2,2-bis(methylthio)propane

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

excretion

Principles of method if other than guideline:

The objective of this study was to characterise the routes and rates of excretion of BMTP and/or its radiolabelled metabolites in urine, faeces and expired air.

GLP compliance:

no

Remarks:

This is a non-regulatory study for which a claim of GLP compliance has not be made. However, the laboratory procedures were fully commensurate with International Standards of GLP.

Species:

rat

Strain:

other: Han Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: no data
- Weight at study initiation: 208 and 238 g
- Housing:3 per cage prior to dosing
- Diet (ad libitum): SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services)
- Water (ad libitum): mains water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 42 and 65
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was supplied pre-formulated, ready to administer to the animals.

Duration and frequency of treatment / exposure:

Single administration

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

Nominal radioactive dose: 5 MBq/kg

No. of animals per sex per dose / concentration:

3

Control animals:

no

Details on study design:

- Dose selection rationale: from subacute repeated dose toxicological studies by oral route, a NOAEL was established at 160 mg/kg bw/day.

Details on dosing and sampling:

- Excretion Balance Investigation
Following dose administration, rats were placed in all-glass metabolism cages suitable for the separate collection of urine and faeces.
Urine and faeces were collected over the following time intervals after dose administration:
• 0-6, 6-24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours.
Urine collection vessels were rinsed with a small volume of water (2 to 5 mL) which was added to the urine sample.
At each collection, cage debris was removed and the cages were rinsed with a small volume of water. The aqueous cage washings were retained separately for each animal and time point. Cage debris was pooled for each animal over the entire collection period. Following the final water wash, methanol was used to rinse the cages.
In order that any radioactivity excreted in expired air may be collected, air (dried over drierite or calcium chloride and carbon dioxide removed with soda lime) was drawn through the cage by vacuum. Air exiting the cage was passed through two CO2 traps in series, each containing a solution of 2-ethoxyethanol : ethanolamine (3:1 v/v), duplicate carbon traps and a cold trap, to collect any potential volatile materials. The cold trap contained ethanol, cooled over wet-ice, which was replaced at the end of each working day.
Expired air trapping solutions and carbon traps were collected at the following time intervals after dose administration:
• 0-6, 6-24, 24-48 and 48-72 hours.
Radioactivity levels in expired air traps were so low (<0.5% of the administered radioactivity in the 48-72 hours collection period) that traps were discontinued after this time.
Following the final excreta collection, a sample of blood was taken by cardiac puncture (whilst under terminal anaesthesia), after which the animals were killed by cervical dislocation and exsanguination following isoflurane anaesthesia. Carcasses were retained for analysis.
Radioactivity was determined in urine, faeces, cage debris, cage washings (aqueous and organic), expired air traps, whole blood and carcasses, using the methods presented in the Sample Preparation section.

- Sample Preparation
Volumes and/or weights of biological samples were measured, where appropriate.
. Urine, cage washings, liquid expired air trap contents
Weighed aliquots were added to liquid scintillant for analysis by LSC.
. Faeces and cage debris
Faeces and cage debris were homogenised in deionised water. Aliquots of the homogenates (ca 0.1g, weighed) were taken and solubilised using Soluene 350 (Perkin Elmer LAS (UK) Ltd), with subsequent addition of liquid scintillant and analysis by LSC.
. Blood
Aliquots (ca 0.1g, weighed) were solubilised using Solvable (Perkin Elmer LAS (UK) Ltd), with subsequent addition of liquid scintillant and analysis by LSC.
. Carbon traps
Carbon trap contents were combusted in an oxygen atmosphere and the resultant gasses trapped using Carbosorb® and Permafluor®, followed by analysis by LSC.
. Carcasses
Carcasses were digested in a solution of potassium hydroxide in methanol (approximately 40%, w/v). Liquid scintillant was added to the samples prior to analysis by LSC.

- Liquid Scintillation Counting
A suitable scintillation counter was used. Radioassays were performed on duplicate weighed aliquots (all samples).
Efficiency correlation curves were prepared and routinely checked by the use of sealed [14C]-toluene and Ultima Gold™ quenched standards (Perkin Elmer LAS (UK) Ltd).
The limit of quantification for each batch of samples analysed was taken as twice the mean background disintegration rate obtained from vials containing an equivalent volume of an appropriate solvent in liquid scintillant

Type:

other: Overall recovery

Results:

97.0%

Type:

other: Excretion in urine

Results:

73.3%

Type:

other: Cage washes

Results:

15.8%

Type:

other: Ecretion in faeces

Results:

1.3%

Type:

other: Excretion in expired air

Results:

< 5%

Animal Observations

During the course of the study, no overt pharmacological or toxicological signs that could have been attributed to the administration ofBMTPwere observed in the test animals.

