Butyl 3-mercaptopropionate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2022 - 06 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 B.40 BIS

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

14 June 2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl 3-mercaptopropionate

EC Number:

240-343-5

EC Name:

Butyl 3-mercaptopropionate

Cas Number:

16215-21-7

Molecular formula:

C7H14O2S

IUPAC Name:

butyl 3-sulfanylpropanoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm model is one of the five RhE test methods recommended by the OECD 439 guideline. The test has been validated for in vitro irritancy testing in an international validating process, and it is considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM Reconstructed Human Epidermis
- Tissue batch number(s): 36139
- Keratinocyte strain: 00267
- Date of initiation of testing: 03 May 2022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the completion of the 60 minutes of exposure, test item was washed out from the tissues by rinsing with a constant stream of sterile DPBS. Care was taken to remove any residual of the test item from the tissues’ surface.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours ±5 minutes
- Wavelength: 570 nm without using a reference filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was evaluated for its potential to interfere with MTT assay. 25 mg of the test item was added with 1 mL of the MTT medium and incubated in an incubator (37±1°C, 5±1% CO2, 90±10%RH) for 60 minutes. Untreated MTT medium was used as a control. During the incubation period, the solution turned into purple, therefore the test item was considered to be MTT reducer.
Negative control (DPBS) on killed tissues: 2
Test item on killed tissues (NSMTT control): 2

Test for potential to cause colour interference
25 mg of the test item was added with 0.3 mL of deionized water in a transparent test-tube. The mixture was incubated in an incubator (37±1°C, 5±1% CO2, 90±10%RH) for 60 minutes. At the end of the incubation period, the mixture was shaken, and presence and intensity of the staining (if any) was evaluated. No color change was noted, therefore, the test substance was presumed to have no potential to stain the tissue.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritatingto skin if the mean tissue viability after 1 hour exposure is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

24± 2 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

84

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Viability results

Tissue treated	Optical density		Viability%
	Tissue number	OD mean (corrected)	Individual	Mean
Negative control (DPBS)	1	1.782	101.6	100
	2	1.812	103.3
	3	1.668	95.1
	Historical control range	1.338-1.778	---	---
Positive control (5% SDS)	1	0.070	4.0	3.5
	2	0.067	3.8
	3	0.046	2.6
	Historical control range	0.006-0.160	---	---
Test item on killed tissues (NSMTT control)	1	0.108	6.1	7.1
	2	0.141	8.0
Negative control (DPBS) on killed tissues	1	0.035	2.0	2.2
	2	0.044	2.5
Test item on living tissues	1	1.514	86.3	88.9
	2	1.654	94.3
	3	1.510	86.1
Test item Non Specific MTT corrected	1	1.429	81.5	84.0
	2	1.569	89.4
	3	1.425	81.2

 

 

VALIDITY OF THE TEST

Assay acceptance criterion 1: Negative Control (NC)

The absolute optical density of the NC tissues (treated with sterile DPBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.

The mean OD measured in case of the NC was 1.754, therefore the assay meets the criterion that the mean OD570 of the NC tissues is ≥ 1.0 and ≤ 2.8.

 

Assay acceptance criterion 2: Positive Control (PC)

A 5% SDS (in H2O) solution is used as PC and tested concurrently with the test chemicals.

Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of PC should be within 95 ± 1% confidence interval of the historical data.

The mean viability of the PC tissues was measured as 3.5% of the NC tissue’s viability, therefore the assay meets the acceptance criterion that the mean viability of PC tissues expressed as % of the NC tissues is < 20%.

 

Assay acceptance criterion 3: Standard Deviation (SD)

Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.

The assay meets the acceptance criterion, as the mean SD calculated from individual % tissue viabilities of the three identically treated replicates is ≤ 18% in case of all compounds examined during the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with the test item, the mean cell viability was 84.0% after 60-minute exposure, compared to the concurrent negative control. This is above the threshold of 50%, therefore the test item was considered as being Non-irritant according to the UN GHS/EU CLP Classification.

Executive summary:

An in vitro skin irritation test with the test item BuMP was performed in a reconstructed human epidermis model. EpiDerm™ is designed to predict and classify the irritancy 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 irritability of the test item was evaluated according to the OECD No. 439 guideline.
Three tissues per exposure time were treated for 60 minutes. Exposure of test material was terminated by rinsing with DPBS solution. The viability of each tissue was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using an extractant (isopropanol) and then quantified spectrophotometrically at 570±10 nm.
DPBS and aqueous SDS solution treated tissues were used as negative and positive controls, respectively (three tissues/compound). During this present study, the test item was shown to act directly on MTT (MTT reducer), therefore additional controls (non-specific MTT, or NSMTT controls) were used to detect and correct for the test item interference with the viability measurement. For this purpose, the test item was applied on two frozen-killed tissues, and further two frozen-killed tissues were treated with the negative control. For each treated and control tissue, cell viability was expressed as a % relative to the concurrent negative control.
Following exposure with the test item, the mean cell viability was 84.0% after 60-minute exposure, compared to the concurrent negative control. This is above the threshold of 50%, therefore the test item was considered as being Non-irritant according to the UN GHS/EU CLP Classification. The experiment met all the validity criteria; therefore, the study was considered to be valid.
In conclusion, based on the result of this present study, the test item BuMP is not irritant to skin.