Reaction mass of pentyl ester butanoic acid and 2-methylbutyl ester butanoic acid

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 May 2019 - 10 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pentyl butyrate

EC Number:

208-739-2

EC Name:

Pentyl butyrate

Cas Number:

540-18-1

Molecular formula:

C9H18O2

IUPAC Name:

pentyl butyrate

Constituent 2

Reference substance name:

2-methylbutyl butyrate

EC Number:

256-973-9

EC Name:

2-methylbutyl butyrate

Cas Number:

51115-64-1

Molecular formula:

C9H18O2

IUPAC Name:

2-methylbutyl butyrate

Test material form:

liquid

Specific details on test material used for the study:

Name: Reaction mass of 2-methylbutyl butyrate and pentyl butyrate
Description: Clear colourless to pale yellow liquid
Purity: 99.8%
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Source strain:

not specified

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin:
EPISKIN™(SM) (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-019, Expiry Date: 13 May 2019) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity and irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Suggested expiry date: 13 May 2019

Quality Control:
EPISKIN™(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKIN™(SM) Test Kits used in the present study) and are documented in Appendix 2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of test item was added to each skin unit

50 µL of negative control (Physiological saline (0.9% (w/v) NaCl solution)) or positive control (glacial acetic acid) were added to each skin unit

Physiological saline (0.9% (w/v) NaCl solution):
Manufacturer: B. Pharmaceuticals SA
Batch No.: 90352Y05-2
Expiry Date: December 2021
Grade: sterile

Glacial acetic acid:
Supplier: VWR International Ltd.
Batch No.: 17H074111
Expiry date: 05 July 2020

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (23.6-24.7°C)

Duration of post-treatment incubation (if applicable):

After 4 hours incubation time , the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours
(±1 hour) at 37 °C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37 °C in an incubator with 5% CO2 for 3 hours, protected from light, in a > 95% humidified atmosphere.

At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 µL acidified isopropanol (one tube corresponded to one well of the assay plate).

The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight (corrosivity part of the test) or for two hours (irritation part of the test) at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Number of replicates:

2 per timepoint

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

129.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

119.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

124.1

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:

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in the results. The OD values for the test item treated skin samples showed 124.1% relative viability compared to the negative control.

Validity criteria:
After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the blank samples (acidified isopropanol) were 0.046 and 0.047.
The mean OD value of the two negative control tissues was in the recommended range (0.766).
The two positive control treated tissues showed 0.1% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 8.1%
The difference of viability between the two negative control tissue samples in the MTT assay was 2.3%.
High viability results (>>100%) do regularly occur in cases where the test item causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.
Following exposure with Pentyl Butyrate (Sum of Isomers), the mean cell viability was 124.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Any other information on results incl. tables

Table 1. Optical Density (OD) and the calculated relative viability % of the samples (Corrosivity test)

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative Control: Physiological saline (0.9% (w/v) NaCl)	1	0.822	0.775	101.2
	2	0.804	0.757	98.8
	Mean	-	0.766	100
Positive Control: Glacial acetic acid	1	0.048	0.002	0.2
	2	0.046	0	0
	Mean	-	0.001	0.1
Test Item: Pentyl Butyrate (Sum of Isomers)	1	1.036	0.99	129.2
	2	0.959	0.912	119.1
	Mean	-	0.951	124.1

Notes:

1. Mean blank value was 0.046.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Pentyl Butyrate (Sum of Isomers), the mean cell viability was 124.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Executive summary:

An in vitro skin corrosivity and irritation test of Pentyl Butyrate (Sum of Isomers) was performed in a reconstructed human epidermis model. EPISKIN^TM(SM) is designed to predict and classify the corrosivity and 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 (detailed in section 3.6). The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines [1, 2].

 

Disks of EPISKIN^TM(SM) were treated withthe test itemand incubated for 15 minutes (three units for irritation testing) and 4 hours (two units for corrosivity testing) 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% CO_2,in a > 95% humidified atmosphere (irritation testing). 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% CO₂protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline(0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and 5% (w/w) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing.For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, 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 irritant to skin.

 

Following exposure withPentyl Butyrate (Sum of Isomers), the mean cell viability was 124.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.