions:

Under the conditions of the conducted EpiOcular test, the test substance showed irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.
Based on the weight of evidence approach in consideration of the results of the BCOP Assay (please refer to reference 7.3.2-2) and the Epiocular test (reference 7.3.2-1), the test substance meet the classification criteria as eye irritant according to Regulation(EC) No. 1272/2008.

CLP: Eye Irrit. Cat. 2, H319