Tetraamminezinc(2+) carbonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 December 2016 - 17 November 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 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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Tetraamminezinc(2+) carbonate

EC Number:

254-099-2

EC Name:

Tetraamminezinc(2+) carbonate

Cas Number:

38714-47-5

Molecular formula:

Zn(NH3)4 CO3

IUPAC Name:

Tetraamminezinc(2+) carbonate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: freshly slaughtered cattle, age of the animals: minimum 12 months, maximum 60 months

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

Details on study design:

SELECTION AND PREPARATION OF 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 with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer.

QUALITY CHECK OF THE ISOLATED CORNEAS
Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: de-ionized water

POSITIVE CONTROL USED: 100% ethanol

APPLICATION DOSE AND EXPOSURE TIME
Before application, the medium in the anterior chamber was removed using a syringe. 750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes.

TREATMENT METHOD: closed chamber

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)
- POST-EXPOSURE INCUBATION: 2 hours at about 32 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: before measurement, each cornea was observed visually and observations were recorded

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITARIA:
The following decision criteria apply:

IVIS Prediction
< 1.5 No classification for eye irritation (1)
1.5 – 4.5 Borderline (1,3)
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another suitable method is required (1,2)
45 - 65 Borderline (1,3)
> 65 Ocular corrosive or severe irritant
(1) According to OECD Guideline 437 (adopted July 2013), this prediction is possible, however, due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances not leading to the prediction “ocular corrosive or severe irritant” are
generally examined in the EpiOcular test as well.
(2) The test method according to the OECD Guideline 437 revised and adopted in 2013 does not allow for the evaluation of eye irritation. I.e., the result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study are needed.
(3) The borderline“-evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was determined statistically using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

1st experiment

Value:

23.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2.4

Positive controls validity:

valid

Remarks:

24.1

Results 2

Irritation parameter:

other: permeability score

Run / experiment:

1st experiment

Value:

0.067

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

0.931

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

1st experiment

Value:

24.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2.4

Positive controls validity:

valid

Remarks:

38 (PC1) and 107.4 (PC2)

Applicant's summary and conclusion

Interpretation of results:

other: not eye damaging (Cat.1)