2,3-dihydro-2,2,6-trimethylbenzaldehyde

Endpoint summary

Description of key information

Skin irritation, in vitro: irritating, OECD TG 439, 2015

Skin corrosion, in vitro: non-corrosive, OECD TG 430, 2019

Eye irritation, in vitro: irritating, OECD TG 438, 2015

Eye irritation, in vitro: irritating, OECD TG 491, 2019

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From January 02, 2019 to January 11, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

isolated skin discs

Source species:

rat

Details on animal used as source of test system:

Species: Rat (Rattus norvegicus)
Strain: Wistar
Source: Bred and reared at INTOX PVT. LTD.
Age at start of study: 22 days
No. of animals and sex: 5 females

Justification for test system used:

Rat is the rodent species and Wistar is the strain recommended by the test guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

Preparation of the skin discs: Animals were humanely sacrificed by CO2 asphyxiation, when they were 28 days old. The dorso­ lateral skin of each animal was then removed and stripped of excess subcutaneous fat by peeling it away from the skin. Skin discs, with a diameter of approximately 20 mm each, were prepared using scissors. As many as 4-5 skin discs were obtained from a single rat skin.
Fitting of skin disc to PTFE tubes: Each skin disc was placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface is in contact with the tube. A rubber "O" ring was press-fitted over the end of the tube to hold the skin in place and excess tissue was trimmed away.
The tube was supported by a rubber cork inside a receptor chamber containing MgS04 solution (154 mM). The skin disc was fully submerged in the MgS04 solution.
Before testing begins, the electrical resistance of skin discs was measured as a quality control procedure for each animal skin.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

150 µL test item was applied uniformly to the epidermal surface inside the tube.
The control items, 150 µL each of analytical grade water and 1OM hydrochloric acid (HCL) as negative and positive controls respectively were applied uniformly.

Duration of treatment / exposure:

Test item and control items were applied for 24 hours at 22 °C. Thereafter the test and control items were removed by washing with tap water until no further material could be removed.

Duration of post-treatment incubation (if applicable):

1 night

Number of replicates:

3

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Value:

6.91

Vehicle controls validity:

not applicable

Remarks:

No vehicle

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

dye content (µg/disc)

Value:

40.13

Vehicle controls validity:

not applicable

Remarks:

No vehicle

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test item will be considered non-corrosive to skin:
1. if the mean TER value obtained for the test item is greater than (>) 5 kn .
2. if the mean TER value obtained for test item is less than or equal to (≤) 5 kn and the skin discs show no obvious damage (e.g. perforation), and mean disc dye content is less than 1OM HCI positive control obtained concurrently.

The test item will be considered to be corrosive to skin:
1. if the mean TER value obtained for test item is less than or equal to (≤) 5 kn and the skin discs are obviously damaged (e.g. perforated)
2. if the mean TER value obtained for test item is less than or equal to (≤) 5 kn and the skin discs show no obvious damage (e.g. perforation) but the mean disc dye content is greater than or equal to (≥) the mean disc dye content of the 1OM HCI positive control obtained concurrently.

The mean resistance of the skin in the negative control group was found to be 13.39 kΩ and the mean dye content of skin disc was 34.69 µg/disc, which were found to be accepted.

The mean resistance of the skin in the positive control group was found to be 1.28 kΩ and the mean dye content of skin disc was 61.50 µg/disc, which were found to be accepted, thereby validating the experimental procedure.

The mean TER value obtained for test item was 6.91 kΩ (which was greater than 5 kΩ) and the skin discs showed no obvious damage (e.g. perforation) and the mean disc dye content was 40.13 µg/disc, which was lesser than the mean disc dye content of the 1OM HCI positive control obtained concurrently.

Interpretation of results:

GHS criteria not met

Conclusions:

In-Vitro Skin Corrosion Study of Safranal by Transcutaneous Electrical Resistance Test Method (TER), using Skin Discs of Rats was carried out in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 430 - In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER), adopted by the council on 28 July 2015.

The mean TER value obtained for test item Safranal was 6.91 kΩ (which was greater than 5 kΩ) and the skin discs showed no obvious damage (e.g. perforation). The mean disc dye content was 40.13 µg/disc, which was lesser than the mean disc dye content of the 1OM HCI positive control obtained concurrently.

According to the criteria of acceptance, under the experimental conditions of the study 'In-Vitro Skin Corrosion Study of Safranal by Transcutaneous Electrical Resistance Test Method (TER)', it is concluded that the test item Safranal is considered as non-corrosive to rat skin as per the United Nations(UN) Globally Harmonised System (GHS) for classification of chemicals.

Executive summary:

In-Vitro Skin Corrosion Study of Safranal by Transcutaneous Electrical Resistance Test Method (TER), using Skin Discs of Rats was carried out in compliance with the Organization for Economic Co-operationand Development (OECD) Guidelines for Testing of Chemicals, Section 4, No.430-In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER), adopted by the council on 28 July 2015 and as per mutually agreed Study Plan.

