Diphenyl methylphosphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-08 to 2012-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to Guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

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)

Test material

Test material information

Constituent 1

Reference substance name:

Diphenyl methylphosphonate

EC Number:

231-388-1

EC Name:

Diphenyl methylphosphonate

Cas Number:

7526-26-3

Molecular formula:

C13H13O3P

IUPAC Name:

diphenyl methylphosphonate

Details on test material:

- Name of test material (as cited in study report): Diphenyl methylphosphonate
- Analytical purity: 99.88 %
- Stability under test conditions: not indicated by the sponsor
- Storage condition of test material: at room temperature, under nitrogen

Test animals / tissue source

Species:

cattle

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

freshly isolated bovine cornea

Test system

Vehicle:

physiological saline

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): application of 0.75 mL test item dissolved in saline (20 % (w/v))
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): application of 0.75 mL test item dissolved in saline (20 % (w/v))
- Concentration (if solution):0.9 % (w/v)
- Lot/batch no. (if required): 310112

Duration of treatment / exposure:

240 min (+/- 15 min). At the beginning of the test, the basal opacity of all corneae was recorded (t0). After 240 minutes of incubation with the test item, the opacity was measured again (t240).

Number of animals or in vitro replicates:

3 freshly isolated bovine eyes were used per group (positive control, negative control, test item).

Details on study design:

Positive Control
10 % (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9 % (w/v) NaCl in deionised water (saline, produced in-house, lot no. 310112) served as positive control.

Negative Control
Saline (produced in-house, lot no. 310112) served as negative control.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

6.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Evaluation of Results

Opacity

The change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 - t0), for each individual cornea. The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability

The corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

In Vitro Irritation Score Calculation

The following formula was used to determine the in vitro irritation score of the negative control:

In vitro Irritation Score = opacity value + (15 * OD490 value)

The following formula was used to determine the in vitro irritation score of the positive control and the test item:

In vitro Irritation Score = (opacity value - opacity value mean negative control) + (15 * corrected OD490 value)

The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values.

Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437:

In vitro Irritation Score	In vitro Irritation Scale(according to OECD 437)
≥ 55.1	Corrosive/ Severe irritant CLP/EPA/GHS(Cat 1)

Data Recording

The data generated were recorded in the raw data file. The results were presented in tabular form, including experimental groups with the test item, negative, and positive controls. The data of permeability measurement were registered and transferred into an Excel sheet.

Historical Data

	Positive Control	Negative Control
Mean in vitro Irritation Score	173.79	1.74
Standard Deviation	43.90	0.74
Values of 138 studies with solid test items performed in 2008 to 2012

Results

Results after 240 Minutes Incubation Time
Test Group	Opacity value = Difference (t240 -t0) of Opacity		Permeability at 490nm (OD490)		In vitro Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	1	0,67	0,048	0.050	1.72	1.42	Non corrosive / non severe irritant
	1		0,050		1.75
	0		0,053		0.80
Positive Control	230.33*		0.035*		230.85	234.77	Corrosive / severe irritant
	257.33*		0.020*		257.63
	214.33*		0.100*		215.83
Test Item	6.33*		0.046*		7.02	6.50	Non corrosive / non severe irritant
	3.33*		0.054*		4.14
	8.33*		0.000*		8.33
*corrected values

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item Diphenyl methylphosphonate is not corrosive / not severely irritating to the eye (CLP/EPA/GHS (Cat 1)).

Executive summary:

In compliance with GLP criteria and according to OECD guideline 437 as well as EU guideline No 1152/2010: B.47, this in vitro study was performed to assess the corneal irritation and damage potential of Diphenyl methylphosphonate by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20 % (w/v) solution in saline (0.9 % (w/v) NaCl in deionised water) of the test item Diphenyl methylphosphonate, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained MEM medium supplemented with sodium bicarbonate and L-glutamine and 1 % fetal calf serum (FCS) (complete medium = cMEM). After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10 % (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Diphenyl methylphosphonate did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritation score was 6.50. According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

In conclusion, according to the current study and under the experimental conditions reported, the test item Diphenyl methylphosphonate is not corrosive / not severe irritant to the eye (CLP/EPA/GHS (Cat 1)).