Esterification product of sunflower-oil fatty acids, with 1,4:3,6-dianhydro-D-glucitol

Endpoint summary

Description of key information

According to the results of the two key studies for skin and eye irritation and CLP criteria, the test item LAB 448 did not required classification for eye and skin irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 4 January 2018 to 15 February 2018

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch E7064
- Expiration date of the lot/batch: January 2019
- Purity test date: 23 August 2017 retested July 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8°C), protected from humidity (tight closed container), under inert gas (e.g. N2)
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no vehicle was used

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was used neat, as supplied.
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) the test item was used neat, as supplied

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

other: EpiSkinTM model ; three-dimensional human epidermis model

Cell source:

other: Adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen

Source strain:

other: not applicable

Justification for test system used:

The irritation potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EPISKINTM (SM) reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Model
- Tissue batch number(s): 18-Ekin-002 and 17-EKIN-0034
- Production date: not specified
- Shipping date:not specified
- Delivery date: not specified
- Date of initiation of testing: 10 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21.3-24.5°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the 15 minutes incubation time, the EPISKIN TM (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).
- Observable damage in the tissue due to washing: No damage
- Modifications to validated SOP: No modification of SOP was performed

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours ± 5 minutes
- Spectrophotometer: Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until
August 2018)
- Wavelength: 570 nm
- Filter: No filter was used
- Filter bandwidth: no filter was used
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Conform
- Barrier function: Conform
- Morphology: Well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: Absence of contaminaton
- Reproducibility: Conform

NUMBER OF REPLICATE TISSUES: triplicates were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Source: SkinEthic, France, Batch No.: 17-EKIN-034, Expiry Date: 28 August 2017) 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 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 25 August 2017 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.
- N. of replicates : 2
- Method of calculation used:
Non-specific MTT reduction calculation (NSMTT%):
NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD

If NSMTT% is ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues

The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test item is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 : pretest (with MTT direct reduction test and colour interference test) and main test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and after 42 hours post exposure incubation period is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and after 42 hours post exposure incubation period is greater than 50%
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: Not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 µL
- Concentration (if solution): pure

VEHICLE
No vehicle was sued

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of PBS
- Concentration (if solution): pure

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 5% (w/v) SDS solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates were used

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

91

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.018, Non Specific Colour % (NSCkilled%) was calculated as 2.5% . Because the NSCliving% was not used, therefore correction with NSCkilled% was not necessary.

- Colour interference with MTT: As colour change was observed after three hours of incubation of the test item in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability.
Based on the observed mean OD (0.005), the calculated NSMTT% is 0.7%. This was considered to be significant, thus correction with NSMTT was made.


DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control condition showed ability of the test item to measure positve response

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.714). Standard deviation of the viability results for negative control samples was 5.2%.
- Acceptance criteria met for positive control: The positive control treated tissues showed 3.6% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.3%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 10.6%.

Table: Optical Density (OD) and thecalculatedrelativeviability% of thesamples

 

	Optical Density (OD)			TODTT	Viability
Substance		Measured	Blankcorrected		(% RV)
NegativeControl	1	0.782	0.735	--	103.0
Phosphatebufferedsaline	2	0.718	0.671	--	94.0
	3	0.783	0.736	--	103.1
	mean	--	0.714	--	100.0
Positive Control:	1	0.068	0.021	--	3.0
5% (w/v) SDS solution	2	0.067	0.020	--	2.8
	3	0.083	0.036	--	5.1
	mean	--	0.026	--	3.6
	1	0.684	0.637	0.632	88.5
	2	0.637	0.590	0.585	81.9
	3	0.784	0.737	0.732	102.6
	mean	--	0.655	0.650	91.0

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).

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental condition of the study, following exposure with LAB 4448, the mean cell viability was 91.0% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin according to CLP criteria.

Executive summary:

This GLP-compliant in vitro study was performed in order to assess the potential skin irritation effect of the registered substance LAB4448 using EpiSkinTM model, according to OECD TG 439 method.

Disks of EPISKINTM (SM) (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, in a >95% humidified atmosphere. 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, in a >95% humidified atmosphere, 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). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 min 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.

Under the experimental condition of the study, following exposure with LAB 4448, the mean cell viability was 91.0% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin according to CLP criteria.

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:

From 6 January 2016 to 20 January 2016

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor
- Expiration date of the lot/batch:August 2017
- Purity test date: August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature and protected from light
- Stability under test conditions: not applicable, the test condition period was only 10 minutes
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was used as received
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: Not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) Neat, as supplied

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Spear Products
- Number of animals: 9 corneas were used
- Characteristics of donor animals (e.g. age, sex, weight): not detailed
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported in HBSS medium with penicillin-streptomycin on the 7 January 2016 at the test facility
- Time interval prior to initiating testing: immediately after receipt
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: streptomycin and penicillin were used in medium

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): neat

VEHICLE
No vehicle was used

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3 replicates per condition

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in specially designed holders that were separated into anterior and
posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder was incubated at 32ºC (± 1º) and allowed to equilibrate for at least one hour but not longer than two hours.

