Ammonium 6H-dibenzo[c,e][1,2]oxaphosphinin-6-olate 6-oxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2011 - September 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully Guideline- and GLP-compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD-guideline 437, Bovine Corneal Opacity and Permeability Test Method (BCOP) , 7. September 2009

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

DOPO-OX-Ammonium

IUPAC Name:

DOPO-OX-Ammonium

Details on test material:

Trade name: DOPO-OX-Ammonium.
Chemical name: 9,10-dihydro-10-hydroxy-9-oxa-10-phosphaphenanthren-10-on- resp. 10-oxid-ammonium salt, corrected later to: 9,10-dihydro-10-hydroxy-9-oxa-10-phosphaphenanthren-10-oxid-ammonium salt
Article no: 21908200.
Molecular formula: C12H12NO3P.
Charge. No.:DAS-1101.
Appearance: White to slightly yellow solid.
Conditions of storage: At room temperature.
Expiry date: The sponsor guarantees stability during test period under storage conditions.
Certificate of Analysis available.
The analysis of the test substance for identity and purity is at the responsibility of the sponsor.

Test animals / tissue source

Species:

other: in vitro system: bovine corneas

Strain:

other: Isolated corneas from the eyes of cows and bulls aged between 24 – 26 month and free of macroscopically visible defects.

Details on test animals or tissues and environmental conditions:

Isolated corneas from the eyes of cows and bulls aged between 24 - 26 month and free of macroscopically visible defects from the slaughterhouse
"Klaus Grandis", Ungerbachstraße 10, 2860 Kirchschlag, Austria were used. The study required a total of 9 corneas (3 for the test substance, 3 for
the negative control and 3 for the positive control) and additionally 3 corneas for the benchmark control.

Test system

Vehicle:

water

Remarks:

deionised water

Controls:

yes

Amount / concentration applied:

Application amount: 750µL.
Test substance: inhomogeneous suspension of 20% (w/v) in deionised water.
Positive control: Imidazole solution of 20% (w/v) in deionised water.
Negative control: deionised water.
Benchmark control: 0.9 % sodium chloride.

Duration of treatment / exposure:

4 hours.

Observation period (in vivo):

Endpoint measurement of opacity change and permeability values.

Number of animals or in vitro replicates:

9 corneas (3 for the test substance, 3 for the negative and 3 for the positive control) and additionally 3 corneas for the benchmark substance.

Details on study design:

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined after equilibration. 750 µL of
the test substance preparation were topically administered to 3 isolated bovine corneas to the epithelial surfaces for 4 hours and the final opacity
was measured. Then 1 ml of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.
Three groups of 3 corneas each served as positive, negative and benchmark controls. All control substances were administered under identical
conditions as the test substance. The following solutions served as control substances:
Negative control: sterile aqua dest.
Positive control: 20 % imidazole.
Benchmark control: 0.9 % sodium chloride.
Finally the IVIS (In Vitro Irritancy Score) was calculated as follows:
IVIS = mean opacity value + (15 x mean permeability).
The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity
value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS ≥ 55.1 is defined as ocular corrosive or severe
irritant. The positive and negative control groups were simultaneously used for other, concurrently performed studies.

Results and discussion

In vivo

Results

Irritation parameter:

other: IVIS (in vitro irritancy score)

Basis:

mean

Time point:

other: 4 hours

Score:

39.3

Reversibility:

not specified

Remarks on result:

other: not ocular corrosive or severe irritant

Irritant / corrosive response data:

The IVIS of DOPO-OX-Ammonium was 39.3. Furthermore, an increase in opacity and no increase in permeability was observed.

Other effects:

The validity of the opacitometer is given since the measurements of the control filters were within the range of the historical data.
The validity of the cornea holder is given because the cornea holder control value Io was within the range of the historical data.
The correlation coefficient of the fluorescein dilutions was 0.995. Since the correlation coefficient was > 0.99 the linearity was given.
The mean IVIS of the corneas, treated with 20 % imidazole (PC) was 96.7, which is within the range of the historical data.
The mean opacity and the mean permeability (not corrected for negative control opacity and permeability values, but corrected for background
opacity and permeability values) of the corneas, treated with deionised water (NC) were 8.9 and -0.002 respectively.
The opacity of the negative control (8.9) slightly exceeds the upper limit of the historical data (8.5). This deviation is considered to be of no relevance for the outcome of this study since the deviation is very small in reference to a small amount of historical data (n=11) and that the test system is
biological material and high temperature during slaughter causes cell damage as in house practical experience showed.
Furthermore, the benchmark control (0.9 % NaCl) which is recommended as negative control substance by the “Draft Guidance Document in the
Supplement of Test Guidelines 437 and 438” had an IVIS (not corrected for negative control opacity and permeability values) of 0.8 which is in the
range of negative controls.
The mean IVIS of the corneas, treated with 0.9 % Sodium chloride was -8.1 (0.8 not corrected for negative control opacity and permeability values),
no historical data are available.

Any other information on results incl. tables

Results:

Opacity, permeability and IVIS values

Opacity, permeability (1 x value measured and 15 x values for IVIS calculation) and IVIS values of test substance, negative and positive controls. Individual data, means and standard deviations (SD).The mean opacity and mean permeability values of the positive control, the test substance and the benchmark substance were corrected for background and negative control opacity and permeability values. The negative control values in this table are listed not corrected for negative control opacity but for background permeability.

