Sodium glyoxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 24 April 2017. Experimental completion date: 12 May 2017

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

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)

Deviations:

yes

Remarks:

In a previous studies with this substance (Envigo CRS GmbH project No. 1810302, Skin Irritation Test) a sample of the test had to be archived. This deviation was considered to have not affected the integrity or validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

Deviations:

yes

Remarks:

In a previous studies with this substance (Envigo CRS GmbH project No. 1810302, Skin Irritation Test) a sample of the test had to be archived. This deviation was considered to have not affected the integrity or validity of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium glyoxylate

EC Number:

220-298-8

EC Name:

Sodium glyoxylate

Cas Number:

2706-75-4

Molecular formula:

C2H2O3.Na

IUPAC Name:

sodium oxoacetate

Specific details on test material used for the study:

Identification: Safelink SPM-01
Batch: G150201
Purity: 99.9% HPLC
Appearance: White, crystalline (detected by Envigo CRS laboratory staff)
Expiry Date: 17 November 2017
Storage Conditions: At room temperature

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Cell Culture
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm Ø).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the assay medium. After one hour, the Assay Medium was replaced by 1 mL of fresh assay medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (about 16.5 hours).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Approximately 50 mg of the test item was tested topically on duplicate EpiOcular™ tissues.

Negative Control: 50 µL deionised water

Positive Control: 50 µL Methyl acetate

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

The tissues were incubated for approximately 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2

Number of animals or in vitro replicates:

The test item, positive control and negative control were all carried out in duplicate

Details on study design:

EpiOcular Kit Components Needed for the Assay
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free D-PBS Dulbecco´s Phosphate Buffered Saline

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
60 mL Extractant solution (Isopropanol) For extraction of formazan crystals

EXPERIMENTAL DESIGN AND STUDY CONDUCT
The negative control did not meet the acceptance criteria in a 1st experiment. Therefore, the test was performed twice. Only the outcome of the 2nd experiment is reported, but the raw data of both experiments are archived.

Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca2+Mg2+free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
Test item exposure: After the 30 minute Ca2+Mg2+free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 6 hours.
At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca2Mg2+-free DPBS (brought to room temperature).
Three clean beakers containing a minimum of 100 mL each of Ca2+Mg2+-free DPBS were used per test item. The test item utilized a different set of three beakers. 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 tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) 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 are mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then be rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for approximately 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were immediately extracted (shaken for about 2 hours at room temperature). The tissues were not be pierced. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically without piercing. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability

Run / experiment:

Mean

Value:

2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show 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 2.8% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.

Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.466 and 1.544).
• The mean relative viability of the positive control is below 50% of the negative control viability (28.0%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 0.0% and 4.1%) in the same run (for positive and negative control tissues and tissues of single test items).

Any other information on results incl. tables

 Results

Results after treatment for 6 hours withSafelink SPM-01and 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 Tissues	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]*
Blank	0.038	0.039	0.038	0.000
Negative Control	1.544	1.516	1.530	1.492	1.462	102.0	4.1	100.0
	1.466	1.475	1.471	1.432		98.0
Positive Control	0.421	0.457	0.439	0.401	0.409	27.4	1.2	28.0
	0.454	0.459	0.456	0.418		28.6
Test Item	0.079	0.079	0.079	0.041	0.040	2.8	0.0	2.8

* = Relative absorbance [rounded values] = 100 x ((absorbance _{test item/ positive control} / absorbance _{negative control})

Discussion

This in vitro study was performed to assess the eye irritation potential of Safelink SPM-01 by means of the Human Cornea Model Test.

Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured, did not dye water or isopropanol, and did not prove to be a MTT reducer.

About 50 mg of the test item and each 50 µL of the controls, respectively, were applied to each of duplicate EpiOcular™ tissue for 6 hours.

Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 28.0%, thus the validity of the test system is ensured.

The acceptance criteria were met.

Relevant irritating effects were observed following 6 hours incubation with Safelink SPM-01. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 2.8% compared with the value of the negative control (threshold for irritancy: ≤ 60%).

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

A limitation of the Test Guideline OECD 492 is that it does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B), as defined by UN GHS

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Safelink SPM-01 possesses an eye irritating potential.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of Safelink SPM-01 by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Also its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) 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 were well within the required acceptability criterion of 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 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 Safelink SPM-01. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (2.8%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, Safelink SPM-01 possesses an eye irritating potential.