(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), compound with 2-aminoethanol (1:[?])

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), compound with 2-aminoethanol (1:?)

EC Number:

814-283-0

Cas Number:

42220-47-3

Molecular formula:

C4 H13 N1 O7 P2

IUPAC Name:

(1-hydroxyethane-1,1-diyl)bis(phosphonic acid), compound with 2-aminoethanol (1:?)

Details on test material:

Physical state/ Appearance:liquid/yellowish, clear

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0016044582
- Content: 91.4 /100 g (100 g/100 g minus water content)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: No analysis of test substance preparation was performed because the test substance was applied minimally moistened with deionized water or PBS.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
pH value: Ca. 6 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
Physical state / color: Liquid, high viscous to solid, waxy / colorless to light yellowish to white
Handling of the test substance: The solid test substance was grinded with pestle and mortar before application.
Storage conditions: Room temperature

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof (slaughterhouse) Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: none

Test system

Vehicle:

water

Remarks:

deionized

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750µL
- Concentration: 20% (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

The negative control and the positive control were removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
Because the test substance could not be removed by using a syringe the epithelium was rinsed with the open chamber method.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION, PREPARATION AND QUALITY CHECK OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour.

TREATMENT METHOD: [closed chamber / open chamber]
The test-substance preparation (non-surfactant) was applied directly to the epithelial surface of the cornea by using a pipette (open chamber method).

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was spectrophotometrically measured. Three aliquots per cornea were transferred to a 96 well microtiter plate (Sunrise Absorbance Reader) and the optical density (OD490) was determined. An aliquot was diluted 1:5 in Eagle’s MEM (without phenol red) and analogously measured (positive control and test substance).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria were modified and amended with a borderline evaluation based on additional internal validation data: The borderline evaluation was statistically determined by using historic BASF data and hence considers the variance of the test method.

IVIS Prediction
< 1.5 No classification for eye irritation
1.5 – 4.5 Borderline
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another suitable method is required
45 - 65 Borderline
> 65 Ocular corrosive or severe irritant

The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437. There is no relevance for the outcome of this study since the IVIS is greater than 65.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: mean opacity value

Run / experiment:

Mean value from three replicates.

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: mean permeability value

Value:

0.001

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: In Vitro Irritancy Score

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Opacity score of the test substance, the NC and the PC

 

Test substanceidentification	Cornea-No.	Initialopacity	Finalopacity	OpacityChange	Corrected Opacity Change*	Mean	SD
	7	6.3	3.7	-2.5	0.0
16/0277-1	8	5.2	3.9	-1.4	0.0	0.0	0.0
	9	5.4	5.4	0.0	0.0
	1	4.5	18.6	14.2	NA
NC	2	4.4	13.3	9.0	NA	16.2	8.4
	3	3.1	28.5	25.4	NA
	4	3.9	105.7	101.7	85.6
PC	5	3.9	97.1	93.2	77.0	83.8	6.0
	6	6.8	111.7	104.8	88.7

 

  Permeability score of the test substance, the NC and the PC

 

Test substance identification	Cornea- No.	Mean OD490	Dilution Factor	Mean Corrected OD490*	Mean	SD
	7	0.001	1	0.000
16/0277-1	8	0.002	1	0.001	0.001	0.001
	9	0.004	1	0.002
	1	0.001	1	NA
NC	2	0.002	1	NA	0.002	0.001
	3	0.003	1	NA
	4	0.457	5	2.283
PC	5	0.386	5	1.926	1.787	0.578
	6	0.231	5	1.153

 

* Negative values are set to zero for further calculation

In Vitro Irritancy score (IVIS) of the test substance, the NC and the PC

Test substance identification	Cornea No	Opacity per Cornea	Permeability per cornea	IVIS per cornea     per group                mean        SD
16/0277-1	7	0.0	0.000	0.0
	8	0.0	0.001	0.0	0.0	0.0
	9	0.0	0.002	0.0
NC	1	14.2	0.001	14.2
	2	9.0	0.002	9.0	16.2	8.4
	3	25.4	0.003	25.5
PC	4	85.6	2.283	119.8
	5	77.0	1.926	105.9	110.6	8.0
	6	88.7	1.153	106.0

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed for the BCOP test, the test substance does not cause irreversible eye effects