Excretion Balance

All values quoted in this section are means for the two animals used for this study.

Overall recovery of radioactivity was 97.0% over the 168 hour collection period, with excretion principally in the urine (73.3%) and cage washes (15.8%, probably mostly derived for urine). Only low levels of radioactivity were present in faeces (1.3%) and cage debris (0.1%). Radioactivity was collected in the expired air traps (principally in the ethoxyethanol : ethanolamine traps) but this accounted for less than 5% of the administered dose.

Radioactivity remained detectable in both blood (0.984 µg equiv/g) and the carcass (2.68 µg equiv/g; 1.7% of the administered dose) at 168 hours post-dose.

Conclusions:

Nearly 100% of the dose was absorbed, the principal route of excretion was in the urine, with only low levels recovered in either faeces or expired air.

Executive summary:

The excretion of radioactivity has been investigated following oral administration of [14C]-BMTP to male rats. Two male rats of the Han Wistar strain received a single oral administration of [14C]-BMTP, by gavage at a nominal dose level of 160 mg/kg. Urine and faeces collected at sampling periods up to 168 hours. Expired air was drawn through a series of traps, designed to collect volatile components, at times up to 72 hours. Following each urine/faecal collection, cages were washed with a small volume of water and any cage debris removed. Blood was collected at 168 hours post-dose, prior to the animals being humanely killed and carcasses retained. Radioactivity in blood, excreta and carcasses was determined by liquid scintillation counting. The mean recovery of radioactivity was 97.0% over the collection period. The majority of the radioactivity recovered was in the urine (73.3%) and cage washes (15.8%), with limited levels in faeces (1.3%) and cage debris (0.1%). Expired air traps contained radioactivity but this accounted for less than 5% of the administered dose. Low levels of radioactivity were retained in both blood (0.984 µg equiv/g) and the carcass (2.68 µg equiv/g; 1.7% of the dose). In conclusion, nearly 100% of the dose was absorbed, the principal route of excretion was in the urine, with only low levels recovered in either faeces or expired air.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Objective of study:

absorption

toxicokinetics

Principles of method if other than guideline:

The objectives of this study were to measure the absorption and pharmacokinetics of BMTP following single and repeat oral administration to the rat.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Han Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited
- Age at study initiation: no data
- Weight at study initiation: 245-278g males, 230-260 females
- Housing: up to 5 per cage according to sex
- Diet ad libitum): SQC Rat and Mouse Maintenance Diet No 1, Expanded
- Water (ad libitum): mains water
- Acclimation period: 6 to 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 30 and 65
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The dose formulations were prepared by Quotient Bioresearch (Radiochemicals) Ltd.
The homogeneity of the formulations was assessed by subjecting at least three aliquots (suitably diluted) to liquid scintillation counting both pre- and post-dose for single dose phases and on day 1 and day 14 for the repeat dose phase.
The supplier has supplied information indicating that the radiochemical purity of the formulated test material declined from 93.7% to 87.3% over a 28 day period.

Duration and frequency of treatment / exposure:

single administration or 14 daily doses

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

single administration and 14 daily doses

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

single administration

No. of animals per sex per dose / concentration:

4 males + 4 females (single doses)
8 males (repeated dose)

Control animals:

no

Details on study design:

Dosing Details
Animals received either a single administration or 14 daily doses of [14C]-BMTP (radiodiluted to the appropriate specific activity with non-labelled BMTP). All doses were by the oral route, administered by gavage at a nominal dose volume of 5 mL/kg.

Body Weights
Body weights were recorded the day after arrival and before dose administration. For repeat dose animals, body weights were recorded prior to dosing on day 1, day 7 and day 14.

Details on dosing and sampling:

Following dosing, animals were returned to holding cages. Samples of blood (nominally 150 µL) were withdrawn from each animal, via a peripheral vein, at each of the following times after dose administration:
Single dose groups Pre-dose, 30 minutes, 1, 2, 4, 6, 8, 12, 24 and 48 hours.
Repeat dose groups Animals were divided between two sub-groups of 4. Blood was sampled from each sub-group alternately:
Tmax from single dose phase after doses 1-13 (4 hours post-dose, as determined from group A data);
30 minutes, 1, 2, 4, 6, 8, 12, 24 and 48 hours after dose 14.
Radioactivity concentrations were determined in blood samples by solubilisation followed by liquid scintillation counting.