 

Safranal was evaluated for its corrosive potential by its ability to produce a loss of normal stratum corneum integrity and barrier function, which was measured as a reduction in the TER below the threshold level (cut-off value) of 5 kΩ for rat skin, and from any increase in the ionic permeability for sulforhodamine B dye.

 

The skin discs were taken from humanely sacrificed rats aged 28 days and were applied with 150 µI of test item, in triplicate. Concurrent triplicate sets of skin discs were applied with analytical grade water (150µI) as negative control and with 10M hydrochloric acid (150µI) as positive control.

 

The test and control items were applied for up to 24 hours at 22 °C to the epidermal surface of skin discs in a two compartment test system in which the skin disc functionedastheseparationbetween thecompartments.The test / control items were then removed by washing with a jet of tap water until no further material could be removed. The skin impedance was measured as TER by using Aplab Autocompute LCR-Q meter 4910. The electrodes of LCR-Q meter were placed on either side of the skin disc to measure the resistance of skin disc at a frequency of 100 Hz and using series values. Following TER measurement, the skin was carefully examined for obvious damage.After visual examination, 10% (w/v) sulforhodamine B was applied to the epidermal surface of each skin disc for 2 hours, which was followed by the measurement of dye content in each skin disc. The results have been tabulated below.

End-point	Values	Test Item	Negative Control Item	Positive Control Item
Mean TER (kn)	Observed	6.91	13.39	1.28
	Acceptable range	-	10 to 25	0.5 to 1.0
Mean dye content (µg/disc)	Observed	40.13	34.69	61.50
	Acceptable range	-	15 to 35	40 to 100

The mean resistance of the skin in the negative control group was found to be 13.39 kΩ and the mean dye content of skin disc was34.69 µg/disc,which were found to be accepted.

The mean resistance of the skin in the positive control group was found to be 1.28 kΩ and the mean dye content of skin disc was 61.50 µg/disc, which were found to be accepted, thereby validating the experimental procedure.

The mean TER value obtained for Safranal was 6.91KΩ which was greater than 5 kΩ, the skin discs showed no obvious damage (e.g.perforation) and mean disc dye content was found to be 40.13 µg/disc which was lesser than that observed in skin discs treated with the positive control item.

 

According to the criteria of acceptance, under the experimental conditions of the study 'In-Vitro Skin Corrosion study of Safranal by Transcutaneous Electrical Resistance Test Method (TER)', it is concluded that the test item Safranal is considered as non-corrosive to rat skin as per the United Nations (UN) Globally Harmonised System (GHS) for classification of chemicals.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 30, 2015 to July 21, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in 2015 at recognised contract research organisation.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPISKIN-SM, three-dimensional human epidermis model

Justification for test system used:

The EPISKIN-SM model has been validated for irritation testing in an international validation study [9] 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)

Details on test system:

EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-015, Expiry Date: 20 April 2015) is a three-dimensional human epidermis model. Adult humanderived 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) [7]. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.: 14-EKIN-044, Expiry Date: 24 November 2014) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for approximately 48 hours. At the end of the incubation the water was discarded and the dead epidermis units were frozen on 21 November 2014. Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

10 µL of test item were applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

Duration of treatment / exposure:

42 hours

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

1.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

According to EU classification, the irritancy potential of test substances is predicted for distinguishing between skin irritating (R38) and no-label (non-skin irritating) test substances OECD TG 404, OECD 439 & Method B.4 of Annex V to Directive 67/548/EEC [1,4,12,13]. The irritation potential of test substances can be also classified according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals [10]. In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test item. The test item is considered to be irritant to skin (Category 2, H315), 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 prediction model (PM) is described below:
- If Mean tissue viability % is ≤ 50%: Irritant (I) with EU (67/548/EEC); Category 2 with UN GHS / CLP
- If Mean tissue viability % is > 50%: Non Irritant (NI) with EU (67/548/EEC); No Category with UN GHS / CLP

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In conclusion, in this in vitro EPISKIN model test with SAFRANAL, the results indicate that the test item is irritant to skin.

Executive summary:

An in vitro skin irritation test of SAFRANAL test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the 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 (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes 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. 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. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). An additional disk was used to provide an estimate of colour contribution from the test item. Furthermore, three additional test item treated and three negative control treated killed epidermis units were used to determine the MTT interacting potential of the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. 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.

