Dizinc pyrophosphate

Endpoint summary

Description of key information

Skin irritation: OECD 439: not irritant

Eye irritation: OECD 437: not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun-14 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health and Consumer Protection, Hamburg, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation being used as a replacement for the Draize Skin irritation test. Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiDerm™ model
- Tissue batch number(s): EPI-200, Lot no. 25829
- Date of initiation of testing: 20 June 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment/exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: All tissues were washed carefully with DPBS after the exposure period.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues/killed tissues: killed tissues
- Procedure used to prepare the killed tissues: incubating in distilled water at 37 °C for 42 h
- N. of replicates: 3
- Method of calculation used: direct comparison of treated to untreated water-killed tissues

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be a non-irritant to skin if the viability of three individual tissues after 60 minutes exposure period to the test substance followed by 42 hours post-exposure incubation is greater than 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- OTHER EFFECTS:
- Direct-MTT reduction: The results of the direct interference evaluation procedure showed that no interference due to direct reduction of MTT by the test substance occurred.
- Colour interference with MTT: An assessment found the test item had no potential to cause colour interference with the MTT endpoint. It was therefore considered unnecessary to use the results of the colour correction tissues for quantitative correction of results or for reporting purposes

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg applied uniformly over an area of 0.63 cm²

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): DPBS 30 µL
- Concentration (if solution): 100%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): aqueous sodium dodecyl sulphate 30 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

35 min at 37 °C and 25 min at RT

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-min exposure followed by 42-hour post-exposure incubation period

Value:

101.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

6.5%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 for the negative control treated tissues was 1.396 and the standard deviation determined for all triplicates was below 18%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.5% relative to the negative control treated tissues. The positive control acceptance criteria was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The relative mean tissue viability for the test item treated tissues was 101.6% of the negative controls after a 60-min exposure period and 42-hour post-exposure incubation period. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was <18%. The test item acceptance criteria was therefore satisfied.

 Mean OD540 Values and Viabilities for the Negative Control, Positive Control, and Test Items

Item	OD540 of tissues	Mean OD540 of triplicate tissues	%CV of mean OD540	Relative individual tissue viability (%)	Relative mean viability (%)	%CV of Relative mean viability (%)
Negative Control	1.313	1.396	7.6	94.1	100*	7.6
	1.360			97.4
	1.516			108.6
Positive Control	0.083	0.092	10.4	5.9	6.5	10.9
	0.090			6.4
	0.102			7.3
Test Item	1.349	1.418	5.3	96.6	101.6	5.2
	1.408			100.9
	1.497			107.2

* = The mean viability of the negative control tissues is set at 100%

OD = Optical density

CV = Coefficient ovf variation

Interpretation of results:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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)

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.

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.

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

Interpretation of results:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Reliable data is available in order to examine the skin irritation potential of the test substance (LPT, 2017). The EpiDerm model according to OECD 439 was performed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues. The test substance was applied as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium. The mean viability of cells exposed to the test substance was 101.6% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. The test substance was considered to be non-cytotoxic and predicted to be non-irritant to skin. The mean optical density (OD) of 3 negative control tissues was 1.396 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 6.5% of the negative control and fulfilled the acceptance criterion of ≤ 20%. The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria were fulfilled.

In conclusion, the test substance did not show irritant properties.

Eye irritation:

Reliable data is available in order to examine the eye irritation potential of the test substance (LPT, 2017). The BCOP assay according to OECD 437 was performed.

Three corneas were used for each treatment group (test item, solvent control and positive control). The solid test item was dissolved in a 0.9% sodium chloride solution with a final concentration of 20% test substance as recommended in the test guideline 437 for non-surfactant solids. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm. The acceptance criteria of validity were fulfilled in this test.

Following treatment with the test substance a mean opacity of 1.062 ± 0.900 and a mean permeability value of <0.01 compared to the negative control were determined. The calculated IVIS of 0.627 ± 0.911 is below the cut-off value of 3 (CLP no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified as a severe irritant and is not corrosive according to CLP classification.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.