Butyl 3-mercaptopropionate

Endpoint summary

Description of key information

not irritating to skin (OECD TG 439)

not irritating to eye (OECD TG 438)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

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 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

Irritation / corrosion parameter:

% tissue viability

Value:

84

Negative controls validity:

valid

Positive controls validity:

valid

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

dated 14 February 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
Only the eyes of healthy animals considered suitable for entry into the human food chain were used Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (20.6 ºC to 21.2 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
Cornea integrity was checked by applying one small drop of fluorescein 2 % (w/v) solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03 g or 30 μL per cornea

Duration of treatment / exposure:

exposure period of 10 seconds

Duration of post- treatment incubation (in vitro):

The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.

Number of animals or in vitro replicates:

3 (positive control + test item), 1 (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

EQUILIBRATION AND BASELINE RECORDINGS
- acclimatisation for approximately 45 to 60 minutes at 32 ± 1.5 °C
- At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.


REMOVAL OF TEST SUBSTANCE
- rinsed thoroughly with 20 mL saline solution at ambient temperature

SCORING SYSTEM:
- Mean corneal swelling (%)
For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12 %) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.
- Mean maximum opacity score
Score Observation
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent area; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment
Score Observation
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: decision criteria as indicated in the TG was used

Irritation parameter:

percent corneal swelling

Remarks:

up to 75 min

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

cornea opacity score

Value:

0.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

fluorescein retention score

Value:

0.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Other effects / acceptance of results:

Overall ICE Class: 2x I, 1x II

Positive Control: Benzalkonium chloride solution (5%)

Observation	Value	ICE class
Mean maximum corneal swelling at up to 75 min	25%	III
Mean maximum corneal swelling at up to 240 min	42%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4 was observed in three eyes at 75 minutes after the post-treatment rinse. Loosening of the epithelium was noted in two eyes out of three eyes at 30 and 75 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

 

Negative Control: NaCl (9 g/L saline)

Observation	Value	ICE class
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	2%0	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class	3xI

Interpretation of results:

GHS criteria not met

Conclusions:

In this ICET, the overall ICE classes of the test item were twice I and once II. According to the guideline OECD 438, BuMP is categorized as “No Category”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item BuMP by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 μL/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test item exposure) were determined at each of the above time points.
The test item, the positive control [Benzalkonium chloride solution (5 %)], and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way to evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study.
After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.
In this ICET, the overall ICE classes of the test item were twice I (based on the corneal swelling of 2 % within 240 minutes, based on the corneal opacity score of 0.2) and once II (based on the fluorescein retention of 0.8).
The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. So, the positive and negative controls showed the expected results. The experiment was considered to be valid.
In this ICET, the overall ICE classes of the test item were twice I and once II. According to the guideline OECD 438, the test item is categorized as “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

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.

 

Eye irritation

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item BuMP by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 μL/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test item exposure) were determined at each of the above time points.
The test item, the positive control [Benzalkonium chloride solution (5 %)], and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way to evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study.
After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.
In this ICET, the overall ICE classes of the test item were twice I (based on the corneal swelling of 2 % within 240 minutes, based on the corneal opacity score of 0.2) and once II (based on the fluorescein retention of 0.8).
The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. So, the positive and negative controls showed the expected results. The experiment was considered to be valid.
In this ICET, the overall ICE classes of the test item were twice I and once II. According to the guideline OECD 438, the test item is categorized as “No Category”.

 

 

Justification for classification or non-classification

Based on the available data, BuMP is nor classified for skin or eye irritation.