Diammonium dihydrogenpyrophosphate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

Ex vivo corrosion study performed to support in vivo data submitted.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 18/07/2017 - 20/07/2017 Date of Signature: 28/11/2017

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium dihydrogenpyrophosphate

EC Number:

237-059-9

EC Name:

Diammonium dihydrogenpyrophosphate

Cas Number:

13597-86-9

Molecular formula:

H4O7P2.2H3N

IUPAC Name:

diazanium;phosphono phosphate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Not supplied
- Expiration date of the lot/batch: 05 October 2018
- Purity test date: Not supplied

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C, in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

other: sodium chloride 0.9% w/v

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% w/v

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% w/v
- Lot/batch no. (if required): 3012488
- Purity: 0.9%

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

For the permeability of the corneas to sodium fluoroscein the corneas were incubated for 90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.


NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
No (see below)

SOLVENT CONTROL USED
Yes

POSITIVE CONTROL USED
Yes


TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes - 90 min incubation with sodium fluoroscein.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of the Labtech LT-4500 microplate reader.

- Others: no histology required. The condition of the cornea was visually assessed post treatment.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) . Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA: Decision criteria as per the TG. The test item was classified according to the following prediction model:
IVIS UN GHS EU CLP (Regulation (EC) No 1272/2008)
≤ 3 No Category Not classified for irritation
>3 ≤ 55 No prediction can be made No prediction can be made
> 55 Category 1 Category 1 H318: Causes serious eye damage

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

average of 3 replicates

Value:

7.2

Vehicle controls validity:

valid

Positive controls validity:

not valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.

- Acceptance criteria met for positive control: The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the following table:

Treatment	Cornea Number	Opacity				Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	4	4	0		0.003
	3	2	6	4		0.001
	6	4	4	0		0.011
				1.3*		0.005¨		1.4
Positive Control	7	1	97	96	94.7	1.605	1.600
	10	1	93	92	90.7	1.965	1.960
	11	2	102	100	98.7	2.165	2.160
					94.7·		1.907·	123.3
Test Item	13	1	11	10	8.7	0.089	0.084
	14	2	8	6	4.7	0.035	0.030
	15	2	10	8	6.7	0.005	0.000
					6.7·		0.038·	7.2

 

OD= Optical density           * = Mean of the post-treatment -pre‑treatment values             ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition

The condition of each cornea was as follows:

Treatment	Cornea Number	Observation Post Treatment
Negative Control	2	Clear
	3	Clear
	6	Clear
Positive Control	7	Cloudy
	10	Cloudy
	11	Cloudy
Test Item	13	Cloudy
	14	Cloudy
	15	Cloudy

 

The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	7.2
Negative Control	1.4
Positive Control	123.3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not considered to be irritating to the eyes.

Executive summary:

SUMMARY

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye

damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS UN GHS EU CLP (Regulation (EC) No 1272/2008)

≤ 3 No Category - Not classified for irritation

>3; ≤ 55 No prediction can be made

> 55 Category 1 - Category 1 H318: Causes serious eye damage

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	7.2
Negative Control	1.4
Positive Control	123.3