Toxicokinetic parameters:

Cmax: 42.1 µg equiv/g

Remarks:

males, 100 mg/kg single

Toxicokinetic parameters:

Cmax: 47.9 µg equiv/g

Remarks:

females, 100 mg/kg single

Toxicokinetic parameters:

Cmax: 135 µg equiv/g

Remarks:

males, 300 mg/kg single

Toxicokinetic parameters:

Cmax: 136 µg equiv/g

Remarks:

females, 300 mg/kg single

Toxicokinetic parameters:

Cmax: 73.4 µg equiv/g

Remarks:

males, 100 mg/kg/d 14-day

Metabolites identified:

no

Group A (Single Dose, 100 mg/kg)

Following oral administration of [14C]-BMTP to male and female rats, peak concentrations occurred between 2 and 6 hours for males (mean = 4 hours) and between 6 and 12 hours for female animals (mean = 9.5 hours). Some differences were also evident in both the maximal concentration measured (42.1 and 47.9 µg equiv/g in males and females, respectively) and AUC(0-t) (527 and 858 h*µg equiv/g, males and females, respectively) but these differences were not considered sufficiently significant to justify the use of both sexes in the repeat dose phase of the study (group C).

The terminal elimination phase could not be defined for most animals, but from those where this was determined, there was little difference between male and female animals (10.6 and 12.9 hours, respectively).

Group B (Single Dose, 300 mg/kg)

Following oral administration of [14C]-BMTP to male and female rats, peak concentrations occurred somewhat later than animals dosed at the lower dose level: 8 to 12 hours for males (mean = 9.0 hours) and 12 to 24 hours for females (mean = 15.0 hours). This slower attainment of maximal concentrations, relative to the lower dose level, may have been indicative of some delay in the absorptive processes. There was no difference in C values between the sexes (135 and 136 µg equiv/g) but AUC(0-t) values were higher for female animals (3670 h*µg equiv/g) than males (2130 h*µg equiv/g). Terminal half-lives could not be defined for any animal in this group.

After normalising data for the doses administered, there was no difference in maximal concentrations for animals dosed at either 100 mg/kg or 300 mg/kg.

Ratios of peak levels (group B : group A) were 1.07 for males and 0.95 for females. However, the equivalent ratios for AUC(0-t) were 1.35 and 1.42, respectively, possibly indicating a slower (or saturated) elimination at the higher dose, although this could not be demonstrated from the data as half-lives could not be determined for group B animals.

Group C (Repeat Dose, 100 mg/kg)

Male animals received 14 daily oral administrations of [14C]-BMTP. Mean daily peak concentrations (taken at 4 hours post-dose) increased over the course of the dosing period, rising from 46.1 µg equiv/g on day 1 to 73.4 µg equiv/g after the final dose. After the final dose, all pharmacokinetic parameters were determined from the mean data determined after the day 14 dose. After the final dose, the peak concentration occurred at 4 hours, but representing a 1.59-fold increase on the value 4 hours after the first dose. The AUC(0-t) value for the final day was 1200 h*µg equiv/g, a value approximately 2.3 times higher than after a single dose.

The progressive increase in Cmax over the dosing period and elevated AUC(0-t) possibly resulted from the incomplete elimination of the previous dose before the administration of the next one, as shown by a residual concentration of 3.7 µg equiv/g 24 hours after a single dosing compared to 17 µg equiv/g 24 hours after the 14th dosing. In addition, the terminal elimination half-life was substantially longer (33.2 hours after repeat dosing, compared to 10.6 hours after single administration); a distribution phase half-life, using concentrations at 6, 8 and 12 hours after the 14th dose, was estimated at 5.6 hours. These data may be indicative of saturation of metabolic pathways or elimination, or the generation of one or more long-lived metabolites.

