Ammonium dihydrogen citrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 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:

26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium dihydrogen citrate

EC Number:

224-694-1

EC Name:

Ammonium dihydrogen citrate

Cas Number:

4450-94-6

Molecular formula:

C6H8O7.H3N

IUPAC Name:

azanium; 3-carboxy-3,5-dihydroxy-5-oxopentanoate

Constituent 2

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:

liquid

Details on test material:

Identification: Ammonium di hydrogen citrate
CAS number: 4450-94-6
Appearance: Clear colourless liquid

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL per cornea

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS
The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Physiological saline.

POSITIVE CONTROL USED
Ethanol.

APPLICATION DOSE AND EXPOSURE TIME
750 μL for 10 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: two
- POST-EXPOSURE INCUBATION: 120 ± 10 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Nafluorescein was evaluated. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
- Others: Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value).

DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgeory 1);
For a test substance that induces an IVIS >3 and ≤ 55, no prediction on irritant potency can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Single run

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas were clear after the 10 minutes of treatment with Ammonium di hydrogen citrate.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (Ethanol) was 48 and within two standard deviations of the current historical positive control mean.
It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

According to Regulation (EC) No 1272/2008.

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD 437 guideline and GLP principles, it is concluded that the substance is not irritant or corrosive for the eye.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with the substance according to OECD guideline 437 and GLP principles. The substance was applied undiluted (750 μL/ cornea, n=3).

The mean in vitro irritancy score of the positive control (ethanol) was 48, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.1. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The substance did not induce ocular irritation through opacity or permeability, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment. Since the substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to Regulation (EC) No 1272/2008.