Benzaldehyde, 3-ethoxy-4-(.beta.-D-glucopyranosyloxy)-

Endpoint summary

Description of key information

No irritation to skin or eye observed in the in vitro studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - September 2013

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)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE
- Tissue batch number(s): 13 RHE 0814
- Production date: 16 September 2013
- Delivery date: 17 September 2013
- Date of initiation of testing: 17 September 2013

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1mL PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hrs
- Spectrophotometer: ELx800 absorbance microplate reader supplied by Biotek
- Wavelength: 570

NUMBER OF REPLICATE TISSUES: 3

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 and 42 hrs post-treatment incubation is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hrs post-treatment incubation is greater than 50%
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

test item: 16 mg

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 runs

Value:

99.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item must not be classified in category 2 "irritant". No signal word or hazard statement is required.

Executive summary:

In accordance with the Regulation EC No. 1272/2008, the test item must not be classified in category 2 "irritant". No signal word or hazard statement is required.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS
- Tissue batch number(s): 100-AC1624-1
- Production date: 26 August 2013
- Delivery date: 27 August 2013
- Date of initiation of testing: 27 August 2013

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times with 1 mL PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hrs
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment with 3-minute exposure, 1 experiment with 1-hr exposure

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT; 2 hours with isopropanol

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure, mean of experiment 1 and 2

Value:

104.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure, mean of experiment 1 and 2

Value:

88.9

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:

Positive control - tissue viability: 2.8% after 3-min exposure, 0.2% after 1-hr exposure

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item must not be classified in Category 1 "Corrosive". No signal word or hazard statement is required.

Executive summary:

In accordance with the Regulation EC No. 1272/2008, the test item must not be classified in Category 1 "Corrosive". No signal word or hazard statement is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of test item was applied to an eyeball

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes +/- 5 minutes

Number of animals or in vitro replicates:

3 eyeballs for test item and postive control
1 eyeball for negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg). Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 31 July 2013 at 9.00 a.m. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with isotonic saline. The eyes were enucleated at Phycher on 31 July 2013 between 10:30 am and 10:50 am.

The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane fumly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion ofthe optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the super:fusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the isotonic saline drip. The chambers of the superfusion apparatus was temperature controlled at 32°C ± 1.5°C. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected. Once all eyes have been examined and approved, the eyes were incubated approximately 45 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that end point.

TREATMENT
Test item
The test item was applied for 10 seconds and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature, except for the eyes No.9, 10 and 11 which were rinsed with 25 mL of isotonic saline. The eye (in its holder) was subsequently returned to the super:fusion apparatus in the original upright position.
Controls
Concurrent negative control (physiological saline - Cooper, Batch No. 19FC12GA) and positive control (Sodium Hydroxide Batch No.MKBF9973V) were included in this experiment.

OBSERVATION PERIOD
Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180 and 240 minutes (+/- 5 minutes) after the post-treatment rinse.
The endpoints evaluated were corneal opacity, swelling, fluorescein retention and morphologicla effects. All of the endpoints, with the esception of fluorescein retention (which was determined only at pretreatment and 30 minutes after test item exposure) were determined at each of the above time points.

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean score

Value:

9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Other effects / acceptance of results:

Result for positive control: 3 x IV
Result for negative control: 3x I

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the test item must not be classified in category 1 "irreversible effects on the eye".

Executive summary:

In accordance with the Regulation (EC) No. 1272/2008, the test item must not be classified in category 1 "irreversible effects on the eye".

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

October 2017

Deviations:

no

GLP compliance:

no

Remarks:

Study performed without GLP, however according to the SOP of the laboratory.

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.

The EpiOcular™ Eye Irritation Test (EIT) predicts the acute eye irritation potential of a chemical by measurement of its cytotoxic effect on the EpiOcular™ cornea epithelial model.

RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT [3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide]; into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. Chemicals not requiring classification and labelling according to UN GHS are identified as those that do not decrease tissue viability below a defined threshold (i.e., tissue viability > 60%, for UN GHS No Category).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

25 minutes post-exposure immersion; 6 hrs +/- 15 minutes post-exposure incubation

Duration of post- treatment incubation (in vitro):

18 hrs +/- 15 minutes

Number of animals or in vitro replicates:

2 replicates for each test chemical and each control substance in each run

Details on study design:

- RhCE tissue construct used, including batch number : EpiOcular™ tissues (OCL-200) Batch 27042

- Doses of test chemical and control substances used : neat test item (50 mg) and positive control - methyl actetate, negative control - sterile deionised water

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : exposure - 6 hrs +/- 15 minutes at standard culture conditions, 18 hrs +/- 0.25 hrs post-incubation with assay medium

- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2

- Wavelength: OD at 570 nm

- Test acceptance criteria:
Negative control: If the mean OD value of the tissues is ≥ 0.8 and <2.5 at 570 nm and the variability between 2 tissue replicates < 20%;
positive control: If the mean viability, expressed as % of the NC, is < 50 % and the variability between 2 tissue replicates < 20%;
test item: The variability between 2 tissue replicates < 20%

Irritation parameter:

other: tissue viability %

Run / experiment:

1

Value:

87.22

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

other: tissue viability %

Run / experiment:

2

Value:

83.61

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:

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

Interpretation of results:

GHS criteria not met

Conclusions:

According to EpiOcular EIT, Ethyl Vanilin Glucoside (EVG) is not classified for eye irritation or serious eye damage and defined as UN GHS "No Category".

Executive summary:

According to EpiOcular EIT, Ethyl Vanilin Glucoside (EVG) is not classified for eye irritation or serious eye damage and defined as UN GHS "No Category".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available