Phosphoric acid, mono- and di-C12-14-alkyl esters

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th September 2019 - 10th October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Updated 09 October 2017

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)

Version / remarks:

EC No. 440/2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Historical Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160.

Version / remarks:

Adopted July 6, 2018 Paris.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Constituent 2

Reference substance name:

C12-C14 alcohol

IUPAC Name:

C12-C14 alcohol

Constituent 3

Reference substance name:

Mono (C12-C14 alkyl) phosphate ester

IUPAC Name:

Mono (C12-C14 alkyl) phosphate ester

Constituent 4

Reference substance name:

Di (C12-C14 alkyl) diphosphate ester

IUPAC Name:

Di (C12-C14 alkyl) diphosphate ester

Constituent 5

Reference substance name:

Tri (C12-C14 alkyl) triphosphate ester

IUPAC Name:

Tri (C12-C14 alkyl) triphosphate ester

Constituent 6

Reference substance name:

C12-C14 alkyl pyrophosphate esters

IUPAC Name:

C12-C14 alkyl pyrophosphate esters

Constituent 7

Reference substance name:

Ortho-phosphoric acid

IUPAC Name:

Ortho-phosphoric acid

Constituent 8

Reference substance name:

unknown

Molecular formula:

unknown

IUPAC Name:

unknown

Test material form:

solid: bulk

Specific details on test material used for the study:

CAS Number: 68412-59-9
Batch: 1023S17201
Purity: 100%
Physical state/Appearance: White solid
Expiry Date: 09 July 2020
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

physiological saline

Remarks:

sodium chloride 0.9% w/v

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of Substance 20% w/v solution in sodium chloride 0.9% w/v;
0.75 mL of negative control;
0.75 mL of positive control;

Duration of treatment / exposure:

240 min at 32 ± 1 ºC.

Duration of post- treatment incubation (in vitro):

Following opacity measurements the corneas were used further for pereability measurement. The corneas were rinsed to remove the test/control substances and further incubated for 90 min with sodum fluorescein at 32 ± 1 ºC.

Number of animals or in vitro replicates:

triplicate

Details on study design:

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 75 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading.
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the test substsance, negative and positive control.

Treatment of Corneas.
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the substance preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire
cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein.
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations.
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology.
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data Evaluation.
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement.
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement.
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score.
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation.
The condition of the cornea was visually assessed post treatment.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

ca. 87.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

IVIS = 2.1

Positive controls validity:

valid

Remarks:

IVIS=108.1

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In vitro Irritation Score^1, 2
Negative control	2.0	0.003	2.1
Positive control (20% Imidazole)	85	1.538	108.1
Substance	86	0.095	87.4

¹    Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and substance.

²    In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Corneal epithelium post-treatment condition was visually examined. For positive control and substance treated corneas appeared cloudy; the negative control treated corneas appeared clear.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The mean in vitro irritancy score (IVIS) of the test substance is 87.4. Therefore, the substance is classified to cause serious eye damage (Category 1).

Executive summary:

In a reliable in vitro eye irritation study, conducted according to 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’, the ability of the substance to induce opacity and permeability in an isolated bovine cornea were determined. The substance was applied 20% w/v solution (in sodium chloride 0.9% w/v) at 750 µL onto corneas (n=3) for a period of 240 min at 32 ± 1 ºC, followed by rinsing of the substance. A post-treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein (following further 90 min incubation) was evaluated. According to opacity and permeability measurements, the substance resulted in a mean in vitro irritancy score (IVIS) of 87.4. In conclusion, since the substance induced an IVIS > 55, the substance is classified to cause serious eye damage.