Summary of mean pharmacokinetic parameters
	Group A		Group B		Group C (day 14)
	100 mg/kg		300 mg/kg		100 mg/kg/day
Parameter	Males	Females	Males	Females	Males
Cmax(µg equiv/g)	42.1	47.9	135	136	73.4
Tmax(h)	4	9.5	9	15	4
t½(h)	10.6	12.9	NR	NR	5.58 / 33.2 *
AUC(0-t)(h*µg equiv/g)	527	858	2130	3670	1200
AUC(0-inf)(h*µg equiv/g)	560	990	NR	NR	1780
Cmax/D	0.42	0.48	0.45	0.45	0.73
AUC(0-t)/D	5.26	8.59	7.08	12.2	12.0
Gender ratios (male : female):
Cmax	0.88	-	0.99	-	-
AUC(0-t)	0.61	-	0.58	-	-
Dose proportionality ratios (group B : group A)
Cmax/ D	-	-	1.07	0.95	-
AUC(0-24)/ D	-	-	1.35	1.42	-
Accumulation ratios (group C : group A):
Cmax	-	-	-	-	1.74
AUC(0-t)	-	-	-	-	2.28

NR – Not reported (no clear terminal elimination phase).

* Half- life estimates for distribution phase (using 6, 8, 12 hour data) and terminal phase respectively.

Conclusions:

• There was an indication of a gender difference after single administration at both the 100 and 300 mg/kg dose levels. Maximal concentrations occurred later in females and exposure to test substance related radioactivity (as determined by AUC) was also somewhat higher in female animals.
• Maximal concentrations at the higher dose level were later relative to those at the low dose, indicating a possible delay in absorption.
• After single doses, for both genders, Cmax and exposure were essentially dose proportional.
• Concentrations of radiolabelled test substance related material following repeat administration were slightly higher, with both Cmax and especially AUC(0-t) elevated after 14 doses, relative to single administration.
• [14C]-BMTP was quickly and significantly absorbed. The residual radioactivity in blood 48 hours after single and repeated administrations were between 5 and 16% of the Cmax, indicating no tendency for bioaccumulation.

Executive summary:

The absorption and toxicokinetics of [¹⁴C]-2,2-Bis(methylthio)propane ([¹⁴C]-BMTP) was evaluated following single and repeat oral administration to the rat. Sixteen healthy male and eight healthy female Han Wistar rats were used. Each animal received a single oral gavage dose of [¹⁴C]-BMTP (group A: 100 mg/kg; group B: 300 mg/kg; 4 males and 4 females for each group) or 14 daily oral gavage doses (group C: 100 mg/kg/day; 8 males). Blood samples were taken as follows from groups A and B (single dose) animals:

·          pre-dose, 30 minutes; 1, 2, 4, 6, 8, 12, 24, 48 hours.

Group C animals(14 daily dose)were split into two cohorts of four male animals each.

For cohort 1, blood samples were taken as follows:

·          4 hours following doses on days 1, 3, 5, 7, 9, 11, 13.

·          Following day 14 dose: 1, 4, 8, 24 hours.

For cohort 2, blood samples were taken as follows:

·          4 hours following doses on days 2, 4, 6, 8, 10, 12.

·          Following day 14 dose: 0.5, 2, 6, 12, 48 hours.

Radioactivity concentrations were determined in blood samples by solubilisation followed by liquid scintillation counting.

Mean pharmacokinetic parameters were as followed.

	Group A		Group B		Group C (day 14)
	100 mg/kg		300 mg/kg		100 mg/kg/day
Parameter	Males	Females	Males	Females	Males
Cmax(µg equiv/g)	42.1	47.9	135	136	73.4
Tmax(h)	4	9.5	9	15	4
t½(h)	10.6	12.9	NR	NR	5.58 / 33.2 *
AUC(0-t)(h*µg equiv/g)	527	858	2130	3670	1200
AUC(0-inf)(h*µg equiv/g)	560	990	NR	NR	1780
Cmax/D	0.42	0.48	0.45	0.45	0.73
AUC(0-t)/D	5.26	8.59	7.08	12.2	12.0
Gender ratios (male : female):
Cmax	0.88	-	0.99	-	-
AUC(0-t)	0.61	-	0.58	-	-
Dose proportionality ratios (group B : group A)
Cmax/ D	-	-	1.07	0.95	-
AUC(0-24)/ D	-	-	1.35	1.42	-
Accumulation ratios (group C : group A):
Cmax	-	-	-	-	1.74
AUC(0-t)	-	-	-	-	2.28

NR – Not reported (no clear terminal elimination phase).

* Half- life estimates for distribution phase (using 6, 8, 12 hour data) and terminal phase respectively.

Following single oral administration of [¹⁴C]-BMTP to male and female rats and fourteen daily oral administrations of [¹⁴C]-BMTP to male rats:

·        There was an indication of a gender difference after single administration at both the 100 and 300 mg/kg dose levels. Maximal concentrations occurred later in females and exposure to test substance related radioactivity (as determined by AUC) was also somewhat higher in female animals.

