Sorbitan, tridocosanoate

Endpoint summary

Description of key information

Skin irritation (OECD 439): not irritating

Eye irritation (OECD 437): not irritating

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:

25 - 28 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: Keratinocyte Strain: 4F0219

Justification for test system used:

reccomended in TG OECD 439

Vehicle:

unchanged (no vehicle)

Remarks:

Tissue was wetted with 25 µL of PBS prior to exposure

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm TM Reconstructed Human Epidermis, EPI-200, EPI-212
- Tissue batch number(s): 27124
- Production date: 27 Sep 2017 (Date of Certificate of Analysis)
- Date of initiation of testing: 25 Sep 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C, humid, 5% CO2 (incubator), then room temperature
- Temperature of post-treatment incubation: not provided

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissue was rinsed 15 times with PBS(-), then they were 3 times completely submerged in 150 mL PBS(-) and once rised from inside and outside with PBS(-); Remaining PBS(-) was removed from the inside and outside of the tissue insert with a sterile cotton swab before transferring the tissues into the wells with fresh medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 180 ± 5 min
- Spectrophotometer: FLUOstar OPTIMA, BMG LABTECH
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.802 ± 0.057
- Barrier function: test was passed
- Morphology: well-differentiated epidermis consisting of a viable basal layer, intermediate spinous and granular layers and a functional stratum corneum
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive or irritant to skin if the mean relative viability after 60 minutes exposure is less or equal to 50% of the negative control.
- The test substance is considered to be non-corrosive or non irritant to skin if the viability after 60 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration: 5%

Duration of treatment / exposure:

60 min (35 min at 37°C and 25 min at room temperature)

Duration of post-treatment incubation (if applicable):

24 ± 2 h, after transfer to lower wells containing fresh medium further 18 ± 2 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of positive control (5% SDS)

Value:

1.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of test substance

Value:

98.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 2.348)
- Acceptance criteria met for positive control: yes (mean viability = 1.9%)
- Acceptance criteria met for variability between replicate measurements: yes (0.2 - 3.2%)

Table 1: Results MTT assay after 3 h ± 5 min exposure

Substance	Tissue No.	OD570	OD570 (mean per tissue)	OD570 (mean of 3 tissues)	Cell viability [%]	Cell viability (mean) [%]	Standard Deviation of cell viability [%]
Negative Control	1	2.409	2.385	2.348	101.6	100.0	1.4
		2.36
	2	2.376	2.338		99.6
		2.299
	3	2.364	2.322		98.9
		2.280
Positive Control	1	0.046	0.046	0.044	2.0	1.9	0.2
		0.046
	2	0.041	0.041		1.7
		0.04
	3	0.047	0.045		1.9
		0.042
Test substance	1	2.381	2.348	2.313	100.0	98.5	3.2
		2.314
	2	2.389	2.364		100.7
		2.338
	3	2.261	2.227		94.8
		2.192

 

Interpretation of results:

other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

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:

25 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

09 Oct 2017

Deviations:

no

GLP compliance:

yes

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Hokkaido Livestock Corporation, Douo Plant, Hayakita Factory
- Number of eyeballs: 30, 12 used in the study
- Characteristics of donor animals: 12 months to less than 60 months old, healthy
- Storage, temperature and transport conditions of ocular tissue: The collected eyeballs were immersed in Hanks' balanced salt solution, and stored and transported at less than 20°C until use in the test (temperature range during transportation: 12 - 14°C).
- Time interval prior to initiating testing: same day
- Indication of any existing defects or lesions in ocular tissue samples: no

Vehicle:

water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 10% (w/v)

VEHICLE
- Amount(s) applied: 8 mL
- Concentration: undiluted
- Lot/batch no.: K7D7

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS : A careful macroscopic examination was performed on all eyes to detect the presence of any defects (corneal opacity, scratches, neovascularization etc). Any eyes with defects were discarded. The corneas were dissected with a 2 to 3 mm rim of the sclera remaining, and were immersed in Hanks' balanced salt solution (HBSS). The corneae were directly used in the BCOP test on the same day.

