3-methyl-1,3-butanediol-1-ethylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-08 to 2016-06-08

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,3-butanediol-1-ethylate

EC Number:

816-856-0

Cas Number:

5205-01-6

Molecular formula:

C7H14O3

IUPAC Name:

3-methyl-1,3-butanediol-1-ethylate

Specific details on test material used for the study:

Batch number: 161121
Purity: 86.5%
Expiry date: May 2019
Appearance: Clear colourless liquid
Storage conditions: Room temperature in the dark under nitrogen

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not stated

Source strain:

other: Not applicable

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: MatTek in vitro life science laboratories, Slovakia
- Tissue batch number: 28646
- Assay medium lot number: 081618TMB

ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls. As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of the test item were mixed per 300 µL Aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated for 1 hour at 37°C, 5.0% CO2 / 95% air. If the test item was classified as non-corrosive and colouring was detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbed light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues per exposure period treated with 50 µL of the test item (TVT). All steps were then performed exactly as described in the chapter below, except for the MTT-staining of the test item treated tissues, which were incubated in medium without MTT.

EXPERIIMENTAL PROCEDURE
Upon receipt of the EpiDerm, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37°C, 5.0% CO2 / 95% air for at least 1 hour. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 minute experiment was placed back into the incubator. The other plate was used for the 60 minute treatment. Approximately 1 hour before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.

60 minute experiment: tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval was kept between dosing. Then the 6-well plate was incubated at 37°C, 5.0% CO2 / 95% air.

3 minute experiment: tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 seconds was kept between dosing.
After the 3 minute application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed approximately 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well holding plate containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well MTT assay plate containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 hours at 37°C, 5.0% CO2 / 95% air.

60 minute experiment: after the 60 minute application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed approximately 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well holding plate containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
The inserts were dried again and transferred into a prepared 24-well MTT assay plate containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 hours at 37°C, 5.0% CO2 / 95% air.

3 minute and 60 minute experiment: after the 3 hour MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. The inserts were transferred into 12-well extraction plates. Two millilitres of isopropanol were pipetted into each skin tissue insert, thus the insert was covered from both sides to extract the complete formazan. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out over night without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. The inserts were discarded and the extraction plates were placed on a shaker for 15 minutes.
For each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

EVALUATION CRITERIA
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 minutes and 60 minutes treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%) according to the following Prediction Model:

STEP 1:
Mean tissue viability (% negative control) < 50% after 3 min exposure = Corrosive

Mean tissue viability (% negative control) => 50% after 3 min exposure AND < 15% after 60 min exposure = Corrosive. A combination of optional Sub-categories 1B and 1C

Mean tissue viability (% negative control) => 50% after 3 min exposure AND =>15% after 60 min exposure = Non corrosive

STEP 2:
Mean tissue viability (% negative control) < 25% after 3 min exposure = Optional Sub-category 1A

Mean tissue viability (% negative control) => 25% after 3 min exposure = A combination of optional Sub-categories 1B and 1C

ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all the following criteria were met:
- mean absolute OD570 nm of the two negative control tissues of the 3 minute and 60 minute treatment periods was between 0.8 and 2.8,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period was <15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically was =<30%.

QUANTITATIVE MTT ASSESSMENT
If NSCliving was =< 5% relative to the negative control of living epidermis, no correction of the results was necessary.
If NSCliving was > 5% and =< 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected.
If NSCliving was > 30% relative to the negative control of living epidermis, the test item was considered as incompatible

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL (79.4 µL/cm2)

Duration of treatment / exposure:

3 and 60 minutes

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 97.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

ca. 80.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Pre-Experiments

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 50 µL test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equalled 0%.

 

Main Experiment:

Results

 

Table 1: Experimental results

Parameter	Negative Control (distilled water)	Test article (IPD-AC)	Positive Control (KOH)
3 minute exposure
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected) ±SD	1.635 ±0.051	1.598 ±0.053	0.160 ±0.018
Mean Relative Tissue Viability [%]	100	97.7	9.8
Coefficient Of Variation [%]***	3.1	3.3	11.0
3 minute exposure
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected) ±SD	1.679 ±0.069	1.355 ±0.036	0.106 ±0.008
Mean Relative Tissue Viability [%]	100	80.7	6.3
Coefficient Of Variation [%]***	4.1	2.7	7.7

 

A summary of the results for both the 3 and 60 minute in vitro skin corrosion experiments are shown above.

 

Mean absolute OD in the negative control group for both treatment periods were deemed acceptable. The mean cell viability in the positive control group in the 60 minute exposure was <15%. The coefficient of variation between the two tissues treated was <30%.

 

Table 2: Test acceptance criteria

	Value	Cut off	Pass/fail
Mean Absolute OD570 nm NK (3 min Experiment)	1.680	0.8= NK =>2.8	Pass
Mean Absolute OD570 nm NK (60 min Experiment)	1.723	0.8=< NK=>2.8	Pass
Mean Relative Tissue Viability [%] of PC (60 min experiment)	6.3	<15%	Pass
CV [%] (in the range of 20 – 100% viability)	2.7 – 4.1	=<30%	Pass

The acceptance criteria were met and the study was accepted as valid.

The potential of the test item to induce skin corrosion was analysed using the three-dimensional human skin model EpiDerm, comprising a reconstructed epidermis with a functional stratum corneum.

 

In the present study 3-Methyl-1,3-butanediol-1-acetate was applied topically to the EpiDerm tissue for 3 and 60 minutes followed by immediate determination of cytotoxic effects via MTT reduction assay.

 

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 50 µL test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

 

The test item exhibited no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

 

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was =50% (97.7%) after the 3 minute treatment and =15% (80.7%) after the 60 minute treatment.

 

The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was >0.8 and =2.8 for each exposure period (1.680, 1.723). The mean relative tissue viability (% negative control) of the positive control was <15% (6.3%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was <30% (2.7% - 4.1%). The negative and positive control treatments met the acceptance criteria (see Table 4), thereby demonstrating the sensitivity and specificity of this assay.

 

It is concluded that 3-Methyl-1,3-butanediol-1-acetate was not corrosive following exposure to a reconstituted three-dimensional human epidermis model (EpiDerm^TM). These conditions included exposure to 3-Methyl-1,3-butanediol-1-acetate for 3 and 60 minutes, with tissue viabilities of 97.7% and 80.7%, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as not corrosive to skin.

Executive summary:

An in vitro skin corrosion study has been conducted with IPD-AC in accordance with OECD test guideline 431 (2016) and in compliance with GLP. The test substance was applied topically to reconstructed human epidermis model. Negative (distilled water) and positive (KOH) controls were tested concurrently. The skin tissues were tested in duplicated for 3 and 60 minutes with the test substance or control. The materials were then removed and the tissues incubated for 3 hours. Cell viability was determined by the enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells). The optical density was measured at 570 nm. Viability was determined relative to the negative control tissue.

It is concluded that 3-Methyl-1,3-butanediol-1-acetate was not corrosive following exposure to a reconstituted three-dimensional human epidermis model (EpiDerm^TM). These conditions included exposure to 3-Methyl-1,3-butanediol-1-acetate for 3 and 60 minutes, with tissue viabilities of 97.7% and 80.7%, respectively.