Reaction mass of dodecyl (S)-2-(((S)-2-hydroxypropanoyl)oxy)propanoate and dodecyl (S)-2-hydroxypropanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2018 to 12 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dodecyl (S)-2-(((S)-2-hydroxypropanoyl)oxy)propanoate and dodecyl (S)-2-hydroxypropanoate

Molecular formula:

C12H26O - C21H38O7

IUPAC Name:

Reaction mass of dodecyl (S)-2-(((S)-2-hydroxypropanoyl)oxy)propanoate and dodecyl (S)-2-hydroxypropanoate

Test material form:

liquid

Details on test material:

- Appearance / physical state: Clear, colourless, liquid
- Storage conditions: Room temperature

In vitro test system

Test system:

human skin model

Source species:

other: human

Cell type:

non-transformed keratinocytes

Cell source:

other: accredited institutions after consent obtained from donor or donor's next of kin

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

INTRODUCTION
- The objective of this study was to evaluate the test item for its ability to induce skin corrosion.
- For this purpose, test material was topically applied on a human three dimensional epidermal model.

TEST ITEM CHARACTERISATION
- The study sponsor provided information on identity, purity, composition and stability of the test item.

RESERVE SAMPLES
- For each batch (lot) of test item, a reserve sample was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

TEST ITEM INVENTORY AND DISPOSITION
- Records of the receipt, distribution, and storage of test item(s) were maintained.
- With the exception of reserve samples, all unused Sponsor-supplied test item were discarded or returned to the Sponsor after completion of the scheduled program of work.
- Records of the decisions made were kept at the Test Facility.

TEST SYSTEM
- The test is based on the experience that corrosive chemicals show cytotoxic effects following short-term exposure to the stratum corneum of the epidermis. The test is designed to predict and classify the skin corrosion potential of a test item by assessment of its effect on a three dimensional human epidermis model.
- The test consists of topical application of test material on the skin tissue for 3-minutes and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.
- EpiDerm Skin Model (EPI-200, Lot no. 27667, kit H and G, see Appendix 4, attached).
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

TEST FOR INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly
reduce MTT.
- The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

TEST FOR COLOUR INTERFERENCE BY THE TEST ITEM
- The test material was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure.
- To assess the colour interference, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and checked to establish whether a blue / purple colour change was observed.

TEST FOR REDUCTION OF MTT BY THE TEST ITEM
- The test material was checked for possible direct MTT reduction before the study was started.
- To assess the ability of the test item to reduce MTT, 50 μL of the test item or 50 μL Milli-Q
water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. At the end of the exposure time the mixture was checked to establish whether a blue / purple colour change or a blue / purple precipitate was observed.

TEST SYSTEM SET UP
- A diagram of the application procedure is shown in Figure 1 (attached).
- On the day of receipt, the tissues were kept on agarose and stored in the refrigerator.
- On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.
- Supplemented DMEM (Dulbecco’s Modified Eagle’s Medium) serum free was supplied by MatTek Corporation.
- MTT concentrate (5 mg/mL; MatTek Corporation) was diluted (1:5) with MTT diluent (supplemented DMEM; MatTek Corporation).
- All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 % (actual range 47 to 84 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 37.3 °C).
- Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

TEST ITEM PREPARATION
- No correction was made for the purity/composition of the test item.
- The liquid test item was applied undiluted (50 μL) directly on top of the tissue.

APPLICATION / TREATMENT OF THE TEST ITEM
- The skin tissues were kept in the refrigerator from the day they were received.
- The next day, at least 1 hour before the assay was started, the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see figure 1, attached).
- The plates were incubated for approximately 2.5 hours at 37.0 ± 1.0 °C.
- The medium was replaced with fresh DMEM just before the test item was applied.
- The test was performed on a total of 4 tissues per test item together with a negative control and positive control.
- Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.
- Undiluted test item (50 µL) was added into the 6-well plates on top of the skin tissues.
- For the negative and positive controls, 2 tissues were treated with 50 μL Milli-Q water
(negative control) and 2 tissues were treated with 50 μL 8N KOH (positive control) for both
the 3-minute and 1-hour time point.
- After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
- The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5% CO2.
- After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with a TECAN Infinite M200 Pro Plate Reader.

ACCEPTANCE CRITERIA
- The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be < 15 %.
c) In the range 20 to 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
- All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with
the test item is decreased below 15 %.
- A test item is considered non-corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

CALCULATION OF CELL VIABILITY
- Optical Density readings were transferred into Microsoft Excel to allow further calculations
to be performed.
- The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw) using the equation ODc = ODraw – ODbl
- The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
- The %Viability for each sample and positive control is calculated using the equation %Viability = (ODc/mean ODlt_u+MTT) * 100.

COMPUTERISED SYSTEMS
- Critical computerized systems used in the study are listed below:
a) REES Centron (version SQL 2.0) for temperature and humidity (laboratory facilities data collection).
b) Magellan Tracker (version 7.0) for optical density measurement.
- All computerised systems used in the conduct of the study have been validated. When a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Two

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minute exposure

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS
- The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed, it was concluded that the test item did not interfere with the MTT endpoint.
- The mean absorption at 570 nm measured after treatment with test material and controls are presented in Table 1 (attached). The individual OD570 measurements are presented in Appendix 2 (attached).
- Mean tissue viability obtained after 3-minute and 1-hour treatments with test material compared to the negative control tissues is shown in Table 2 (attached).
- Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained both after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95 %. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1-hour treatment, the substance is considered to be not corrosive.
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range (See Appendix 3, attached).
- The mean relative tissue viability following the 1-hour exposure to the positive control was 4.7%. In the range of 20 to 100% viability the Coefficient of Variation between tissue replicates was ≤ 14%, indicating that the test system functioned properly (see Table 3, attached).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was determined to be not corrosive in the in vitro skin corrosion test under the experimental conditions described.

Executive summary:

GUIDELINE

The test item was evaluated for its ability to induce skin corrosion on a human three-dimensional epidermal model (EpiDerm (EPI-200)) in accordance with OECD Guideline 431 “In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method” (adopted 29 July 2016) and EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test" Official Journal of the European Union No. L142, 31 May 2008.

 

METHODS

The supplied batch of test material was a clear colourless liquid. The test item was applied undiluted (50 μL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 4.7% after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20 to 100 % viability the Coefficient of Variation between tissue replicates was ≤ 14 %, indicating that the test system functioned properly. Skin corrosion was expressed as the remaining cell viability after exposure to the test item.

 

RESULTS

The relative mean tissue viability obtained both after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95%. Because the mean relative tissue

viability for the test material was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, the substance was considered to be non-corrosive.

 

CONCLUSION

The test item was determined to be not corrosive in the in vitro skin corrosion test under the experimental conditions described.