Calcium dipropionate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 2009-09-07

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:

, 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Calcium dipropionate

EC Number:

223-795-8

EC Name:

Calcium dipropionate

Cas Number:

4075-81-4

Molecular formula:

C3H6O2.1/2Ca

IUPAC Name:

calcium dipropanoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Calcium dipropionate
- Physical state: solid, white (powder)
- Storage condition of test material: at room temperature, protected from light (further detailed: Harlan CCR SOP SUBST.doc)

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:

other: 0.9% (w/v) NaCl in deionised water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
Prior to the application a 20% (w/v) solution of the test item (711.47 mg) in 0.9% (w/v) NaCl in deionised water (3.56 mL) was prepared using ultrasonic technique.

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

OUTLINE OF STUDY
The anterior compartment received the test item suspension or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 141112)) or positive control (10% (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 181012)) at a volume of 0.75 mL each on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath in a horizontal position. The test item, positive control and negative control were tested in triplicate.
The incubation time lasted 240 minutes.
After the test item or control items, respectively, were rinsed off from the application side with 0.9% (w/v) NaCl in deionised water, fresh cMEM was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

OPACITY MEASUREMENT
The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test items and the negative and positive controls. The opacity was measured again after treatment with the test item, positive control and negative control (t240).

PERMEABILITY DETERMINATION
After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values. Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Results and discussion

In vitro

Results

Irritation parameter:

other: IVIS

Value:

13.01

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

not corrosive/non severe irritant

In vivo

Irritant / corrosive response data:

Relative to the negative control, the test item calcium dipropionate caused a slight increase of the corneal opacity, but permeability effects could not be observed. The calculated mean in vitro irritation score was 13.01 (threshold for corrosivity / severe irritancy: ≥ 55.1). According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

Any other information on results incl. tables

Table 1: Results after 240 minutes incubation time

Test group	Opacity value = Difference (t240 – t0) of opacity		Permeability at 490 nm (OD490)		In vitro irritation score	Mean in vitro irritation score	Proposed in vitro irritation scale
		Mean		Mean
Negative control	1	1.00	0.048	0.049	1.72	1.74	Non corrosive / non severe irritant
	1		0.044		1.66
	1		0.056		1.84
Positive control	215.00*		0.007*		215.10	212.55	Corrosive / severe irritant
	213.00*		0.013*		213.19
	209.00*		0.024*		209.36
Calcium dipropionate	10.00*		0.012*		10.18	13.01	Non corrosive / non severe irritant
	13.00*		-0.002*		12.97
	16.00*		-0.007*		15.89

*corrected values

- With the negative control neither an increase of opacity nor permeability of thecorneae could be observed (mean in vitro irritation score 1.74).

- The positive control caused distinct opacity on the corneae (mean in vitro irritation score 212.55) corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Table 2: Historical data

	Positive control	Negative control
Mean in vitro Irritation Score	176.71	1.78
Standard Deviation	42.65	0.75
Range of in vitro irritation scores	99.4 - 292.3	0.41 - 2.99

Values of 138 studies with solid test items performed until November 2012

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive / not severe irritant to the eye

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item calcium dipropionate is not corrosive / not severely irritating to the eye (CLP/EPA/GHS (Cat 1)).