Octadecene, reaction products with hexadecene, hydrogenated

Endpoint summary

Description of key information

In vitro EPISKIN^TM(SM) model test with SynNova Base Oil, the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.

In vitro eye irritation assay in isolated chicken eyes with SynNova Base Oil, the test item is non-irritant, UN GHS Classification: No Category.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion, other

Remarks:

In Vitro Combined corrosivity & irritation study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2019 to 12 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

OECD No. 431, “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method” adopted 29 July 2016.

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), ANNEX III, B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Model Tesrt” as amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

OECD No. 404, “Acute Dermal Irritation/Corrosion” adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Details on animal used as source of test system:

Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-014, Expiry Date: 08 April 2019 in case of corrosivity test; Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-015, Expiry Date: 15 April 2019 in case of irritation test) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity and irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKINTM(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity and irritation testing in an international validation study [13, 15] and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

PERFORMANCE OF THE STUDY
Procedures described were performed under aseptic conditions for irritation testing (in sterile hood using sterile equipment).

Pre-incubation
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2 in a > 95% humidified atmosphere.

Application
Test Item
The Assay Medium (corrosivity testing) and the Maintenance Medium (irritation testing) were pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.
In case of the corrosivity test, 50 μL of test item was applied evenly to each of two test units.
In case of the irritation test, 20 μL of test item was applied evenly to each of three test units. (10 μL of test item was not sufficient to cover the epidermal surface.)
Negative and positive controls
50 μL of negative control (Physiological saline (0.9% (w/v) NaCl solution)) or positive control (glacial acetic acid) were added to each skin unit by using a suitable pipette in the corrosivity test. 20 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette in the irritation test.
The plates with the treated epidermis units were incubated for the exposure time of 4 hours (±10 minutes) at room temperature (21.4-25.6°) in the corrosivity test and 15 minutes (± 0.5 minute) at room temperature (23.1-25.9°C) in the irritation test.

Rinsing
After 15 minutes incubation time (in the irritation test) or 4 hours incubation time (in the corrosivity test), the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

Incubation during the irritation test
After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (±1 hour) at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

MTT test
In both tests, MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±5 minutes), protected from light, in a > 95% humidified atmosphere.

Formazan extraction
In both tests, at the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight (corrosivity test) or for about two hours (irritation test) at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel in each case.

Cell viability measurements
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 13 August 2018, calibration is valid until August 2020) at the required wavelength on each day before use.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

In case of the corrosivity test, 50 μL of test item was applied evenly to each of two test units.
In case of the irritation test, 20 μL of test item was applied evenly to each of three test units.

Duration of treatment / exposure:

After 15 minutes incubation time (in the irritation test) or 4 hours incubation time (in the corrosivity test), the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible.

Duration of post-treatment incubation (if applicable):

Incubation during the irritation test
After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (±1 hour) at 37°C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

Number of replicates:

In this assay, two replicates per time point were used for test item (in the corrosivity part of the test) and three replicates per time point were used for test item (in the irritation part of the test). Two negative controls and two positive controls were included in the corrosivity test and three negative controls and three positive controls were included in the irritation test.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosivity Test - Test item

Value:

98.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Irritation Test - Test item

Value:

100.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corrosivity testing:
The mean OD value for the test item treated skin samples showed 98.1% relative viability.
Irritation testing:
The OD values for the test item treated skin samples showed 100.5% relative viability.

VALIDITY OF THE TEST
General validity criteria:
After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues in the corrosivity and irritation test was in the recommended range (1.095 and 0.775).
The mean OD value of the blank samples (acidified isopropanol) in the corrosivity and irritation test was 0.047 and 0.049.
High viability results (>100%) do regularly occur in cases where the test item causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.

Specific criteria for corrosivity testing:
The positive control treated tissue showed 0.2% viability demonstrating the proper performance of the assay.
Note: Only one positive control sample was used for counting because the skin tissue of the other sample had been destroyed by the positive control material. This sample was excluded from the mean OD calculation.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 9.1%.
The difference of viability between the two negative control tissue samples in the MTT assay was 10.1%.

