Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10th December 2013 (Single Day Study)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Substance in 80% solution with propylene glycol in order to make the test feasable. The solvent is not thought to affect the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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)

Deviations:

no

Principles of method if other than guideline:

Substance in 80% solution with propylene glycol in order to make the test feasable. The solvent is not thought to effect the results.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

EC Number:

939-981-3

IUPAC Name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Constituent 2

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

IUPAC Name:

propylene glycol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: Not supplied
- Purity test date: 14th May 2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test material prepared with 80% of substance and 20% propylene gylcol

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: The eyes from adult cattle were obtained from freshly slaughtered adult catlle from a local abattoir. The eyes are a by-product. Once excised, they were placed in Hanks' Balanced Salt Solution (HBSS) supplemented with antibiotics (Penicillin/Streptomycin). The eyes were transported on ice packs to the laboratory where the corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.75mL of the test item/control was applied in each treatment.

Duration of treatment / exposure:

10 minutes at 32°C +/- 1°C

Duration of post- treatment incubation (in vitro):

Medium of the anterior chamber was removed post-treatment and replaced with Sodium Flourescein. 1mL of sodium flourescien solution at as concentration of 4mg/mL was used for each one. The holders were then incubated for 90 minutes at 32°C +/- 1°C

Number of animals or in vitro replicates:

3 replicates per treatment, tottaling 9 corneas.

Details on study design:

SELECTION AND 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 (epithelial side uppermost) in a dish containing Hanks' Balanced Salt Solution until they were mounted in Bovine Corneal Opacity and permeability holders.
The anterior and posterior chambers Of each Bovine Corneal Opacity and permeability holder were filled with complete Eagle's minimum essential medium and plugged. The holders were incubated at 32°C +/- 1°C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

The medium from both chambers of each holder was replaced with fresh complete Eagle's minimum essential medium. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Corneas were also allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES

3 replicates for each test group.

NEGATIVE CONTROL USED

0.9% w/v sodium chloride solution

POSITIVE CONTROL USED

Ethanol

APPLICATION DOSE AND EXPOSURE TIME

0.75mL of the test item/control was applied in each treatment. The exposure was for 10 minutes at 32°C +/- 1°C.

TREATMENT METHOD: [closed chamber / open chamber]

Closed Chamber

POST-INCUBATION PERIOD: yes - Opacity readings were taken before the post-incubation period. The medium of the anterior chamber was removed post-treatment and replaced with Sodium Flourescein. 1mL of sodium flourescien solution at as concentration of 4mg/mL was used for each one. The holders were then incubated for 90 minutes at 32°C +/- 1°C

REMOVAL OF TEST SUBSTANCE
Medium of the anterior chamber was removed post-treatment and replaced with Sodium Flourescein. 1mL of sodium flourescien solution at as concentration of 4mg/mL was used for each one. The holders were then incubated for 90 minutes at 32°C +/- 1°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others Histopathology: Corneas were retained after tersting for possible conduct of histopathology. Each indiviudual cornea was placed into a labelled tissue casette with a histology sponge top protect the endothelialsurface. The casette was then immersed in 10% neutral buffered formalin.
Visual Observation: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: A test item that induces an IVIS of ≥55.1 us defined as an ocular corrosieve or severe irritant and will be labelled EU DSD (67/548ECC) R41 and EU CLP/UN GHS Category 1.

CRITERION FOR AN ACCEPTABLE TEST:

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Item

Value:

56.4

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control

Value:

2.2

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

not measured/tested

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive Control

Value:

40.5

Vehicle controls validity:

not examined

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

not measured/tested

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

Positive Control

Value:

20.7

Vehicle controls validity:

not examined

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

not measured/tested

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

Test Item

Value:

31

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The negative control had no visible effects in all 3 replicates. The positive control made the test subject appear cloudy in all 3 replicates. The test iteem produced areas of brown staining in all 3 replicates.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 22.4 to 60.3. As the score was 40.5 the positive control acceptance criterion was satisfied.
- Range of historical values: 22.4 to 60.3

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the conditions of the test, the test item is considered to be an ocular corrosive or severe irritant.

Executive summary:

Under the conditions of the test, the test item is considered to be an ocular corrosive or severe irritant.