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Toxicological information

Eye irritation

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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 guidelineopen allclose all
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
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

Constituent 1
Chemical structure
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:
diammonium dihydrogenpyrophosphate
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