L-cystine dihydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 January 2017 - 01 March 2017

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

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

L-cystine dihydrochloride

EC Number:

250-391-9

EC Name:

L-cystine dihydrochloride

Cas Number:

30925-07-6

Molecular formula:

C6H12N2O4S2.2ClH

IUPAC Name:

L-cystine dihydrochloride

Test material form:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterisation of the test system:
- Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
- Lot No.: 23759
- Keratinocyte strain: 4F1188
- Supplier: MatTek In Vitro Life Science Laboratories

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg per tissue

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes at room temperature, 18 hours at 37 ± 1.5 °C

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37°C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37°C and 5% CO2 overnight (about 16.7 hours) without medium exchange.

Pre-Treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37°C and 5% CO2 for 30 minutes (± 2 minutes).

Exposure and Post-Treatment:
After the 30 minute DPBS pre-treatment, the solid test item, the negative and the positive control were tested by applying 50 mg (test item) or µL (controls) topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37°C and 5% CO2 for 6 h.
At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.
After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes at room temperature.
After the 25 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37°C) assay medium for 18 h at 37°C and 5% CO2.

- RhCE tissue construct used, including batch number:

Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 23759
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used: 50 mg (test item), 50 µL (controls)
- Duration and temperature of exposure, post-exposure: exposure: 6 hours at 37 °C; post-exposure: 25 min at room temperature + 18 h at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

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

- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan:
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is ≤ 60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:

1. The negative control OD >0.8 and <2.5,

2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: below 50% of control viability

3. Acceptable variability between tissue replicates: < 20 %

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: tissue viability %

Remarks:

tissue 1

Run / experiment:

1

Value:

3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

other: tissue viability %

Remarks:

tissue 2

Run / experiment:

1

Value:

2.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1 Tissue viability

Treatment group	Absorbance well1 (tissue 1/2)	Absorbance well 2 (tissue 1/2)	Mean absorbance (tissue 1/2)	Mean absorbance minus mean blank (tissue 1/2)	Mean tissue absorbance*	Relative absorbance [%] (tissue 1/2)**	Difference of rel. absorbances [%]	Viability [% of negative control]**
Blank	0.039	0.039	0.039	0.000
Negative control	1.216	1.232	1.224	1.185	1.291	91.9	16.3	100.0
	1.415	1.453	1.434	1.396		108.1
Positive control	0.114	0.118	0.116	0.078	0.104	6.0	4.2	8.1
	0.169	0.171	0.170	0.131		10.2
Test item	0.089	0.066	0.077	0.039	0.033	3.0	0.9	2.6
	0.067	0.065	0.066	0.027		2.1

*       Mean of two replicate wells after blank correction

**     Relative absorbance (rounded values) =  100 x (absorbance treated group / absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 (irreversible effects on the eye) or Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of the present study, the test item did show an eye hazard potential. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 1 or Category 2).

Executive summary:

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test.

The white to colourless test item did not prove to be an MTT reducer in the MTT pre-test, and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

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 (between 1.216 and 1.453) 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 the negative control value in the relative absorbance (8.1%) thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (values between 0.9% to 16.3%) for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (2.6%).