Dihydrogen bis[P,P-dioctyl diphosphato(2-)-O'',O''''][hydroxyacetato(2-)-O1,O2]titanate(2-), branched and linear

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Mar 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 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:

adopted in 2017

Deviations:

yes

Remarks:

The positive control had an IVIS that was slightly greater than two standard deviations of the historical mean. The assay was considered to be acceptable since the IVIS still demonstrated the anticipated outcome.

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Dihydrogen bis[P,P-dioctyl diphosphato(2-)-O'',O''''][hydroxyacetato(2-)-O1,O2]titanate(2-), branched and linear

EC Number:

271-571-3

EC Name:

Dihydrogen bis[P,P-dioctyl diphosphato(2-)-O'',O''''][hydroxyacetato(2-)-O1,O2]titanate(2-), branched and linear

Cas Number:

68585-64-8

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

({bis[(2-ethylhexyl)oxy]phosphoryl}oxy)[(2-{[({bis[(2-ethylhexyl)oxy]phosphoryl}oxy)(hydroxy)phosphoryl]oxy}-4-oxo-1,3-dioxa-2-titanacyclopentan-2-yl)oxy]phosphinic acid; {[({bis[(2-ethylhexyl)oxy]phosphanyl}oxy)({[({bis[(2-ethylhexyl)oxy]phosphoryl}oxy)(hydroxy)phosphoryl]oxy})[(2-hydroxyacetyl)oxy]titanio]oxy}({bis[(2-ethylhexyl)oxy]phosphoryl}oxy)phosphinic acid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from an abattoir
- Characteristics of donor animals: >35 weeks of age
- Storage, temperature and transport conditions of ocular tissue: Bovine eyes were transported to the MB Research in a refrigerated container containing Hanks’ Balanced Salt Solution (HBSS) with penicillin-streptomycin.
- Time interval prior to initiating testing: The bovine eyes were transported to MB Research on 15 Mar 2018, within 24 hours of harvest and directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were examined after receipt from the abattoir. Any cornea with visible evidence of neovascularization, pigmentation, opacity or scratches was discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: The test article was used as received.

NEGATIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: Minimal Essential Media (MEM), MEM solution was prepared by stirring together one jar (9.3 g) of MEM powder (sufficient to make one liter of solution), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 mL of fetal bovine serum (FBS) and then bringing the mixture to a final volume of 1000 mL with distilled water.

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 100 % (v/v) ethanol

Duration of treatment / exposure:

10 ± 1 min

Duration of post- treatment incubation (in vitro):

2 h (incubated at 32 ± 1°C in MEM solution)

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : Any cornea with visible evidence of neovascularization, pigmentation, opacity or scratches was discarded. Corneas free of visible defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The excised corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in specially designed holders (MC2, formerly Electro-Design – the manufacturer of the Op-KIT opacitometer) that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium.

QUALITY CHECK OF THE ISOLATED CORNEAS
After placing the cornea in the holders, each cornea was visually inspected again to ensure there were no defects. Prior to collection of the pretest opacity scores, the OP-KIT opacitometer (Electro-Design Corporation, RIOM, France) was calibrated. A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied by the opacitometer. Fresh MEM was added before taking these baseline opacity readings. Any cornea with a value greater than 7 units was discarded.

NUMBER OF REPLICATES : 3 corneae per test group

NEGATIVE CONTROL USED : Minimal Essential Media (MEM) , Lot No. SLBV3954, Sigma-Aldrich

POSITIVE CONTROL USED : 100% ethanol, Lot No. SHBH4983V, Sigma-Aldrich

APPLICATION DOSE AND EXPOSURE TIME : 0.75 mL of test article, ethanol, or MEM solution was applied to the cornea for 10 ± 1 min

TREATMENT METHOD: closed chamber: negative and positive control, open chamber: test substance

POST-INCUBATION PERIOD: at least 1 h, but not longer than 2 h

REMOVAL OF TEST SUBSTANCE : Number of washing steps after exposure period: the test article, ethanol, or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.