Specific criteria for irritation testing:
Standard deviation of the viability results for negative control samples was 4.0%.
The positive control treated tissues showed 3.9% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.4%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 10.8%.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Corrosivity testing:

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability (%RV)
		Measured	Blank corrected
Negative Control: Physiological saline (0.9% (w/v) NaCl)	1 2	1.086 1.197	1.039 1.150	94.9 105.1
	Mean	--	1.095	100.0
Positive Control: Glacial acetic acid	1 2	*0.046 0.049	*-0.001 0.002	*-0.1 0.2
	Mean	--	0.002	0.2
Test item: SynNova Base Oil	1 2	1.170 1.072	1.123 1.025	102.6 93.6
	Mean	--	1.074	98.1

Notes:

1. Mean blank value was 0.047

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. *: Negative values of positive controls were excluded from the calculation

 

Irritation testing:

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability (%RV)
		Measured	Blank corrected
Negative Control: Phosphate buffered saline	1 2 3	0.789 0.849 0.833	0.740 0.800 0.784	99.5 103.2 101.2
	Mean	--	0.775	100.0
Positive Control: 5% (w/v) SDS solution	1 2 3	0.078 0.083 0.077	0.029 0.034 0.028	3.8 4.4 3.6
	Mean	--	0.031	3.9
Test item: SynNova Base Oil	1 2 3	0.911 0.744 0.828	0.862 0.695 0.779	111.2 89.7 100.5
	Mean	--	0.778	100.5

Notes:

1. Mean blank value was 0.049

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

 

HISTORICAL CONTROL DATA

 

CORROSIVITY TESTING

(updated 15 November 2018)

	Negative control (Physiological saline)	Positive control (Glacial acetic acid)
Minimum optical density (OD)	0.590	0.000
Maximum optical density (OD)	1.516	0.051
Mean optical density (OD)	0.849	0.012
Standard Deviation (SD)	0.137	0.009
Number of cases	182	179

OD: Optical density (absorbance)

SD: Standard deviation

Note: All optical density (OD) values measured are background corrected values (measured at 570±30 nm).

 

IRRITATION TESTING

(updated 15 November 2018)

	Negative control (PBS)	Positive control (5% (w/v) SDS solution)
Minimum optical density (OD)	0.573	0.019
Maximum optical density (OD)	1.362	0.354
Mean optical density (OD)	0.784	0.059
Standard Deviation (SD)	0.122	0.038
Number of cases	338	333

PBS: Phosphate buffered saline

SDS: Sodium dodecyl sulphate

OD: Optical density (absorbance)

SD: Standard deviation

Note: All OD values (measured at 570±30 nm) are background corrected values.

Interpretation of results:

GHS criteria not met

Conclusions:

Corrosivity testing: Following exposure to SynNova Base Oil for 4 hours, the mean cell viability was 98.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.
Irritation testing: Following exposure to SynNova Base Oil for 15 minutes, the mean cell viability was 100.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.
The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKINTM (SM) model test with SynNova Base Oil, the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.

Executive summary:

An in vitro skin corrosivity and irritation test of SynNova Base Oil was performed in a reconstructed human epidermis model. EPISKIN^TM(SM) is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines.

 

Disks of EPISKIN^TM(SM) were treated with the test item and incubated for 15 minutes (irritation testing) and 4 hours (corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere (irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Corrosivity testing:

Following exposure with SynNova Base Oil for 4 hours, the mean cell viability was 98.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

 

Irritation testing:

Following exposure with SynNova Base Oil for 15 minutes, the mean cell viability was 100.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

 

The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN^TM(SM) model test with SynNova Base Oil, the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: 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:

key study

Study period:

08 April 2019

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Guidelines for the Testing of Chemicals No. 438 (2018)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, the Annex to Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL of the test item was applied onto the entire surface of the cornea.
One negative control eye was treated with 30 μL of physiological saline
Three positive control eyes were treated with 30 μL 5% (w/v) Benzalkonium chloride solution.

Duration of treatment / exposure:

exposure period of 10 seconds from the end of the application

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

7 replicates (3 test item/3 positive control/ 1 negative control)

Details on study design:

SELECTION AND PREPARATION OF EYES FOR THE TEST
Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and were placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Identification
The eyes were identified by chamber number, marked on the door of the chamber.

THE BASELINE ASSESSMENTS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change during acclimatization by more than 5% between the -45 minute and the zero time. No changes in thickness (0.0%) were observed in the eyes used in the study. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

TEST PROCEDURE
Treatment
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In the study, 30 μL of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea. Three test item treated eyes were examined in the study.
One negative control eye was treated with 30 μL of physiological saline; three positive control eyes were treated with 30 μL 5% (w/v) Benzalkonium chloride solution.

Test item removal
The time of application was observed, then after an exposure period of 10 seconds from the end of the application, the cornea surface was rinsed thoroughly with 20 mL physiological saline* at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.
*Note: Physiological saline (Manufacturer: B. Braun Pharmaceuticals SA, Lot number: 84222Y05-1, Expiry date: 30 September 2021) was used for rinsing.

OBSERVATION
Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 75 min - Test item

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 240 min - Test item

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MORPHOLOGICAL EFFECTS
Severe loosening of epithelium was observed on one eye at 120 minutes and on one eye at 180 minutes after the post-treatment rinse in case of the positive control material.

