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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-19 January 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 2-oxoglutarate
EC Number:
206-167-8
EC Name:
Disodium 2-oxoglutarate
Cas Number:
305-72-6
Molecular formula:
C5H6O5.2Na
IUPAC Name:
disodium 2-oxopentanedioate
impurity 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Oxidane
Test material form:
solid: crystalline
Remarks:
Crystalline powder
Details on test material:
Batch No: 14152300
Storage: 15 – 25 °C

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: ABP, Perth, PH1 3XB
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Cow eyes were collected from freshly slaughtered cattle, and placed into containers containing Hank’s balanced salt solution (HBSS). Transport to test facility was on the same day as slaughter. Cold packs were used to keep the contents cool.
- Time interval prior to initiating testing: Corneas were prepared and used for testing on the day of collection
- indication of any existing defects or lesions in ocular tissue samples: Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use.Any eyes showing defects were rejected from further use.
- Indication of any antibiotics used: Not specified

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration Test Item: 20.08 %, w/v in physiological saline
NEGATIVE CONTROL: Physiological saline
- Amount(s) applied: 750 µL
- Concentration: Sodium Chloride 0.9%, w/v
- Batch no.: 16B29T2C
POSITIVE CONTROL : Imidazole
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20%, w/v in physiological saline
- Batch no.: SLBP2962V
Duration of treatment / exposure:
4 h ± 10 min
Duration of post- treatment incubation (in vitro):
no post-treatment incubation
Number of animals or in vitro replicates:
Three Corneas per test item and control
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
Upon receipt, eyes were rinsed with HBSS and examined for defects (scratches, opacity or neovascularisation) prior to use. Any eyes showing defects were rejected from further use. Corneas from undamaged eyes were dissected, leaving a sclera margin of ca 3 mm. Collected corneas were placed epithelial side down in HBSS at ambient temperature until use. Prior to mounting corneas the width of the cornea was measured using a ruler.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were then mounted, epithelial side forward, into pre-warmed corneal holders. Once mounted, both chambers of the cornea holders were filled with pre-warmed EMEM (Eagles Minimum Essential Medium) without phenol red. The posterior chamber was filled first to encourage the cornea to return to its original curvature, and care was taken to avoid the introduction of bubbles into the medium. Holders were then allowed to equilibrate for >1 h in an incubator.
Unless otherwise stated, all incubations of corneas were conducted in an incubator set to maintain 32°C (actual temperatures have been recorded in the raw data). At the end of the equilibration period, the medium in both chambers was replaced with fresh, EMEM without phenol red. Baseline opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Damaged corneas (opacity >7 opacity units) were rejected from further use.

TREATMENT METHOD: closed chamber. Prior to dosing, cornea holders were tipped forward to avoid contact between the dosing solution and the corneal epithelium. alpha-Ketoglutarate disodium salt (20%, w/v in physiological saline; 750 µL) was applied to the surface of three corneas using syringe. The negative control, physiological saline (750 µL) and the positive control, imidazole (20%, w/v in physiological saline; 750 µL), were also applied to the surface of three corneas each, using a syringe. The anterior chambers were then sealed with tape. To begin exposure, the corneal holders were tilted to a horizontal position, taking care to ensure that the entire epithelial surface was covered with the dosing solution. The dosed units were then returned to the incubator for 4 h ± 10 min.

REMOVAL OF TEST SUBSTANCE
EMEM with and without phenol red were pre-warmed prior to use. Following the exposure incubation, the test items and control solutions were removed from the anterior chambers through the holes using a vacuum pump. Corneas were then rinsed three times with EMEM with phenol red (ca 5 mL per rinse), removing the EMEM each time, then rinsed once with EMEM without phenol red (ca 5 mL), which was also removed. Finally, both chambers were refilled with EMEM without phenol red, and any bubbles introduced into the medium were removed.