·        Maximal concentrations at the higher dose level were later relative to those at the low dose, indicating a possible delay in absorption.

·        After single doses, for both genders, C_maxand exposure were essentially dose-proportional.

·        Concentrations of radiolabelled test substance related material following repeat administration were slightly higher, with both C_maxand especially AUC_(0-t)elevated after 14 doses, relative to single administration.

[14C]-BMTP was quickly and significantly absorbed. The residual radioactivity in blood 48 hours after single and repeated administrations were between 5 and 16% of the Cmax, indicating no tendency for bioaccumulation.

Reference 3

Endpoint:

dermal absorption, other

Remarks:

QSAR

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on IR&CSA, Chapter R.14, Occupational exposure assessment Update to change the scope of the guidance from exposure estimation to exposure assessment

Principles of method if other than guideline:

IH SkinPerm (v2.04) is a mathematical tool for estimating dermal absorption. The rate of mass build-up (or loss) on the skin comes from the deposition rate onto the skin minus the absorption rate into the Stratum Corneum (SC) and the amount evaporating from the skin to the air.

Species:

other: human

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Details on study design:

DATA INPUT
Molecular weight: 136.3 g/mol
Temperature: 25 °C
Vapour Pressure: 443 Pa
Water solubility: 333 mg/L
Log Kow: 3.3
Density: 987 mg/cm3
Melting point: -23.7°C

SCENARIO PARAMETERS
- Instantaneous deposition
Deposition dose*: 1000 mg
Affected skin area**: 1000 cm²
Maximum skin adherence***: 2 mg/cm²
Thickness of stagnant air****: 1 cm
Weight fraction: 1
Timing parameters
. Start deposition: 0 hr
. End time observation: 8 hr
Report parameters
. Calculation (intervals/hr): 10000
. Report (intervals/hr): 100

- Deposition over time
Affected skin area**: 1000 cm²
Maximum skin adherence***: 1 mg/cm²
Dermal deposition rate: 1 mg/cm²/hr
Thickness of stagnant air****: 1 cm
Weight fraction: 1
Timing parameters
. Start deposition: 0 hr
. Duration of deposition: 8hr
. End time observation*: 8 hr
Report parameters
. Calculation (intervals/hr): 10000
. Report (intervals/hr): 100

*Default value defined according to the internal validation study
**Estimated skin surface of two hands of an adult.
***The skin adherence field is greyed out and a default of -1 is indicated if the substance is a liquid at 25°C. Smart logic is built into IH SkinPerm; the program recognizes whether a substance is a solid or liquid at standard temperature (25°C) based on the physicochemical properties. For substances
that are solids at 25°C a maximum adherence value up to 2 mg/cm² is allowed based on studies of soil-on-skin adherence. If the deposition rate results in an increase above the input figure (0.2-2 mg/cm²), it is assumed that the surplus disappears just by removal from the skin.
*** 3 cm if clothing involved, 1 cm if bare skin involved

Time point:

8 h

Dose:

1000 mg

Parameter:

percentage

Absorption:

10.9 %

Remarks on result:

other: Instantaneous deposition

Time point:

8 h

Dose:

1 mg/cm²/h

Parameter:

percentage

Absorption:

5.2 %

Remarks on result:

other: Deposition over time for 8 hr

Conclusions:

The dermal absorption of 2,2-bis(methylthio)propane10.9 is estimated to be moderate (<= 50%).

Executive summary:

The dermal absorption of 2,2-bis(methylthio)propaned leads to the following results, obtained using the SkinPerm v2.04 model according to the input data:

	Instantaneous deposition	Deposition over time End time observation 8 hr
Total deposition (mg) or deposition rate (mg/cm²/hr)	1000	1
Fraction absorbed (%)	10.9	5.2
Amount absorbed (mg)	109	416
Lag time stratum corneum (min)	4.82
Max. derm. abs. (mg/cm²/h)	0.0261

Description of key information

The data available on 2,2 -bis(methylthio)propane and the estimations from its physico-chemical properties indicate a significant absorption by oral and inhalation routes of exposure with a very low rate of dermal absorption, widespread distribution, rapid metabolism by oxidation of the sulphide functions and/or by hydrolysis to form acetone and methyl mercaptan and no bioaccumulation potential of parent and transformation products.