VALIDITY OF THE TEST
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in the experiment. This study was considered to be valid.

TEST ITEM

Observation	Value	ICE Class
Mean maximum corneal swelling up to 75 min	0.6%	I
Mean maximum corneal swelling up to 240 min	1.7%	I
Mean maximum corneal opacity change	0.00	I
Mean fluorescein retention change	0.33	I
Other Observations	None
Overall ICE Class	3xI

Based on this in vitro eye irritation assay in isolated chicken eyes with SynNova Base Oil, the test item is non-irritant, UN GHS Classification: No Category

 

POSITIVE CONTROL

Observation	Value	ICE Class
Mean maximum corneal swelling up to 75 min	11.5%	II
Mean maximum corneal swelling up to 240 min	28.4%	III
Mean maximum corneal opacity change	4.00	IV
Mean fluorescein retention change	3.00	IV
Other Observations	Severe loosening of epithelium was observed on one eye at 120 minutes and on one eye at 180 minutes after post-treatment rinse.
Overall ICE Class	1xIII 2xIV

The positive control (5% (w/v) Benzalkonium chloride solution) was classified as severely irritating, UN GHS Classification: Category 1.

 

NEGATIVE CONTROL

Observation	Value	ICE Class
Mean maximum corneal swelling up to 75 min	0.0%	I
Mean maximum corneal swelling up to 240 min	0.0%	I
Mean maximum corneal opacity change	0.00	I
Mean fluorescein retention change	0.00	I
Other Observations	None
Overall ICE Class	3xI

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

 

SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II, or 2 endpoints classed as II and 1 endpoint classed as I:	True
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:	True
Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity = 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False
Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False

 

Historical Control Data (updated on 07 November 2018):

 

Negative Control: Physiological Saline

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-3.2%	3.4%
Maximum corneal swelling at up to 240 min	-4.8%	3.4%
Maximum corneal opacity change	0.00	0.50
Fluorescein retention	0.00	0.50
Number of cases	416

 

Positive Control: 5% (w/v) Benzalkonium chloride solution

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-8.5%	27.0%
Maximum corneal swelling at up to 240 min	-10.7%	38.3%
Maximum corneal opacity change	2.50	4.00
Fluorescein retention	1.50	3.00
Number of cases	234

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro eye irritation assay in isolated chicken eyes with SynNova Base Oil, the test item is non-irritant, UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (25 June 2018).

 

After the zero time reference measurements, the eyes were held in horizontal position and 30 μL of the test item was applied onto the centre of the corneas such that the entire surface of the corneas were covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three treated eyes per test item, three positive control treated eyes and one negative control treated eye were examined.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

 

No significant corneal swelling (mean <5.0%) was observed during the four-hour observation period on the test item treated eyes. No corneal opacity change (severity 0.0) was noted on all eyes. No significant fluorescein retention change (severity 0.0 on one eye and severity 0.5 on two eyes) was noted on all eyes. No other corneal effects were observed.

 

Based on this in vitro eye irritation assay in isolated chicken eyes with SynNova Base Oil, the test item is non-irritant, UN GHS Classification: No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin corrosivity and irritation test

SynNova Base Oil was used in a reconstructed human epidermis model. EPISKIN^TM(SM) and is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Disks of EPISKIN^TM(SM) were treated with the test item and incubated for 15 minutes (irritation testing) and 4 hours (corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere (irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Corrosivity testing: Following exposure with SynNova Base Oil for 4 hours, the mean cell viability was 98.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Irritation testing: Following exposure with SynNova Base Oil for 15 minutes, the mean cell viability was 100.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

 

In conclusion, in this in vitro EPISKIN^TM(SM) model test with SynNova Base Oil, the results indicate that the test item is non-corrosive and non-irritant to the skin, UN GHS Classification: No Category.

In vitro eye irritation

Study of the test item was performed in isolated chicken’s eyes.

After the zero time reference measurements, the eyes were held in horizontal position and 30 μL of the test item was applied onto the centre of the corneas such that the entire surface of the corneas were covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three treated eyes per test item, three positive control treated eyes and one negative control treated eye were examined.

 

No significant corneal swelling (mean <5.0%) was observed during the four-hour observation period on the test item treated eyes. No corneal opacity change (severity 0.0) was noted on all eyes. No significant fluorescein retention change (severity 0.0 on one eye and severity 0.5 on two eyes) was noted on all eyes. No other corneal effects were observed.

Based on this in vitro eye irritation assay in isolated chicken eyes with SynNova Base Oil, the test item is non-irritant, UN GHS Classification: No Category.