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity readings were then taken using an Opacitometer (Duratec Analysentechnik GmbH) comprising a Testo 545 luminometer with a photo diode and xenon halogen light source in a custom enclosure. Gross damage or other changes were observed by visual assessment.
- Corneal permeability: Following the measurement of opacity, the EMEM in both chambers of the cornea holder wasremoved. Posterior chambers were refilled with EMEM without phenol red and sealed. Sodium fluorescein solution (5 mg/mL in EMEM without phenol red; ca 1 mL) was added to the anterior chambers. Cornea holders were rotated to the horizontal position, and incubated for 90 min ± 5 min in an incubator set to maintain a temperature of 32°C. At the end of the permeability test, EMEM from the posterior chambers was collected. These samples were stored overnight in a refrigerator set to maintain a temperature of 4°C.
Following overnight storage, triplicate aliquots from each sample (380 µL) were transferred into a 96 well plate, and analysed with a Multiskan Spectrum plate reader at 490 nm. Three blank wells containing EMEM without phenol red (380 µL) were analysed on the same plate, along with two aliquots of a quality control solution (sodium fluorescein, 10 µg/mL).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = Opacity change + (15 x A490 Value)

DECISION CRITERIA:
In accordance with OECD Test Guideline No. 437, irritancy was assigned on the basis of in vitro irritancy scores (IVIS), calculated from the opacity and permeability value for each cornea, as follows:

IVIS<=3: not classified according to GHS/CLP
3IVIS>55: Classified as Eye Damaging Category 1 according to GHS/CLP

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Single experiment
Value:
1.28
Vehicle controls validity:
valid
Remarks:
IVIS: 0.000
Positive controls validity:
valid
Remarks:
IVIS: 116.33
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean post treatment opacity of 2.13 (historical values range 3.83 +/- 1.82) / mean permeability corrected for blank of 0.044 (historical values range 0.045 +/- 0.043). The classification achieved was “No Category”.
- Acceptance criteria met for positive control: yes, mean IVIS 116.33 (historical range: Mean +/- 2SD =66.29 to 169.36). The classification achieved was “Category 1”.
- The BCOP results were similar for all corneas and are presented in full in Table 1 (see Any other information on results incl. tables section).

Any other information on results incl. tables

Table 1                         BCOP Results for Controls and alpha-Ketoglutarate disodium salt (n=3)

 Treatment   

Opacity Post Dose

(Opacity Units, Uncorrected)

  

Corrected* Opacity Change

(Opacity Units)

 
Permeability (A490), Corrected for Blank and Dilution Only  

   

Permeability (A490), Corrected*

  

IVIS

 

IVIS (Mean)

 

UN GHS Category**

Physiological saline (negative control)

6.92

4.61

6.56

0.26

-0.33

0.07

0.044

0.051

0.037

0.000

0.007

-0.007

  0.27

-0.23

-0.04

 0.00

No Category

Imidazole (positive control)

98.53

96.18

87.52

92.35

89.52

80.70

1.858

2.156

1.881

1.813

2.112

1.836

119.55

121.20

108.25

116.33

 Category 1

 alpha-Ketoglutarate disodium salt

6.92

11.36

7.28

0.74

3.79

0.22

0.046

0.003

0.023

0.002

-0.041

-0.021

0.77

3.18

-0.09

1.28

 No Category

* Corrected for Negative Control

**Classifications from OECD Guidelines for the Testing of Chemicals No. 437 (2013)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, alpha-Ketoglutarate disodium salt (CAS No. 305-72-6) was categorised as a non-irritant (No Category/ not classified) according to the UN GHS classifications and the CLP classification system in a BCOP assay following the OECD 437 test guideline.
Executive summary:

The objective of this study was to evaluate the ocular irritation potential of a-Ketoglutarate disodium salt (CAS No. 305-72-6) with the OECD 437 Bovine Corneal Opacity and Permeability (BCOP) assay.

Corneas were dissected from freshly obtained cow eyes, and mounted into corneal holders. Following a pre-dose equilibration period, and measurement of baseline opacity (using an opacitometer), the epithelial sides of the corneas were treated as follows:

alpha-Ketoglutarate disodium salt (20%, w/v in physiological saline; 750 µL), physiological saline or imidazole (20%, w/v in physiological saline) as negative and positive controls, respectively, were each applied to three corneas. Following exposure for 4 h ± 10 min, the test or control items were rinsed off.

The opacity of each cornea was then determined, followed by measurement of permeability by assessment of the passage of sodium fluorescein through the cornea. Irritancy was assigned on the basis of in vitro irritancy scores (IVIS). The results are summarised as follows:

The mean IVIS score for alpha-Ketoglutarate disodium salt was 1.28

In conclusion the BCOP assay categorised alpha-Ketoglutarate disodium salt (CAS No. 305-72-6) as a non-irritant (No Category/not classified) according to the UN GHS classifications and the CLP classification system.