Following exposure with SAFRANAL, the mean cell viability was 1.1% compared to the negative control (after adjustment for colour and non-specific MTT reduction). This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. Note that this test protocol can not identify corrosives but usually only corrosive substances cause effects of this magnitude. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with SAFRANAL, the results indicate that the test item is irritant to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

From January 26, 2015 to August 07, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in 2015 at recognised contract research organisation.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129. Hungary
Chicken heads were collected after slaughter in a commercial abattoir from chickens 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 approximately 2 hours of collection.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied as supplied, no formulation was required.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied in a volume of 30 µL 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.

Duration of treatment / exposure:

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 the test item if possible.

Duration of post- treatment incubation (in vitro):

approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

3

Details on study design:

After the zero reference measurements, the eye was held in horizontal position and 30 μL of SAFRANAL was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline.

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 75 min

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum corneal swelling at up to 240 min

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum corneal opacity

Value:

0.67

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean fluorescein retention

Value:

0.67

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with SAFRANAL, the test item is not classified as severe irritant and not classified as non-irritant.

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 (26th July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 μL of SAFRANAL was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with 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 (Salsol solution, NaCl 0.9% w/v).

No corneal swelling was observed during the four hour observation period. Corneal opacity change (severity 0.5 or 1) and fluorescein retention change (severity 0.5 or 1) was noted on all test item treated eyes. These results are slightly above the maximum threshold for a negative.

Based on this in vitro eye irritation in the isolated chicken eyes test with SAFRANAL, the test item is not classified as a severe irritant and not classified as non-irritant.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From January 12, 2019 to January 21, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 491 (Short Time Exposure In Vitro 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)

Species:

other: Cells

Details on test animals or tissues and environmental conditions:

Cell line medium: SIRC (Statens Seruminstitut Rabbit Cornea) cell line. Minimum essential medium (MEM) supplemented with L-glutamine containing 10% fetal bovine serum (FBS) and Penicillin Streptomycin Solution (1X).
Culture conditions: 37 ± 1 °C, -5% CO2, 90 to 100% Humidity
Cell refreshing agent: Dulbecco' Phosphate Buffer Saline
Cell dispersion agent: 0.25% Trypsin EDTA.

Vehicle:

other: Name: Mineral oil; CAS: 8042-47-5; Batch: MKBX6122V; Date of Expiry: 31-10-2022

Controls:

yes

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

The cells were seeded at the density of 6 x 103 cells per well of 96 well plate and were used four days after seeding for experiment no. 1, 2 and 3 at a culture volume of 200 µL. When cells reached about 80% confluence monolayer then they were treated with 5 and 0.05% concentration of test item.

Duration of treatment / exposure:

The 96 well plates were incubated at room temperature for five minutes.

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

After exposure, cells were washed twice with 200 µL of PBS and 200 µL of MTT solution (0.5 mg MTT per ml of culture medium) was added. After two-hour reaction time in an incubator (37 ± 1°C, ~5% CO2 and 90 to 100% humidity), the MTT solution was decanted. MTT formazan was extracted by adding 200 µL of 0.04 N hydrochloric acid-isopropanol for 60 minutes in the dark at room temperature, and the absorbance of the MTT formazan solution was measured at 570 nm with ELISA plate reader.

Irritation parameter:

other: Relative cell viability

Run / experiment:

Mean for a concentration of test item at 5% (%)

Value:

35.34

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Standard deviations of the final cell viability derived from three experiments was 2.02 for both 5% concentration of test item.

Irritation parameter:

other: Relative cell viability

Run / experiment:

Mean for a concentration of test item at 0.05% (%)

Value:

78.59

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Standard deviations of the final cell viability derived from three experiments was 6.18 for both 0.05% concentration of test item.

Other effects / acceptance of results:

Test results are acceptable when the following criteria are all satisfied:
a) Optical density of the medium control (exposed to culture medium) should be 0.3 or higher after subtraction of blank optical density.
b) Viability of the vehicle control should be 80% or higher relative to the medium control. If multiple vehicle controls are used in each experiment, each of those controls should show cell viability greater than 80% to qualify the test item tested with those vehicles.
c) The cell viability obtained with the positive control (0.01% SLS) should be within two standard deviations of the historical mean. The upper and lower acceptance boundaries for the positive control should be frequently updated i.e., every three months, or each time
an acceptable test is conducted in laboratories where tests are conducted infrequently (i.e., less than once a month). Where a laboratory does not complete a sufficient number of experiments to establish a statistically robust positive control distribution, it is acceptable that the upper and lower acceptance boundaries established by the method developer are used, i.e., between 21.1% and 62.3% according to its laboratory historical data, while an internal distribution is built during the first routine tests.
d) Standard deviation of the final cell viability derived from three independent experiments should be less than 15% for both 5% and 0.05% concentrations of the test item.

