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

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2011 - December 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully Guideline- and GLP-compliant.

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ", 13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”, Paris 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Regulation (EC) 761/2009: B.46 "In vitro skin irritation: Reconstructed Human Epidermis Model Test". 23 July 2009

Deviations:

no

GLP compliance:

yes

Species:

other: in vitro system

Strain:

other: MatTek´s EpiDerm System

Details on test animals or test system and environmental conditions:

MatTek´s EpiDerm System consists of normal, human-derived epidermal keratinocytes which
have been cultured form a multilayered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum
containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture
inserts (MILLICELLs, 10 mm ) and shipped as kits, containing 24 tissues on shipping agarose.

Vehicle:

unchanged (no vehicle)

Controls:

other: n.a.

Amount / concentration applied:

The application spoon was filled with 25 mg finely grounded test substance.
The "spoonful" was levelled by gently scratching the excess material away, avoiding compression.
25 µL H2O were added for wetting of the test substance.

Duration of treatment / exposure:

Epiderm Skin Corrosivity Test:
• 3 minutes
• 1 hour
Epiderm Skin Irritation Test:
• 60 minutes, post-incubation: 42 hours

Observation period:

n.a.

Number of animals:

Epiderm Skin Corrosivity Test
Two tissue replicates were used for each treatment (exposure time), including distilled water
as negative and 8N KOH as positive control.
Epiderm Skin Irritation Test
Three tissue replicates were used, including distilled water as negative and 5 % SDS as
positive control.

Details on study design:

Epiderm Skin Corrosion Test
Two tissue replicates were used for each treatment (exposure time), including deionised water as negative and 8N KOH as positive control.
Exposure times:
• 3 minutes
• 1 hour
50 μL of each reference substance were dispensed directly atop the EpiDerm™ tissue.
The application spoon was filled with 25 mg finely grounded test substance.
The "spoonful" was levelled by gently scratching the excess material away, avoiding compression.
25 µL H2O were added for wetting of the test substance.

Epiderm Skin Irritation Test
Three tissue replicates were used, including distilled water as negative and 5 % SDS as
positive control.
Exposure time:
• 60 minutes, post-incubation: 42 hours
30 μL of each reference substance were dispensed directly atop the EpiDerm™ tissue.
The application spoon was filled with 25 mg finely grounded test substance.
The "spoonful" was levelled by gently scratching the excess material away, avoiding compression.
25 µL H2O were added for wetting of the test substance.


MTT-test
After incubation with the test substance and washing with PBS, the tissues were incubated
with MTT medium at 37°C and 5 % CO2. After 3 hours, the MTT medium was aspirated from
all wells and the tissues were gently rinsed with PBS (2 times). For extraction, the tissues
were incubated with extractant solution (isopropanol) for 2 hours with shaking.
After the extraction period, the tissues were pierced with an injection needle and the extract
(now a blue formazan solution) was allowed to run into the well from which the tissue was
taken. The 24-well plates were placed on a shaker for 15 minutes until the solutions were
homogeneous in colour.
For the Epiderm Skin Corrosivity Test per each tissue 3 × 200 μL aliquots, for the Epiderm Skin Irritation Test 2 × 200 μL aliquots
of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate and the OD was measured using
the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference filter.

Calculations

Cell viability
Cell viability was calculated for each tissue as percent of the mean of the negative control
tissues. The skin corrosivity/irritation potential of the test substance was classified according
to remaining cell viability obtained after test substance treatment with either of the two
exposure times.

Irritation parameter:

other: Mean tissue viability (%)

Basis:

mean

Time point:

other: 3 minutes

Score:

91.3

Reversibility:

other: n.a.

Remarks on result:

other: Non-Corrosive

Irritation parameter:

other: Mean tissue viability (%)

Basis:

mean

Time point:

other: 1 hour

Score:

81.9

Reversibility:

other: n.a.

Remarks on result:

other: Non-Corrosive

Irritation parameter:

other: Mean tissue viability (%)

Basis:

mean

Time point:

other: 60 minutes, 42 hours post-incubation

Score:

97.4

Reversibility:

other: n.a.

Remarks on result:

other: Non-Irritant

Irritant / corrosive response data:

Epiderm Skin Corrosivity Test:
• The mean percentage viability of the treated skin discs after 3 minutes of exposure was
91.3 % which is above the threshold of 50 % for classification.
• The mean percentage viability of the treated skin discs after 1 hour of exposure was
81.9 % which is above the threshold of 15 % for classification.

Epiderm Skin Irritation Test:
• The mean percentage viability of the treated skin discs was 97.4 % which is above the
threshold of 50 % for classification.