QUALITY CHECK OF THE ISOLATED CORNEAS
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied with the opacitometer. Any cornea with a value greater than 7 units was discarded.

NUMBER OF REPLICATES
3 replicates were used per condition

NEGATIVE CONTROL USED
MEM Minimal Essential Medium

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 µL for 10 minutes

TREATMENT METHOD: Close Chamber

POST-INCUBATION PERIOD: Yes 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: he test article, ethanol or MEM solution was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were refilled with fresh MEM solution.

- POST-EXPOSURE INCUBATION: All corneas were incubated at 32ºC (± 1º) for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: using OP-KIT
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score (IVIS) = Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)

DECISION CRITERIA: Criteria from OECD TG 437 was used

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Article - Calculated in vitro irritation score

Value:

1.03

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No damage

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control showed technical proficiency to measure a positive result

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Conform
- Acceptance criteria met for positive control: Conform; The ethanol positive control In Vitro Irritancy Score was 22.56, which fell within the acceptance range of 20.43 – 34.27 (± 2 standard deviations of the historical mean).

Table 1 :Results

Cornea ID	Pretest Opacity Score	10-minute Opacity Score	2-Hour Opacity Score	Corrected Opacity Scores^1		OD490 nm (Permeability)	Corrected Optical Density^2
				10-minute	2-hour
10	0	1	2	1	2	0.018	0.001
11	0	2	1	2	1	0.015	-0.002
12	4	3	3	-1	-1	0.023	0.006
Corrected Mean Optical Density^3=							0.002
2-Hour Corrected Mean Opacity Score^4=							1.00

 

Table 2 Calculated In Vitro Irritation Scores

Test Article	Negative Control	Positive Control
HydraSynolIDL; IsosorbideDilinoleate
(INCI name proposed);	MEM	100% Ethanol
CAS# 1818326-42-9, Lot# CB 15026
1.00 + (15 x 0.002)	-0.33 + (15 x 0.017)	15.33 + (15 x 0.482)
1.00 + 0.03	-0.33 + 0.255	15.33 + 7.23
IVIS =   1.03	IVIS =  -0.07	IVIS =  22.56

 

Positive Control Historical Data
Mean IVIS	27.35
Standard Deviation	3.46
n	28

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, the test item HydraSynol IDL; Isosorbide Dilinoleate induced an IVIS of 1.03. Hence, according to CLP criteria, no classification was required for the test substance for eye irritation.

Executive summary:

This GLP compliant In Vitro study was performed in order to determine the potential for ocular irritation of the test substance HydraSynol IDL; Isosorbide Dilinoleate according to OECD 437 method (BCOP Assay)

Three bovine corneas per group were dosed with 0.75 ml of HydraSynol IDL; Isosorbide Dilinoleate, Minimal Essential Medium as negative control and Ethanol as positive control condition. Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined.

2 hours of post incubation period was performed and measurements of opacity was taken with each treated cornea compared to the blank. After 90 minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a spectrophotometer.

Calculated IVIS values are 1.0, -0.07 and 22.56 respectively for test substance, negative and positive control.

Under the experimental conditions of the study, the test item HydraSynol IDL; Isosorbide Dilinoleate induced an IVIS of 1.03. Hence, according to CLP criteria, no classification was required for the test substance for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

One key study is available for skin irritation assessment : Varga Kanizsai, OECD TG 439, GLP, Klimisch 1

Disks of EPISKIN TM (SM) (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. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Under the experimental condition of the study, following exposure with LAB 4448, the mean cell viability was 91.0% compared to the negative control.

One key study is available for eye irritation assessment : Yasso, OECD TG 437, Klimisch 1, GLP

Three bovine corneas per group were dosed with 0.75 ml of HydraSynol IDL; Isosorbide Dilinoleate, Minimal Essential Medium as negative control and Ethanol as positive control condition. Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined. 2 hours of post incubation period was performed and measurements of opacity was taken with each treated cornea compared to the blank. After 90 minutes, the fluid from the posterior chamber was removed and the amount of dye, which passed through the cornea (permeability), was measured as the optical density at 490 nm by a spectrophotometer. Calculated IVIS values are 1.0, -0.07 and 22.56 respectively for test substance, negative and positive control.

Justification for classification or non-classification

According to the results of the two key studies for skin (Varga Kanizsai, OECD TG 439, GLP, Klimisch) and eye irritation (Yasso, OECD TG 437, Klimisch 1, GLP) , the test item LAB 448, HydraSynol IDL did not induced adverse effect in both of these studies. Hence, the test item LAB 448 did not required classification for eye and skin irritation according to CLP criteria.