1,2,3,4-Butanetetracarboxylic acid, tetramethyl ester, reaction products with 1,2,2,6,6-pentamethyl-4-piperidinol and β,β,β',β'-tetramethyl-2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Oct 2016 - 20 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Batch: 112Z5
- Purity: 100%
- Physical state/Appearance: white powder
- Expiry Date: 30 June 2017
- Storage Conditions: room temperature in the dark

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir

- Number of animals: 3 per control group

- Characteristics of donor animals - age: 12 - 60 months

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were excised and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with penicillin at 100 IU/mL and streptomycin at 100 μg/mL. 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

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75mL
- Concentration (if solution): 20% w/v solution

VEHICLE
- Concentration (if solution): 0.9% w/v sodium chloride solution

Duration of treatment / exposure:

240 min

Duration of post- treatment incubation (in vitro):

240 minutes.

Number of animals or in vitro replicates:

3 per control group

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 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 65 minutes.
At the end of the incubation period each cornea was examined for defects.
Only corneas free of damage were used.

NUMBER OF REPLICATES
3 per control group

NEGATIVE CONTROL USED
- Sodium chloride 0.9% w/v
- Batch: 3011542
- Purity: 0.9%
- Expiry Date: 01 Mar 2017
- Storage Conditions: Room temperature in the dark

POSITIVE CONTROL USED
- Imidazole
- Batch: 1420882
- Purity: 100%
- Expiry Date: 28 February 2021
- Storage Conditions: Room temperature in the dark

APPLICATION DOSE AND EXPOSURE TIME
0.75mL of 20% w/v solution in 0.9% w/v sodium chloride solution. at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 with fresh Eagle’s Minimum Essential Medium (EMEM)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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)
IVIS Classification
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage

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

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

13.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

12.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: Permeability

Run / experiment:

mean

Value:

0.038

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction of eye irritation can be made