Fatty acids, vegetable-oil, Me esters, sulfurized

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27. Nov. 2017 to 01. Dec. 2017

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 Guidelines for the Testing of Chemicals Part 431, adopted 29. Jul. 2016
“In vitro Skin Corrosion: reconstructed human epidermis (RHE) test method”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200-SIT
- Tissue batch number(s): 25861
- Delivery date: 28. Nov. 2017
- Date of initiation of testing: 29. Nov. 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos Reader 2010 Flexi
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
After the treatment with the test item, the mean value of relative tissue viability was in-creased to 102.9 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tis-sue surface.

One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

One plate was used for treatment with the test item:
30 µL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours 45 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

>= 100.4 - <= 107.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue forma-zan. Formazan production was evaluated by measuring the optical density (OD) of the re-sulting solution. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus show-ing the quality of the tissues. The OD was 1.7 (3 minutes experiment) and 1.6 (1 hour ex-periment). The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 6.1 % for the 1 hour treatment. After 3 minutes treatment with the test item, the mean value of relative tissue viability was increased to 102.6 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, the mean value of relative tissue viability was increased to 105.0 %. This value, too, is above the threshold for corrosion potential (15%).

Therefore, Fatty Acids, vegetable-oil, Me-Esters, sulfurized is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDerm^TM were treated with Fatty Acids, vegetable-oil, Me-Esters, sulfurized for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.7. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.9 % (required: ≤ 20%).

The variation within the tissue replicates of negative, control, positive control and test item was acceptable (required: ≤ 18%).

 After the treatment with the test item, the mean value of relative tissue viability was increased to 102.9 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

 

Therefore, Fatty Acids, vegetable-oil, Me-Esters, sulfurized is considered

non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27. Nov 2017 - 01. Dec 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 (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Justification for test system used:

This in vitro study was performed in order to evaluate the potential of Fatty Acids, vegetable-oil, Me-Esters, sulfurized to evoke skin irritation in a reconstructed human Epidermis (RhE) test method.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM-Kit, procured by MatTek
- Tissue batch number(s): 25861
- Delivery date: 28.11.2017
- Date of initiation of testing: 27.11.2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: 37 ± 1°C
- Temperature of post-treatment incubation (if applicable): 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: unkown
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos Reader 2010 Flexi
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive/irritant to skin if the viability after exposure is equal or less than 50% of negative control.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than 50% of negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL
- Concentration:
KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 L

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5%

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours 45 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

102.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
OD of negative control ≥ 0.8 and ≤ 2.8; found 1.7

- Acceptance criteria met for positive control:
% tissue viability of positive control SDS ≤ 20% of negative control; found 2.9%

- Acceptance criteria met for variability between replicate measurements:
SD of mean viability of the tissue replicates (%) ≤ 18
8.4% (negative control)
0.3% (positive control)
3.6% (test item)

Interpretation of results:

GHS criteria not met

Conclusions:

After the treatment with the test item, the mean value of relative tissue viability was increased to 102.9 %. This value is above the threshold for skin irritation potential (50%).
Test items that induce values above the threshold of 50% are considered non-irritant to skin.
Therefore, Fatty Acids, vegetable-oil, Me-Esters, sulfurized is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

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:

24 Oct. 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

other: Bos primigenius Taurus

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750µL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES
For each treatment group (negative control solution, test item and positive control), three replicates were used.
NEGATIVE CONTROL USED
HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20171024

POSITIVE CONTROL USED
Dimethylformamide, DMF, CAS-No. 68-12-2, undiluted, batch no.: 475235719

APPLICATION DOSE AND EXPOSURE TIME
750 μL negative control solution, test item and positive control were applied to each replicate.

TREATMENT METHOD:
closed chamber

POST-INCUBATION PERIOD:
yes; 2 hours

REMOVAL OF TEST SUBSTANCE
After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red. After post-incubation time, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

- Corneal permeability:
Passage of sodium fluorescein dye measured with the aid of microtiter plate photometer (OD492)

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Irritation parameter:

in vitro irritation score

Run / experiment:

For each treatment group (negative control solution, test item and positive control), three replicates were used. Duration of each exposure: 10 minutes

Value:

0.79

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test item Fatty Acids, vegetable-oil, Me-Esters,
sulfurized showed no effects on the cornea of the bovine eye. The calculated IVIS is 0.79.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires
no classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No adverse effects observed in any relevant study.