Other effects:

Epiderm Skin Corrosivity Test:
Assay acceptance criteria according to the protocol used during the ECVAM validation study:
•The mean OD of the tissues, treated with deionised water (negative control) was 1.818 after 3 minutes, and 1.854 after 1 hour of exposure, that is higher than 0.800, as required by the assay acceptance criteria.
•The mean tissue viability of the 1 hour positive control was 8.0%, that is lower than 15.0 %, as required by the assay acceptance criteria.
•The coefficient of variation of the test substance treated skin discs were 0.3 for the 3 minutes and 0.0 for the 1 hour exposure, that is below 0.3 % as required by the assay acceptance criteria.


Epiderm Skin Irritation Test:
Assay acceptance criteria according to the protocol used during the ECVAM validation study:
•The mean OD of the tissues, treated with deionised water (negative control) was 2.003, that is higher than 1.000 and lower than 2.500, as required by the assay acceptance criteria.
•The mean tissue viability of the positive control was 6.3 %, that is lower than 20.0 %, as required by the assay acceptance criteria.
•The standard deviation calculated from individual percental tissue viabilities of the test substance treated skin discs was 0.6 %, that is below 18.0 % as required by the assay acceptance criteria.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the results of this study, the Directive 2001/59/EC and the GHS the test substance "DOPO-OX-AMMONIUM" is considered to be non-corrosive and non-irritant to skin.

Executive summary:

Aim and Method

The EpiDerm Skin Corrosivity/Irritation Test (Model EPI-200) was performed to reveal possible irreversible tissue damages of the skin following the application of "DOPO-OX-AMMONIUM".

The test substance was topically applied for 3 minutes and 1 hour to the epidermal surfaces of three-dimensional human epidermis models, followed by immediate determination of the cytotoxic effect. As there was no corrosive effect observed, the test substance was topically applied for 60 minutes to the epidermal surfaces ofthree-dimensional human epidermis models. After a post-incubation of 42 hours, a cell viability test was performed.

EpiDerm Skin Irritation Test: The test substance was topically applied for 60 minutes to the epidermal surfaces of three-dimensional human epidermis models. After a post-incubation of 42 hours, a cell viability test was performed.
EpiDerm Skin Corrosivity Test: During the post-incubation of 42 hours the test substance was topically applied for 3 minutes and 1 hour to the epidermal surfaces of three-dimensional human epidermis models, followed by immediate determination of the cytotoxic effect.

Investigations performed were in conformance with

·    the Regulation (EC) 440/2008: B.40.BIS. "In vitro skin corrosion: human skin model"

·    the OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ",

·    the OECD Guideline “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”,

·    the Regulation (EC) 761/2009: B.46 "In vitro skin irritation: Reconstructed Human Epidermis Model Test".

Results

EpiDerm Skin Corrosivity Test:

Assay acceptance criteria:

·     The mean OD of the tissues, treated with deionised water (negative control) was 1.818 after 3 minutes, and 1.854 after 1 hour of exposure, that is higher than 0.800, as required by the assay acceptance criteria.

·     The mean tissue viability of the 1 hour positive control was 8.0%, that is lower than 15.0 %, as required by the assay acceptance criteria.

·     The coefficient of variation of the "DOPO-OX-AMMONIUM" treated skin discs were 0.1 for the 3 minutes and 0.1 for the 1 hour exposure, that is below 0.1 % as required by the assay acceptance criteria.

"DOPO-OX-AMMONIUM":

·     The mean percentage viability of the treated skin discs after 3 minutes of exposure was 91.3 % which is above the threshold of 50.0 % for classification.

·     The mean percentage viability of the treated skin discs after 1 hour of exposure was 81.9 % which is above the threshold of 15.0 % for classification.

EpiDerm Skin Irritation Test:

Assay acceptance criteria:

·     The mean OD of the tissues, treated with deionised water (negative control) was 2.003, that is higher than 1.000 and lower than 2.500, as required by the assay acceptance criteria.

·     The mean tissue viability of the positive control was 6.3 %, that is lower than 20.0 %, as required by the assay acceptance criteria.

·     The standard deviation calculated from individual percental tissue viabilities of the "DOPO-OX-AMMONIUM" treated skin discs was 3.6 %, that is below 18.0 % as required by the assay acceptance criteria.

"DOPO-OX-AMMONIUM":

·     The mean percentage viability of the treated skin discs was 97.4 % which is above the threshold of 50 % for classification.

Conclusion

According to the results of this study, the Directive 2001/59/EC and the GHS, the test substance
"DOPO-OX-AMMONIUM" is considered to be non-corrosive and non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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.

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.

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.

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

 

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.

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Key study.

Justification for selection of eye irritation endpoint:
Endpoint conclusion: Not corrosive, not severe irritant.

Justification for classification or non-classification

There is no indication which would justify a classification.