Reaction mass of diammonium (2R,3R)-2,3-dihydroxybutanedioate and diammonium bis[(2R,3R)-2,3-dioxidobutanedioato]diantimonate(2-)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

09 Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of diammonium (2R,3R)-2,3-dihydroxybutanedioate and diammonium bis[(2R,3R)-2,3-dioxidobutanedioato]diantimonate(2-)

EC Number:

943-693-3

IUPAC Name:

Reaction mass of diammonium (2R,3R)-2,3-dihydroxybutanedioate and diammonium bis[(2R,3R)-2,3-dioxidobutanedioato]diantimonate(2-)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 170492F01
- Expiration date of the lot/batch: 22 Jun 2019
- Purity: 100.0 area-% (HPLC fingerprint)
- Content:
21.4 g/100 g Test Item
23.5 g/100 g (NH4)2C4H4O6
30 g/100 g Tartrate
6.7 g/100 g Ammonium
9.1 g/100 g Antimony
53.5 g/100 g Water
- pH value: ca. 9 (undiluted test substance)
- Physical state / color: Liquid / colorless to yellowish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Straße 23, 55232 Alzey, Germany
- indication of any existing defects or lesions in ocular tissue samples: no

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): 750 µL

Duration of treatment / exposure:

approx. 10 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Bovine eyes are obtained as a by-product of freshly slaugthered cattle (age of the animals: minimum 12 months, maximum 60 months).

QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle's MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 554 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: deionized water

POSITIVE CONTROL USED: 100 % ethanol for liquid test substances and surfactants

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 minutes

POST-INCUBATION PERIOD: yes, 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with Eagle's MEM (containing phenol red) and once with Eagle's MEM (without phenol red)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was visually observed an observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: For determination of permeability, the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 +/- 5 minutes in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was spectrophotometrically measured. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3: No UN GHS Category
IVIS > 3; ≤ 55: No prediction can be made
IVIS > 55: UN GHS Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

4.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Opacity score of the test substance, the NC and the PC

Test substance identification	Cornea-No.	Initial opacity	Final opacity	Opacity change	Corrected opacity change	Mean	SD
Test substance	16 17 18	6.1 4.7 6.4	15.7 10.9 15.0	9.6 6.2 8.6	6.4 3.0 5.4	4.9	1.8
NC	10 11 12	3.8 5.6 4.8	4.1 8.4 11.4	0.3 2.8 6.6	NA NA NA	3.3	3.2
PC	13 14 15	4.7 5.3 4.4	35.6 41.0 39.3	31.0 35.7 34.9	27.7 32.4 31.6	30.6	2.5

NC: negative control

PC: positive control

SD: standard deviation

NA: not applicable

Table 2: Permeability score of the test substance, the NC and the PC

Test substance identification	Cornea-No.	Mean OD490	Dilution factor	Mean corrected OD490	Mean	SD
Test substance	16 17 18	0.004 0.008 0.000	1 1 1	0.000 0.002 0.000	0.001	0.001
NC	10 11 12	0.000 0.016 0.004	1 1 1	NA NA NA	0.007	0.008
PC	13 14 15	1.260 1.157 0.993	1 1 1	1.253 1.150 0.986	1.130	0.135

NC: negative control

PC: positive control

SD: standard deviation

NA: not applicable

Table 3: In Vitro Irritancy Score (IVIS) of the test substance, the NC and the PC

Test substance identification	Cornea-No.	Opacity per cornea	Permeability per cornea	IVIS
				Per cornea	Per group
					Mean	SD
Test substance	16 17 18	6.4 3.0 5.4	0.000 0.002 0.000	6.4 3.0 5.4	4.9	1.7
NC	10 11 12	0.3 2.8 6.6	0.001 0.016 0.004	0.3 3.1 6.7	3.4	3.2
PC	13 14 15	27.7 32.4 31.6	1.253 1.150 0.986	46.5 49.7 46.4	47.5	1.9

NC: negative control

PC: positive control

SD: standard deviation

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction can be made for eye irritation according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

The potential of the test substance to cause ocular irritation or serious eye damage to the eyes was assessed by a single topical application of 750 µL undiluted test substance to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test substance for 10 minutes followed by a 2-hours post-incubation period.

In addition to the test substance, a negative control (NC, deionized water) and a positive control (PC, 100 % ethanol) were applied to three corneas each.

Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score (IVIS) of the test substance.

The following results were obtained in the BCOP Test:

Test substance identification	Mean Opacity Value	Mean Permeability Value	Mean IVIS
Test substance	4.9	0.001	4.9
NC	3.3	0.007	3.4
PC	30.6	1.130	47.5

NC: negative control

PC: positive control

IVIS: in vitro irritancy score