Substance	Opacity			Permeability (1x)			Permeability (15x)		IVIS
	Individual	Mean	SD	Individual	Mean	SD	Mean	SD	Individual	Mean	SD
Negative control (Aqua dest.)	6,3	8,9	3,7	0,002	0,000	0,002	0,000	0,029	6,3	8,9	3,7
	13,1			-0,001					13,1
	7,2			-0,001					7,2
Positive control (Imidazole)	53,8	54,8	3,0	2,237	2,792	0,847	41,882	12,700	87,3	96,7	11,1
	52,5			3,767					109,0
	58,2			2,373					93,8
Test Substance	35,8	39,3	4,0	-0,003	-0,002	0,001	-0,032	0,013	35,7	39,3	4,0
	43,6			-0,001					43,6
	38,6			-0,002					38,6
Benchmark control 0.9 % NaCl	-7,1	-8,1	0,9	-0,002	-0,003	0,001	-0,038	0,013	-7,1	-8,1	0,9
	-8,5			-0,003					-8,6
	-8,7			-0,002					-8,8

Opacitometer reference filter values, cornea holder control measurement, negative (NC) and positive (PC) control values

Acceptance criteria of the opacitometer via measurement of the reference filter set. The control measurement was made before determination of the initial and final opacity.

Reference filter	Measured value LUX (before initial/final opacity)	Set point range Lux (historical mean +/- 2xSD)
Empty Filter holder	1001/1001	997 – 1007
F2 (NG11)	544/539	536 – 545
F3 (NG5)	300/298	296 – 301
F4 (NG4)	98/98	97 – 100

Acceptance criteria of the cornea holder via measurement of an empty cornea holder with medium.

Empty cornea holder with medium	Measured value LUX	Set point range Lux
# 16	661	630 - 720

Measured opacity and permeability values of the negative control (NC).

NC	Measured Opacity value (mean +/- SD)	Set point range Opacity upper limit (historical mean +/- SD)	Measured permeability value (mean +/- SD)	Set point range Permeability upper limit (historical mean +/- SD)
Aqua dest.	8,9 +/-3,7	3,5 – 7,5	-0,002 +/-0,002	0,0041 +/-0,0076

Calculated IVIS of the positive control (PC).

PC	Calculated IVIS (mean +/- SD)	Set point range IVIS (historical mean +/- 2xSD)
20 % Imidazole	96,7 +/-11,1	92,5 – 130,1

The historical data:

The historical data includes all generated data of this year. The historical range of the cornea holder is generated by measurement of all holders once of this year.

Acceptance criteria of the opacitometer via reference filter set.

Historical data	Filter holder empty	Filter holder F2 (NG11)	Filter holder F3 (NG5)	Filter holder F4 (NG4)
Min/max values (LUX)	997/1008	536/545	296/301	97/100
Mean (LUX)	1002	540	298	98
+/- SD (LUX)	5	5	2	2
n (number of measurements)	33	33	33	33

 

Acceptance criteria of all 30 cornea holders with medium.

Historical data	Empty filter holder with medium
Range (Lux)	630 - 720

 

Negative control historical data of the upper limits of opacity and permeability.

NC Aqua dest.	Opacity historical upper limit	Permeability historical upper limit
Min/max values (LUX)	1,5/8,5	-0,005/0,021
Mean (LUX)	5,5	0,004
+/- SD (LUX)	2,0	0,008
n (number of measurements)	11	11

Positive control historical data of the IVIS.

PC 20 % Imdiazole	Historical IVIS
Min/max values (LUX)	100,8/127,8
Mean (LUX)	111,3
+/- SD (LUX)	9,4
2 x +/- SD (LUX)	18,8
n (number of measurements)	9

 

Applicant's summary and conclusion

Interpretation of results:

other: DOPO-OX-Ammonium is regarded to be not an ocular corrosive or severer irritant and needs not to be labelled as R41 (EU), Category 1 (EPA and GHS).

Remarks:

Criteria used for interpretation of results: other: Directive 2011/59/EC for classification.

Conclusions:

The IVIS of DOPO-OX-Ammonium was 39.3. Furthermore, an increase in opacity and no increase in permeability were observed.
Thus DOPO-OX-Ammonium is regarded to be not an ocular corrosive or severe irritant, according to the OECD Guideline 437 for the testing of
chemicals “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants”.
According to the results of this study and the Directive 2001/59/EC for classification, the test substance DOPO-OX-Ammonium requires further
testing as outlined in the OECD guideline 405.
According to the results of this study and the Directive 2001/59/EC for classification, the test substance DOPO-OX-Ammonium needs not to be
labelled as R41 (EU), Category 1 (EPA and GHS).

Executive summary:

Aim:

The Bovine Corneal Opacity and Permeability Study (BCOP Test Method) was performed to reveal possible ocular corrosivity and severe irritation of DOPO-OX-Ammonium, according to the OECD Guideline 437 for the testing of chemicals “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants”.

Method:

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µL of the test substance preparation were topically administered to 3 isolated bovine corneas to the epithelial surfaces for 4 hours and the final opacity was measured. Then 1 mL of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.

Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances:

• Negative control:       sterile aqua dest.
• Positive control:        20 % imidazole.

Finally the IVIS (In Vitro Irritancy Score) was calculated as follows:

IVIS = mean opacity value + (15 x mean permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS ≥ 55.1 is defined as ocular corrosive or severe irritant.

Results:

The IVIS for DOPO-OX-Ammonium was 39.3.

IVIS of the negative control was 8.9 and for the positive control 96.7, thus demonstrating the validity of the experiment.

Conclusion:

According to the results of this study and the OECD Guideline 437, the test substance DOPO-OX-Ammonium considered to be not an ocular corrosive or severe irritant. For classification and labelling of DOPO-OX-Ammonium further testing as outlined in the OECD guideline 405 is required.