Methyl 5-mesyl-o-anisate

Endpoint summary

Description of key information

Skin irritation.

Key study. Method according to OECD 439, GLP study. The mean corrected percent viability of the SkinEthic™ RHE treated tissues was 71.8 %, versus 2.9% in the positive control. Therefore, the test item is not irritant to the skin.

Eye irritation.

Weight of evidence: Method according to OECD 438, GLP study. No prediction can be made, since the combinations of the 3 endpoints for the test item were 2 x II, 1 x I.

Weight of evidence: Method according to OECD 437, GLP study. The mean IVIS score for the test item was found to be 0, while the value for the positive control was 163.Therefore, the test item has been identified as not irritant to eye, not requiring classification for eye irritation or serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

Reconstructed Human Epidermis Test Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 19,2016 - November 4, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Test system:

human skin model

Remarks:

SkinEthic RHE® model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

The SkinEthic™ RHE model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as supplied

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:SkinEthic RHE® model
- Tissue batch number(s): 16-RHE-114
- Delivery date: 02 November 2016
Expiration date: November 7, 2016
- Date of initiation of testing: 02 November 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 2h 58 minutes
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength:570 nm
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissue batch: O.D.= 1.2 (CV = 1.3%) specification OD>0.7
Historical control data: mean O.D. range = 0.459 - 1.243
- Barrier function:4.2 h SPECIFICATION 4.0h< ET50<10H
- Morphology: 5 Cell layers, absence of significant histological abnormalities, well differentiated epidermis, specification > 4
- Contamination:no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg of test item (32mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 30 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

ca. 71.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean corrected percent viability of the treated tissues was 71.8 %, versus 2.9% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: A yellow non-homogenous suspension was observed after 3 hours of incubation between 36.9°C and 37.8°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
In water: A white non-homogenous suspension was obtained after 3 hours of incubation between 37.1°C and 37.8°C, 5% CO2.
In isopropanol: A white non-homogenous suspension was obtained after 2 hours of incubation at room temperature. Therefore the test item will not interfere with the MTT assay and there is no need to add nonspecific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals. Summary of proficiency chemicals testing according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the standard deviation of the negative control group was 8.5%, the acceptablility criteria is SD ≤ 18%, OD mean of negative control is 1.185 and the acceptability criteria is OD >0.6 x<1.5
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Summary of results:

	SKIN	OD	Mean OD/disc	Mean OD/product	Viability %	Mean viability %	SD	CRITERIA
Negative control	1	1.091	1.135	1.185	95.8	100.00	8.5
		1.301
		1.118
	2	1.292	1.301		109.8
		1.325
		1.285
	3	1.135	1.118		94.4
		1094
		1.125
Positive control	4	0.034	0.034	0.035	2.9	2.9	0.1	Irritant
		0.035
		0.034
	5	0.038	0.036		3.0
		0.036
		0.034
	6	0.036	0.034		2.9
		0.034
		0.031
Test item PH-15/0504	7	0.806	0.801	0.850	67.6	71.8	3.8	Non irritant
		0.792
		0.805
	8	0.878	0.859		72.5
		0.811
		0.889
	9	0.923	0.890		75.1
		0.886
		0.860

The results were expressed as a viability percentage compared with the negative control

validity %= (ODtest item/ OD negative control )*100

The OD values obtained for each test sample were used to calculate a percentage of viability relative

to the negative control, which was arbitrarily set at 100%.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the treated tissues was 71.8 %, versus 2.9% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

Executive summary:

The aim of the study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). This test was performed according to OECD TG 439 (GLP study). The test item was applied at the dose of 16 mg, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 hours and 30 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.The mean corrected percent viability of the treated tissues was 71.8%, versus 2.9 % in the positive control (5% Sodium Dodecyl Sulfate). The test item has to be considered as Non-irritant to skin, corresponding to UN GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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

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)].

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

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

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

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).

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.

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

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation: Key study: The aim of the study was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). This test was performed according to OECD TG 439 (GLP study). The test item was applied at the dose of 16 mg, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 hours and 30 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The mean corrected percent viability of the treated tissues was 71.8%, versus 2.9 % in the positive control (5% Sodium Dodecyl Sulfate). The test item has to be considered as Non-irritant to skin, corresponding to UN GHS No Category.

Eye irritation: A first study was conducted with the test item according to Isolated Chicken Eye Test Method and the results indicate that no prediction can be made.

Weight of evidence: An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 (GLP study). Three eyeballs per group were isolated from chickens and, after appropriate preparation, were exposed to either 30 mg of the test item, 30 mg of sodium hydroxide (positive control) or 30μL of physiological saline (negative control).According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints for the test item were 2 x II, 1 x I.

Due to that results from this first in vitro study do not allow a conclusive decision on the classification of the substance or the absence of eye irritation potential, an other in vitro study for this endpoint has been considered. The Bovine Corneal Opacity and Permeability study has been conducted.

Weight of evidence: 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. 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, for 4 hours with the open-chamber method. Vehicle and positive controls were applied using the closed-chamber method. After exposure, all items were removed 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).

Justification for classification or non-classification

Based on the available information, the test item is not classified for irritation/corrosion, in accordance with CLP Regulation (EU) No. 1272/2008.