Methyl 5-mesyl-o-anisate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June 15, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

Methyl 5-mesyl-o-anisate

EC Number:

264-267-7

EC Name:

Methyl 5-mesyl-o-anisate

Cas Number:

63484-12-8

Molecular formula:

C10H12O5S

IUPAC Name:

methyl 5-methanesulfonyl-2-methoxybenzoate

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

Test animals / tissue source

Species:

cattle

Strain:

other: Bos taurus (bovine cattle)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Characteristics of donor animals: up to 12 months old, typically, 5 to 8 months old (French Authorities avoid the use of any organs from the head of bovines aged more than 12 months).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported 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 to the laboratory, preparation and selection was performed immediately. The corneas were used within a maximum of 24 hours.
- indication of any existing defects or lesions in ocular tissue samples: No - A macroscopic examination was performed on all eyes to detect the presence of any defects.
- Indication of any antibiotics used: Antibiotic used during transport in buffered Hanks medium [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].

Test system

Vehicle:

other: paraffin oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 µL (± 8 µL) applied on each cornea.
- Concentration (if solution) :20% (w/v) in the paraffin oil

VEHICLE
- Amount(s) applied (volume or weight with unit):750 µL (± 8 µL)

Duration of treatment / exposure:

4 hours (± 5 minutes)

Duration of post- treatment incubation (in vitro):

As the test item is solid no post-exposure incubation was required.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Every eye with some defects was discared.The examination of defects was performed under a lamp, using HBSS. 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. The corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours. 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.

QUALITY CHECK OF THE ISOLATED CORNEAS: A pre-incubation was performed before treatment aplication. Both chambers of the corneal holder were filled with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C). At the end of the pre-incubation period, the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0. Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

NUMBER OF REPLICATES:3

SOLVENT CONTROL USED (if applicable): Yes, paraffin oil

POSITIVE CONTROL USED: Yes, 20% imidazole solution in 0.9% NaCl (w/w).

APPLICATION DOSE AND EXPOSURE TIME: 750 µL (± 8 µL) of 20% (w/w) in the paraffin oil, 4 hours exposure.

TREATMENT METHOD: The open-chamber method was used for the test item to ensure uniform spreading of the test item formulation over the corneas. The closed chamber was used for the vehicle/positive controls.
The medium of the anterior chamber was removed and the each item was applied onto the epithelium of the cornea. The treatment time of each series of three corneas was carefully measured with a chronometer. After application of the items, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for 4h.

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test item formulation was removed with a cotton bud and a pipette of heated cMEM (32°C).The corneas were rinsed four times with pre-warmed cMEM containing phenol red until it was visually confirmed by observing the transparency and the colour changing of the rinsing medium (containing phenol red).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 (OPKIT opacitometer and calibrators (MC2, Clermont-Ferrand, France)) was used to measure light transmission (the level of opacity) through the center of each mounted cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry Cary 100 (OD490)


SCORING SYSTEM: In Vitro Irritancy Score (IVIS). The following formula was used to determine the In Vitro Irritancy Score (IVIS): IVIS = cOPT + (15 x cOD490 nm)
The IVIS was calculated for each test item and positive control cornea. The mean IVIS for each series of three corneas was calculated from the individual scores.

DECISION CRITERIA: as indicated in the TG.
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

ca. 0.016

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, full validation dossier is available in laboratory GLP archive for review or inspection purpose.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean opacity (0.0) and mean OD490 nm of the vehicle control corneas (0.023) should be less than the established upper limit of historical mean ( acceptable range: opacity <= 4, mean OD490 <=0.043).

- Acceptance criteria met for positive control: Yes, the mean In Vitro Irritancy Score (IVIS) of the positive control corneas (163) should fall within two standard deviations of the historical mean (mean+2SD=168.85, acceptable range 113-169).

In vivo

Irritant / corrosive response data:

Due to that the test item induces an IVIS of 0.0 (this is ≤ 3), the test item has no irritation potential to eye. A single experiment composed of three corneas was sufficient for the test item formulation since the resulting classification was unequivocal. Please see "Any other information on results incl. tables" for more details about individual values.

Any other information on results incl. tables

Results:

Vehicle control	Opacity				Permeability
HOLDER	OPT0	OPT2	OPT2-OPT0	NEG. correction	OD490nm	NEG. correction
72	1	0	-1	0	0.031	0.031
77	1	0	-1	0	0.023	0.023
53	1	1	0	0	0.016	0.016
Mean				0.0		0.023
SD				0.0		0.008

Test item	Opacity					Permeability
HOLDER	OPT0	OPT2	OPT2-OPT0	NEG. correction	cOPT	OD490nm	NEG. correction	cOD490nm	IVIS
83	0	0	0	0	0	0.032	0.032	0.009	0
55	1	0	-1	0	0	0.013	0.013	0.000	0
50	2	2	0	0	0	0.063	0.063	0.040	1
Mean					0.0		0.023	0.016	0
SD					0.0		0.008	0.021	0.3

POSITIVE CONTROL	Opacity					Permeability
HOLDER	OPT0	OPT2	OPT2-OPT0	NEG. correction	cOPT	OD490nm	NEG. correction	cOD490nm	IVIS
73	0	110	110	110	110	3.264	3.264	3.241	159
79	1	123	122	122	122	3.228	3.228	3.205	170
61	2	114	112	112	112	3.192	3.192	3.169	160
Mean					114.7			3.205	163
SD					6.4			0.036	6.4

Np- not performed

OD: optical density

cOD-optical density corrected by mean OD value of vehicle control

cOPT: corneal opacity corrected by mean opacity value of vehicle control

OPT0: corneal opcity before treatment

OPT2: corneal opacity after treatment

Neg. correction: all individual values below 0 are set at 0

SD: standart deviations

IVIS:in vitro irritation source

Vehicle control: Parrafin oil

Positive control: 20% imidazole solution in 0.9 & NaCl

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

No irritating

Conclusions:

Under the experimental conditions of this study, the test item has been identified as not irritant to eye, not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Corneas were obtained from freshly slaughtered calves.

A single experiment was performed using three corneas for each treated series ( test item, positive control and vehicle control). Before treatment, opacity measurements were conducted. Then, the test item was applied at the concentration 20% (w/v) in the vehicle (paraffin oil), using a treatment time of 4 hours and the open-chamber method. Vehicle and positive controls were applied using the same treatment time and using the closed-chamber method. After exposure, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. Then the opacity cornea was measured. On the other hand, for measuring the permeability a fluorescein solution was used and the holders were incubated. After this procedure, the OD490 nm was measured and the corneas were examined for irregularities. The mean corneal opacity and the mean IVIS for the test item was 0, whereas the mean permeability was 0.016. All validity criteria were fulfilled. Therefore test item is considered not irritating to eye (no category).