QUALITY CHECK OF THE ISOLATED CORNEAS : Eyes were free of defects.

NUMBER OF REPLICATES : 3

SOLVENT CONTROL USED: yes, distilled water

POSITIVE CONTROL USED : yes, Dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME : 750 μL test item (10%), 10 min

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with phenol red-containing MEM and 1 time with phenol red-free MEM

- POST-EXPOSURE INCUBATION: yes, 2 h

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured using the opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG (OECD 437) was used.

STUDY ACCEPTANCE CRITERIA: The following study acceptance criteria described in OECD TG 437 were adopted:
A test is considered acceptable if the IVIS of the positive control is within 2 standard deviations of the latest historical control mean. A test is considered acceptable if the opacity and permeability values of the negative control are less than the upper limit of opacity and permeability values of the historical control data obtained from the bovine corneas treated with the negative control.

Irritation parameter:

cornea opacity score

Run / experiment:

Negative control (Mean corrected final opacity)

Value:

0.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Remarks:

(Permeability)

Run / experiment:

Negative control (Mean corrected OD490 change)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative control

Value:

0.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Test substance (Mean corrected final opacity)

Value:

-0.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Remarks:

(Permeability)

Run / experiment:

Test substance (Mean corrected OD490 change)

Value:

-0.001

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Test substance

Value:

-0.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Positive control (Mean corrected final opacity)

Value:

95.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Remarks:

(Permeability)

Run / experiment:

Positive control (Mean corrected OD490 change)

Value:

0.77

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control

Value:

107.2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: not demonstrated in the study report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:

Opacity
Negative control: -2.0 - 2.4
Positive control: 38.0 - 111.0

Permeability
Negative control: -0.003 - 0.027
Positive control: 0.171 - 1.708

IVIS:
Negative control: -2.0 - 2.4
Positive control: 42.3 - 128.0 (mean ± 2SD: 67.4 ~ 128.2)

Interpretation of results:

other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vitro

Sorbitan tridocosanoate was investigated for its skin irritation potential in epidermal keratinocytes in vitro using reconstructed human epidermis. The test was conducted with the EpiDerm™ (EPI-200, EPI-212) model according to OECD guideline 439 and in compliance with GLP (Riken Vitamin, 2017a).

25 µL of the undiluted test item, the negative control (phosphate buffered saline, PBS) and the positive control (5% sodium dodecyl sulfate, SDS) were applied topically to the surface of the tissues and incubated for a total of 60 min (35 min at 37 °C followed by 25 min at room temperature). After exposure and 42 h of post-incubation at 37 °C, cytotoxicity was evaluated by MTT reduction. The test item was known to not colour-interfere with water and not directly reduce MTT. All acceptability criteria of the test were met.

When compared to the mean relative tissue viability of the solvent control, the mean relative tissue viability of sorbitan tridocosanoate after 60 min of exposure was 98.5%. This value is above the threshold for irritancy of ≤50%. Therefore, under the conditions of the test, the test item is considered to be non-irritant to the skin.

 

 

Eye irritation in vitro

The eye irritating potential of sorbitan tridocosanoate was investigated using a bovine corneal opacity and permeability (BCOP) test in vitro. The BCOP test was conducted according to OECD guideline 437 and in compliance with GLP (Riken Vitamins, 2017b). After an initial opacity reading, the test item was prepared as 10% (w/v) formulation in distilled water. 750 µL of test substance formulation, solvent (water) or positive control (dimethylformamide) were applied onto the epithelium of each three corneas for 10 min, followed by 2 h of post-exposure incubation. Afterwards, a second opacity reading was performed. In addition, permeability of the corneas was evaluated by measuring the transfer of sodium fluorescein.

All acceptability criteria of the test were met, demonstrating the sensitivity and validity of the test system.

Relative to the negative control, the calculated mean in vitro irritancy score (IVIS) of sorbitan tridocosanoate was -0.7. Based on the experimental findings and under the conditions of the test, the test item is not considered irritant to the eye.

 

Justification for classification or non-classification

The available data on skin irritation/corrosion and eye irritation/corrosion do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.