Interpretation of results:

GHS criteria not met

Conclusions:

'Short Time Exposure In-Vitro Study of Safranal Using SIRC Cell Line for Identification of Serious Eye Damage' was carried out in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 491 - Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, adopted by the council on 25 June 2018 and as per mutually agreed Study Plan.
Under the given experimental conditions of 'Short Time Exposure In-Vitro Study of Safranal Using SIRC Cell Line for Identification of Serious Eye Damage' (OECD Guideline No. 491), it is concluded that this test method has successfully classified the test item into 'No prediction can be made' category as per the United Nations (UN) Globally Harmonised System (GHS) for classification of chemicals.

Executive summary:

'Short Time Exposure In-Vitro Study of Safranal Using SIRC Cell Line for Identification of Serious Eye Damage' was carried out in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 491 - Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, adopted by the council on 25 June 2018 and as per mutually agreed Study Plan.

Safranal was evaluated for its irritancy potential based on its ability to induce cytotoxicity in Short Time Exposure (STE) Test method. The cytotoxic effect of test item on corneal epithelial cells is an important mode of action (MOA) leading to corneal epithelium damage and eye irritation.

After extraction from cells, cell viability in the STE test method is assessed by the quantitative measurement of blue formazan salt produced by the living cells. It is due to enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), also known as Thiazolyl Blue Tetrazolium Bromide. The obtained cell viability is compared to the vehicle control (relative viability) and used to estimate the potential eye hazard of test item.

Optical density of the medium control was found to be 0.367 and after subtraction of blank, optical density was 0.317 which was higher than 0.3.

The relative cell viability obtained for vehicle control (mineral oil) was 87.62%, which was greater than 80% relative to medium control.

The relative cell viability obtained for normal saline (vehicle for Positive control) was 85.96%, which was greater than 80% relative to medium control.

The relative cell viability obtained for 0.01% sodium lauryl sulfate (Positive control) was 25.13%, i.e. between 21.1% and 62.3% according to laboratory historical data established by the method developer.

Standard deviations of the final cell viability derived from three experiments were 2.02 and 6.18 for both 5% and 0.05% concentrations of test item respectively, which is less than 15%.

Cell viability in both the concentrations i.e. 5% and 0.05% was 35.34% and 78.59% respectively, , which was less than 70% for 5% test concentration and greater than 70% for 0.05% test concentration.

Under the given experimental conditions of this study, it is concluded that the test item is considered to fall under 'No prediction can be made' category of UN GHS  classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Key study: An in vitro skin irritation test of SAFRANAL test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the 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 (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Following exposure with SAFRANAL, the mean cell viability was 1.1% compared to the negative control (after adjustment for colour and non-specific MTT reduction). This is below the threshold of 50%, therefore the test item was considered as being irritant to skin. Note that this test protocol can not identify corrosives but usually only corrosive substances cause effects of this magnitude. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with SAFRANAL, the results indicate that the test item is irritant to skin.

Supporting study: In-Vitro Skin Corrosion Study of Safranal by Transcutaneous Electrical Resistance Test Method (TER), using Skin Discs of Rats was carried out in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 430 - In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER), adopted by the council on 28 July 2015.

The mean TER value obtained for test item Safranal was 6.91 kΩ (which was greater than 5 kΩ) and the skin discs showed no obvious damage (e.g. perforation). The mean disc dye content was 40.13 µg/disc, which was lesser than the mean disc dye content of the 1OM HCI positive control obtained  concurrently.

Eye irritation:

Key study: 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 (26th July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 μL of SAFRANAL was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with 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 (Salsol solution, NaCl 0.9% w/v).

No corneal swelling was observed during the four hour observation period. Corneal opacity change (severity 0.5 or 1) and fluorescein retention change (severity 0.5 or 1) was noted on all test item treated eyes. These results are slightly above the maximum threshold for a negative.

Based on this in vitro eye irritation in the isolated chicken eyes test with SAFRANAL, the test item is not classified as a severe irritant and not classified as non-irritant.

Supporting study: 'Short Time Exposure In-Vitro Study of Safranal Using SIRC Cell Line for Identification of Serious Eye Damage' was carried out in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Section 4, No. 491 - Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, adopted by the council on 25 June 2018 and as per mutually agreed Study Plan.

Under the given experimental conditions of 'Short Time Exposure In-Vitro Study of Safranal Using SIRC Cell Line for Identification of Serious Eye Damage' (OECD Guideline No. 491), it is concluded that this test method has successfully classified the test item into 'No prediction can be made' category as per the United Nations (UN) Globally Harmonised System (GHS) for classification of chemicals.

Justification for classification or non-classification

The substance does meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation.

The substance does meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.

 

For skin irritation, the in vitro assay (OECD TG 439) indicates that the substance has the potential to cause irritating effects to the skin but which are insufficient for classification. Based on this study, Safranal is classified skin irritating.

 

For eye irritation, the in vitro assay (OECD TG 438) indicates that the substance is not classified as a severe irritant and not classified as non-irritant. Based on this study, Safranal is classified eye irritating.