Dizinc pyrophosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun - 20 Jul 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:

adopted July 26, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, Behörde für Gesundheit und Verbraucherschutz

Test material

Test material information

Constituent 1

Reference substance name:

Dizinc pyrophosphate

EC Number:

231-203-4

EC Name:

Dizinc pyrophosphate

Cas Number:

7446-26-6

Molecular formula:

H4O7P2.2Zn

IUPAC Name:

dizinc(2+) (phosphonooxy)phosphonate

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, Lindern, Germany
- Characteristics of donor animals: 6 - 12 months old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) containing penicillin (100 IU/mL) and streptomycin (100 µg/mL).
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: Penicillin (100 IU/mL) and streptomycin (100 µg/mL)

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 20% suspension

VEHICLE
- Amount(s) applied: 750 µL
- Concentration: 0.9%
- Lot/batch no.: 170518002

POSITIVE CONTROL
- Amount(s) applied: 750 µL
- Concentration: 20%
- Lot/batch no.: SLBK9670V

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32 °C ± 1 °C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF, Ludwigshafen am Rhein, Germany).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Tecan Sunrise Magellan Version 7.2, Crailsheim, Germany) at 490 nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneas

Value:

0.627

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The corneas treated with the negative control item 0.9 % sodium chloride solution revealed a mean opacity value of 1.209 ± 0.807 and a mean permeability value of 0.031 ± 0.011. The calculated IVIS value of 1.669 ± 0.956 was well below the cut-off value of 3 (UN GHS no category).
- Acceptance criteria met for positive control: The corneas treated with the positive control item 20 % Imidazole in 0.9 % NaCl solution revealed a mean opacity value of 59.694 ± 7.591 and a mean permeability value of 3.011 ± 0.100 compared to the solvent control. The calculated IVIS value of 104.864 ± 8.811 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Any other information on results incl. tables

Table 2: Opacity values

							Cornea No.				Opacity [Opacity Units]							Corrected Opacity
																							Mean of group					Standard deviation
0.9 % NaCl							1				0.558							1.209					1.209					0.807
							2				0.956
							3				2.112
20 % Imidazol							4				53.386							52.177					59.694					7.591
							5				60.757							59.548
							6				68.566							67.357
20 % Test substance							7				2.710							1.501					1.062					0.900
							8				2.868							1.659
							9				1.236							0.027

Table 3: Permeability OD values (490 nm)

					Corne a no.			Permeability [OD]							Mean of Tripli- cates					Corrected Permeability [OD]
																								Per Cornea						Per Group
																								Mean			SD			Mean				SD
0.9 % NaCl					1			0.028							0.027					-				0.027			0.001			0.031				0.011
								0.027												-
								0.027												-
					2			0.022							0.022					-				0.022			0.001
								0.023												-
								0.022												-
					3			0.042							0.043					-				0.043			0.002
								0.042												-
								0.045												-
20 % Imidazol					4			2.908							2.928					2.877				2.897			0.018			3.011				0.100
								2.934												2.903
								2.942												2.911
					5			3.054							3.113					3.023				3.082			0.051
								3.142												3.111
								3.142												3.111
					6			3.010							3.086					2.979				3.055			0.070
								3.100												3.069
								3.148												3.117
20 % Test substance					7			0.007							0.004					-0.024				-0.027			0.003			-0.029				0.002
								0.004												-0.027
								0.002												-0.029
					8			0.000							0.001					-0.031				-0.030			0.001
								0.002												-0.029
								0.001												-0.030
					9			0.000							0.001					-0.031				-0.030			0.001
								0.002												-0.029
								0.001												-0.030

SD: Standard deviation

OD: Optical density

Table 4: IVIS

								Cornea No.					Opacity					Permeability							IVIS
																									Per Cornea				Per Group
																													Mean			SD
0.9 % NaCl								1					0.558					0.027							0.963				1.669			0.956
								2					0.956					0.022							1.286
								3					2.112					0.043							2.757
20 % Imidazol								4					52.177					2.897							95.632				104.864			8.811
								5					59.548					3.082							105.778
								6					67.357					3.055							113.182
20 % Test substance								7					1.501					-0.027							1.096				0.627			0.911
								8					1.659					-0.030							1.209
								9					0.027					-0.030							-0.423

SD: Standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008