Reaction mass of Fatty acids, tallow, Guerbet reaction Products, Ca salts and Fatty acids, tallow, Guerbet reaction Products, Na salts and Fatty acids, tallow, Guerbet reaction Products

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

73138-58-6, 73138-59-7, 73138-60-0

IUPAC Name:

73138-58-6, 73138-59-7, 73138-60-0

Details on test material:

- Name of test material (as cited in study report): LICOWAX R 21 S FL
- Physical state: Slightly yellow flakes
- Analytical purity: 99.6 % (w/w)

Fatty acids, tallow,
Guerbet reaction Products 73138-58-6 21% (w/w)
Fatty acids, tallow,
Guerbet reaction Products, Ca salts 73138-59-7 48% (w/w)
Fatty acids, tallow,
Guerbet reaction Products, Na salts 73138-60-0 31% (w/w)

- Lot/batch No.: DEF2084336
- Expiration date of the lot/batch: 2018-11-13
- Storage condition of test material: Room temperature, protected from light in the tightly closed original container
- Solubility : ≤ 0.01 g/L (20 °C)

In vitro test system

Test system:

human skin model

Source species:

human

Details on animal used as source of test system:

n.a.; in vitro skin model

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

PRE-TEST
The test item was tested for its ability to direct formazan reduction. This pre-test was done before starting the main study.
- 6 well of 12 well plate was filled with 2 mL of MTT solution (0.3 mg/L).
- To above 6 wells, 10 mg of the test item was added to 3 wells and 10 µL of buffer to other 3 wells and mixed well.
- Incubated the mixture for 3 hours at 37°C protected from light.
Since, no change in the color (blue or purple) of MTT solution was observed, the test item was identified to have no interaction with MTT.

MAIN STUDY
The assay medium supplied with the kits was kept at 2-8°C (refrigerator).
a) Maintenance medium was prewarmed to 37°C before using.
b) EPISKIN Standard Model™ kit was kept at room temperature in the microbiological safety cabinet until next step.

PREPARATION AND PRE INCUBATION (DAY 0)
This step was conducted in a microbiological safety cabinet
a) 3 wells of a 12 well assay plates were filled with pre-warmed maintenance medium (2 mL per well).
b) EPISKIN Standard Model™ kit was opened and epidermis units were transferred into maintenance medium filled wells, using sterile forceps.
Labelled plate lids by replicate treatment order as follows
Negative control Positive control Test item
Rep 1 (NCR1) Rep 1 (PCR1) Rep 1 (5740R1)
Rep 2 (NCR2) Rep 2 (PCR2) Rep 2 (5740R2)
Rep 3 (NCR3) Rep 3 (PCR3) Rep 3 (5740R3)
Key: Rep/R: Replicate; NC: Negative control; PC: Positive control; 5740= study number for test item Licowax R 21 S FL
c) Incubated at 37 °C, 5% CO2, in a > 95% humidified atmosphere for 24 hours.

APPLICATION OF TEST ITEM AND RINSING (DAY 1)
a) Pre-warmed maintenance medium (2 mL per well) was filled taking into account of the 3 wells per replicate. Plate lids were labeled with test-item (3 wells per test item), or negative control (3 wells) or positive control (3 wells).
Topical applications: 15 minutes treatment
b) 10 μL of negative control and positive control, 10 mg of the test item were applied on the top of the epidermis.
c) Followed the defined application order (Rep 1, Rep 2 and Rep 3).
d) After application, plate containing the treated epidermis was closed with lid and placed in the same cabinet for 15 minutes (± 0.5 minute) at room temperature.
For positive control, re-spread after 7 minutes contact time. And each tissue applications were done at 1 minute interval.
End of the treatment and removal of the test chemical; After 15 minutes exposure:
e) Treated epidermal units were removed using forceps, and rinsed thoroughly with 25 mL sterile DPBS by filling and emptying the tissue inserts to remove residual material from the epidermal surface.
f) Epidermal units were placed on an absorbent paper and remaining DPBS were removed from the epidermal surface by gently taping, and swept the epidermal surface with a cotton-bud without damaging the epidermis.
g) Blotted tissue units were transferred to the new maintenance medium pre-filled wells.
Post treatment incubation:42 hours
Incubated the treated and rinsed epidermis at 37°C, 5% CO2, 95% humidified atmosphere for 42 hours.

MTT TEST AFTER THE 42 hours INCUBATION PERIOD
Required number of wells of the assay plate were filled with 2 mL per well of 0.3 mg/mL MTT in assay medium.
Treated units were transferred to the MTT filled wells. Plate was covered with lid and incubated for 3 hours at 37°C, 5% CO2, 95% humidified atmosphere.

FORMAZAN EXTRACTION (DAY 3)
a) 1.5 mL of conic “safelock” type micro tubes were labelled appropriately for test item, vehicle control, positive control and their replicate Number (1 tube/epidermis).
Formazan extraction :
b) Tissue units were placed on absorbent paper to dry the tissues.
c) Using the special biopsy punch, a total biopsy of the epidermis was done.
d) Gently separated the epidermis from the collagen matrix with the aid of forceps, and both parts (epidermis and collagen matrix) were placed into the labelled micro tubes.
e) When all the tissues were punched and ready in the micro tubes, 500 μL of acidic isopropanol per tube was added.
f) Each tube was plugged to avoid evaporation and mixed thoroughly using a vortex mixer.
g) All the tissues were correctly immersed in the solvent and extraction start time for each tissue was noted.
h) The tubes were stored for 4 hours at room temperature (recommended 18 to 23°C) and protected from light.
i) Vortex each tube at the middle of the incubation period.
j) Tissues were removed from each tube extraction end time was noted.
k) Extraction solution was homogenized by pipetting 3 times up and down.
l) From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in using spectrophotometer at 560 nm. Acidified isopropanol solution was used as blank.

Control samples:

other: 5% SDS, DPBS

Amount/concentration applied:

10 mg

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

93.78

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The negative controls mean OD was found to be 0.726, which is well within the acceptability range. The % viability of tissue treated with the positive control was 24.13% and it was less than 50% of the negative control, which reflects the irritant nature of positive control and the sensitivity of the tissues used in the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The % viability of tissue treated with the test item was 93.78% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence under the conditions of the study the test item was found to be Non Irritant to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemicals.

Executive summary:

Test item, Licowax R 21 S FL was applied topically to the EPISKIN^TMepidermal model (three epidermis units were used per test item, positive and negative controls) for 15 minutes. Exposure to the test item was terminated by rinsing with Dulbecco’s phosphate buffered saline (DPBS). Epidermis was then incubated at 37°C for 42 additional hours. The viability was assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/mL; 2 mL per well). The precipitated formazan was then extracted using acidified isopropanol (0.5 mL) for 4 hours at room temperature, and quantified spectrophotometrically at 560 nm using 96 well plates (200 μL/well).

 

SDS 5% and DPBS treated epidermis were used as positive (PC) and negative controls (NC) respectively. For each treated tissue the viability was expressed as the % of the negative control tissues (mean).

 

The negative controls mean OD was found to be 0.726, which is well within the acceptability range. The % viability of tissue treated with the positive control was 24.13% and it was less than 50% of the negative control, which reflects the irritant nature of positive control and the sensitivity of the tissues used in the study.

 

The % viability of tissue treated with the test item was 93.78% and it was greater than 50% of the negative control. The variation within the replicates was found to be less than 18%. Hence under the conditions of the study the test item was found to beNonIrritantto skin in accordance with UN GHSas specified in the OECD Guideline for the Testing of Chemicals.