reaction products of 1 mole of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs and 1 mole of cyclohexylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2017 - 22 June 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

Qualifier:

according to guideline

Guideline:

other: OECD guideline 437 (In vitro eye irritation)

Version / remarks:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Source: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Age at study initiation: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
- Transport from Supplier to CiToxLAB France: 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.
- Storage of the corneas: 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:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

other: Reference item: pre-formulation of isopropanol at 1.5% (w/w) in water for injections diluted at the concentration of 10% (w/v) in water for injections.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 10% in vehicle

RE§FERENCE ITEM
- Amount applied: 750 µL
- Concentration: 10% in vehicle

Duration of treatment / exposure:

Exposure period of 10 minutes (± 30 seconds), followed by rinsing

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes)

Number of animals or in vitro replicates:

3 corneas per item

Details on study design:

EYES SELECTION
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. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number. After pre-incuvbation, corneas that showed any macroscopic defect or an opacity value over 7 were discarded.

TREATMENT
Three corneas from the same series were always processed in the same order at each step. The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 10 minutes in a water bath at +32°C (± 1°C).

REMOVAL OF TEST SUBSTANCE
The purpose of rinsing was to eliminate as much items as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item formulation and the reference item were removed from the front opening of the anterior chamber (closed-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 for the test item formulation and three times for the reference item with pre-warmed cMEM containing phenol red (i.e. until the test item formulation 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.

SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. The average change in opacity for the corneas treated with the vehicle control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item formulation, reference item or positive control to obtain a corrected opacity value (cOPT). When the average change in opacity for the corneas treated with the negative/vehicle control was negative, it is considered equal to 0.
The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

- Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item formulation or positive control was calculated by subtracting the average negative (or vehicle) control cornea OD490 nm value from the original OD490 nm value of each cornea. When the average negative/vehicle control cornea OD490 nm value was negative, it is considered equal to 0.

- Scoring:
In vitro irritancy score (IVIS) = cOPT + (15 x cOD490 nm)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

test item

Run / experiment:

mean

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

Reference item

Run / experiment:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

Positive control

Run / experiment:

mean

Value:

50

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS (macroscopic examination):
Vehicle corneas: non
Test item: fluorescein fixation and opacity on each cornea
Reference item: none
Posiive control: incease in thickness, fluorescein fixation and opacity on each cornea

ACCEPTANCE OF RESULTS:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two Standard Deviations of the historical mean,
- the mean opacity and mean OD490 nm of the vehicle control corneas should be less than the established upper limit of historical mean.

Applicant's summary and conclusion

Interpretation of results:

other: No indication of severe irritation/corrosion

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant 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 not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to 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 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 guideline OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice.

 

Method

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.

Since the test item contained 1.5% isopropanol, a 1.5% (w/w) isopropanol solution was prepared in water for injections, and diluted identically to the test item before use, in order to investigate whether possible corneal damages would be related to the test item itself, or to its content in isopropanol.

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

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

The test item, tested at 10% (w/v) in the vehicle (0.9% NaCl), reference item, vehicle and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At 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.

 

Results

Macroscopic examination

Opacity and fluorescein fixation were observed on the corneas treated with the test item formulation. No finding was noted on the corneas treated with the reference 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 formulation-treated corneas was: 16.

The mean In Vitro Irritancy Score (IVIS) of the reference item-treated corneas was: 0.

 

As the IVIS obtained with the reference item was 0, all findings noted on test item formulation-treated corneas were considered to be due to its 98.5% UVCB content, and not to the 1.5% isopropanol content.

 

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).

Conclusion

Under the experimental conditions of this study, the ocular corrosive or severe irritant 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 not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.