Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 August to 10 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Dimethyl adipate

EC Number:

211-020-6

EC Name:

Dimethyl adipate

Cas Number:

627-93-0

Molecular formula:

C8H14O4

IUPAC Name:

Dimethyl adipate

Constituent 2

Reference substance name:

Dimethyl glutarate

EC Number:

214-277-2

EC Name:

Dimethyl glutarate

Cas Number:

1119-40-0

Molecular formula:

C7H12O4

IUPAC Name:

dimethyl glutarate

Constituent 3

Reference substance name:

Dimethyl succinate

EC Number:

203-419-9

EC Name:

Dimethyl succinate

Cas Number:

106-65-0

Molecular formula:

C6H10O4

IUPAC Name:

dimethyl succinate

Test material form:

liquid

Remarks:

Colourless to brown liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable (The test item was applied as supplied, no formulation was required)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable (The test item was applied as supplied)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified (Adult donors)

Source strain:

other: No applicable (human)

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Assessment of the effects of the test on on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and
conversion into a blue formazan salt that is quantitatively measured after extraction from tissues.
- Quality control for skin discs: The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (23.2-27.6°C).
- Temperature of post-treatment incubation (if applicable): 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: one with PBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none
DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] MTT (Concentration of the working solution: 0.3 mg/mL)
- Spectrophotometer:Thermo Fisher Scientific
- Wavelength: 570 nm.
- Filter: Not applicable
- Filter bandwidth: Not applicable
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered to be irritant to skin (Category 2) or corrosive (Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
- The test item is considered to be noni-irritant to skin (Not classified in GHS), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is above (>) to 50% of the negative control.

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

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

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h) at 37°C

Number of replicates:

Three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

70.8

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: none After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- Direct-MTT reduction: After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT.
- Colour interference with MTT: As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Negative and positive controls as well as variability between replicate measurements met the acceptability criteria, therefore the study was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18. The mean OD value of the three negative control tissues was in the recommended range (0.751). Standard deviation of the viability results for negative control samples
was 3.8%.
- Acceptance criteria met for positive control: The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18. The positive control treated tissues showed 8.0% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.3%.
- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.6%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with INNROAD PROTECT, the mean cell viability was 70.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKINTM (SM) model test with INNROAD PROTECT (Batch number: CY71353002), the results indicate that the registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is non-irritant to skin.

Executive summary:

An in vitro skin irritation test of INNROAD PROTECT test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

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). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) t o 5 0% o f t he negative control, the test item is considered to be irritant to skin.

Following exposure with INNROAD PROTECT, the mean cell viability was 70.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with INNROAD PROTECT (Batch number: CY71353002), the results indicate that

the registered substance Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate is non-irritant to skin.