Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 September to 2 November 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl adipate

EC Number:

211-020-6

EC Name:

Dimethyl adipate

Cas Number:

627-93-0

Molecular formula:

C8H14O4

IUPAC Name:

Dimethyl adipate

Constituent 2

Reference substance name:

Dimethyl glutarate

EC Number:

214-277-2

EC Name:

Dimethyl glutarate

Cas Number:

1119-40-0

Molecular formula:

C7H12O4

IUPAC Name:

dimethyl glutarate

Constituent 3

Reference substance name:

Dimethyl succinate

EC Number:

203-419-9

EC Name:

Dimethyl succinate

Cas Number:

106-65-0

Molecular formula:

C6H10O4

IUPAC Name:

dimethyl succinate

Test material form:

liquid

Remarks:

Colourless to brown liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable (The test item was applied as supplied, no formulation was required)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable (The test item was applied as supplied)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Test animals / tissue source

Species:

other: Bovine cattle (Bos Taurus)

Strain:

other: Not appplicable

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): bovine cattle were up to 12 months old (typically, 5 to 8 months old).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
- Time interval prior to initiating testing: Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible.
- indication of any existing defects or lesions in ocular tissue samples: careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded.
- Indication of any antibiotics used: As the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL (± 8 μL)
- Concentration (if solution): Not applicable (the test item was tested undiluted).

VEHICLE: Not Applicable (As the test item was a non-surfactant liquid, it was tested undiluted (i.e. in its original form)).

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes) in a water bath at +32°C (± 1°C).

Number of animals or in vitro replicates:

The test item, the negative and positive control were tested on three corneas each.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.

QUALITY CHECK OF THE ISOLATED CORNEAS
The corneas were carefully examined macroscopically before their assembly in the holders, in order to
detect the presence of any defects. Any corneas with defects were discarded.

NUMBER OF REPLICATES:The test item, the negative and positive control were tested on three corneas each.

NEGATIVE CONTROL USED: 0.9% Sodium Chloride

POSITIVE CONTROL USED: Absolute Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 μL (± 8 μL) applied for 10 minutes (± 30 seconds)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes. If YES please specify duration: 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: On completion of the treatment period, the test item was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured).
Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry / OD490)
- Others (e.g, pertinent visual observations, histopathology): After permeability determination, the corneas were removed from the holders and observed for opaque
spots, other irregularities and any separation of the epithelium.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
No notable opaque spots or irregularities were observed on the negative control-treated corneas.
Opacity and fluorescein fixation were observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The individual and mean opacity and permeability values for the test item, positive control and negative control fulfilled all acceptance criteria.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, INNROAD PROTECT, could not be predicted. Theregistered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to the OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, INNROAD PROTECT, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item, applied undiluted, and both negative and positive controls were applied in a single experiment, using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed

for opaque spots and other irregularities.

Macroscopic examination:

Opacity and fluorescein fixation were observed on the three corneas treated with the test item.

In Vitro Irritancy Score:

All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 32.

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, INNROAD PROTECT, could not be predicted.The registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to the OECD Guideline 437, further testing should be

conducted for classification and labeling purposes.