POST-EXPOSURE INCUBATION: yes, After removal of the test or control substance, the anterior chamber was refilled with MEM and an illuminance measurement was performed after 2 h incubation.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: OP-KIT opacitometer (Electro-Design Corporation, RIOM, France)
- Corneal permeability: passage of sodium fluorescein dye was measured with the aid of a Spectronic 20-D Colorimeter Spectrophotometer, permeability was measured as the optical density at 490 nm (i.e., the OD490 nm)

SCORING SYSTEM: The In Vitro Irritancy Score (IVIS) for the test article was calculated by adding the corrected mean opacity score to 15 times the corrected mean optical density. The calculations to obtain an IVIS for the positive control were performed in the same manner as the test article.
IVIS = Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)
The IVIS for the negative control was calculated by adding the mean opacity score to 15 times the mean optical density. IVIS = Mean Opacity Score + (15 x Mean Optical Density Score)

Individual corrected opacity scores were calculated by subtracting the pretest score from the 10-minute and 2-hour scores. Corrected mean opacity scores were calculated by averaging the individual two-hour corrected opacity scores for a given dose group and subtracting the mean opacity score for the negative control group. A corrected mean opacity score was not calculated for the negative control, rather only the mean of the individual two-hour corrected opacity scores were calculated (with no subtraction of mean opacity score for negative control).

Individual corrected optical densities were calculated by subtracting the mean optical density for the negative control group from the individual optical density values. Individual corrected optical densities were not calculated for the negative control group. Corrected mean optical densities were calculated by averaging the individual corrected optical density values for a given dose group. A corrected mean optical density was not calculated for the negative control, rather only the mean of the individual optical densities was calculated.

DECISION CRITERIA: OECD Guideline No. 437 defines a substance which produces an IVIS of > 55 as Category 1, a substance that causes “Serious eye damage.”

IVIS UN GHS
≤ 3 No Category
> 3 to ≤55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

10 min experiment

Value:

58.91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

other:

Remarks:

The ethanol positive control IVIS was 47.96, which did not fall within the acceptance range of 16.64 – 38.80 (± 2 standard deviations of the historical mean).

Remarks on result:

other:

Remarks:

The assay was considered to be acceptable since the IVIS still demonstrated the anticipated outcome of bovine corneas exposed to 100% ethanol for a 10-minute exposure: a moderately irritating, non-corrosive response. Additionally, the IVIS of the negative control group was within the range of the historical mean.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The MEM negative control IVIS was 0.26, which fell within the acceptance range of 1.27 - 2.29 (± 2 standard deviations of the historical mean).
- Acceptance criteria not met for positive control: The ethanol positive control IVIS was 47.96, which did not fall within the acceptance range of 16.64 – 38.80 (± 2 standard deviations of the historical mean). Although the positive control had an IVIS that was slightly greater than two standard deviations of the historical mean, the assay was considered to be acceptable since the IVIS still demonstrated the anticipated outcome of bovine corneas exposed to 100% ethanol for a 10-minute exposure: a moderately irritating, non-corrosive response. Additionally, the IVIS of the negative control group was within the range of the historical mean.

Any other information on results incl. tables

Table 2: Results In Vitro Irritancy Score (IVIS)

	Test Substance	Negative Control	Positive Control
Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)	57.00 + (15 x 0.127)	0.00 + (15 x 0.017)	36.33 + (15 x 0.775)
	57.00 + 1.905	0.00 + 0.255	36.33 + 11.625
IVIS*	58.91	0.26	47.96

 * In Vitro Irritancy Score (IVIS)= Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)

 

Table 3: Results

Cornea ID	Pretest Opacity Score	10-Minute Opacity Score	2-Hour Opacity Score	Corrected Opacity Scores^1		OD490 nm (permeability)	Corrected Optical Density^2
				10 min	2 h
1 (negative control)	0	0	0	0	0	0	0.012
2 (negative control)	0	0	0	0	0	0	0.016
3 (negative control)	0	0	0	0	0	0	0.019
Corrected Mean Optical Density^3= 0.017
2-Hour Corrected Mean Opacity Score^4= 00.00
4 (positive control)	0	37	35	37	35	0.512	0.495
5 (positive control)	1	39	36	38	35	0.985	0.968
6 (positive control)	0	42	39	42	39	0.880	0.863
Corrected Mean Optical Density^3= 0.775
2-Hour Corrected Mean Opacity Score^4= 36.33
19 (test substance)	0	72	69	72	69	0.139	0.122
20 (test substance)	0	63	73	63	73	0.106	0.089
21 (test substance)	0	28	29	28	29	0.188	0.171
Corrected Mean Optical Density^3= 0.127
2-Hour Corrected Mean Opacity Score^4= 57.00

 ¹Individual Corrected Opacity Score: 10-minute or 2-hour opacity score minus pretest opacity score

²Individual Corrected Optical Density: Individual test article OD minus the mean OD for the negative control group

³Corrected Mean Optical Density: Mean of the individual corrected optical density values for a given dose group

⁴2-Hour Corrected Mean Opacity Score: Mean of the individual 2-hour corrected opacity scores for a given dose group minus the mean opacity score for the negative control group

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS Category 1 (H318) according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) application of the test substance to bovine corneae resulted in a calculated mean IVIS of 58.91.
The test substance caused serious eye damage.