The absorption/excretion, toxicokinetics, metabolism and distribution data of 2,2-bis(methylthio) propane were evaluated from the available toxicological data and the physicochemical properties as suggested by the REACH Guidance Chapter R.7c:

Molecular weight: 136.3 g/mol

Vapour Pressure: 443 Pa

Water solubility: 333 mg/L

Log Kow: 3.3

Density: 987 mg/cm3

Melting point: -23.7°C

ABSORPTION

Oral route

The excretion of radioactivity has been investigated following oral administration of [14C]-BMTP to male rats (Lewsley and Hewitt, 2014). Two male rats of the Han Wistar strain received a single oral administration of [14C]-BMTP, by gavage at a nominal dose level of 160 mg/kg. Urine and faeces collected at sampling periods up to 168 hours. Expired air was drawn through a series of traps, designed to collect volatile components, at times up to 72 hours. Following each urine/faecal collection, cages were washed with a small volume of water and any cage debris removed. Blood was collected at 168 hours post-dose, prior to the animals being humanely killed and carcasses retained. Radioactivity in blood, excreta and carcasses was determined by liquid scintillation counting. The mean recovery of radioactivity was 97.0% over the collection period. The majority of the radioactivity recovered was in the urine (73.3%) and cage washes (15.8%), with limited levels in faeces (1.3%) and cage debris (0.1%). Expired air traps contained radioactivity but this accounted for less than 5% of the administered dose. Low levels of radioactivity were retained in both blood (0.984 µg equiv/g) and the carcass (2.68 µg equiv/g; 1.7% of the dose). In conclusion, nearly 100% of the dose was absorbed, the principal route of excretion was in the urine, with only low levels recovered in either faeces or expired air.

Therefore, the oral absorption of 2,2-bis(methylthio) propane can be assumed to be 100% for risk assessment.

Inhalation route

According to the REACH Guidance, the physicochemical characteristics of 2,2-bis(methylthio) propane (log Pow 3.3) and the molecular mass (136.3 g/mol) are in a range suggestive of absorption as such from the respiratory subsequent to inhalation exposure. This assumption of absorption is supported by the clinical signs of central nervous system depression observed at 20.2 mg/L in the acute inhalation toxicity study (Schuler, 2008). Therefore, the inhalation absorption can be assumed to be 100% for risk assessment.

Dermal absorption

According to the REACH Guidance, the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Between 100 -1000 mg/l absorption is anticipated to be moderate to high. Log P values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal) particularly if water solubility is high.

The rate of absorption was estimated using the IH SkinPerm model (v2.04). For an instantaneous deposition of 1000 mg over 1000 cm² of skin or a deposition over time of 1 mg/cm²/h, the absorption rates were 10.9% and 5.2%, respecticely.

Therefore, according to the REACH Guidance, a default value of 50% skin absorption will be used for risk assessment.

TOXICOKINETIC

The absorption and toxicokinetics of [14C]-2,2-Bis(methylthio)propane ([14C]-BMTP) was evaluated following single and repeat oral administration to the rat (Lewsley and Hewitt, 2015). Sixteen healthy male and eight healthy female Han Wistar rats were used. Each animal received a single oral gavage dose of [14C]-BMTP (group A: 100 mg/kg; group B: 300 mg/kg; 4 males and 4 females for each group) or 14 daily oral gavage doses (group C: 100 mg/kg/day; 8 males). Blood samples were taken as follows from groups A and B (single dose) animals:

·          pre-dose, 30 minutes; 1, 2, 4, 6, 8, 12, 24, 48 hours.

Group C animals (14 daily dose)were split into two cohorts of four male animals each.

For cohort 1, blood samples were taken as follows:

·          4 hours following doses on days 1, 3, 5, 7, 9, 11, 13.

·          Following day 14 dose: 1, 4, 8, 24 hours.

For cohort 2, blood samples were taken as follows:

·          4 hours following doses on days 2, 4, 6, 8, 10, 12.

·          Following day 14 dose: 0.5, 2, 6, 12, 48 hours.

Radioactivity concentrations were determined in blood samples by solubilisation followed by liquid scintillation counting.

Mean toxicokinetic parameters were as followed:

	Group A		Group B		Group C (day 14)
	100 mg/kg		300 mg/kg		100 mg/kg/day
Parameter	Males	Females	Males	Females	Males
Cmax(µg equiv/g)	42.1	47.9	135	136	73.4
Tmax(h)	4	9.5	9	15	4
t½(h)	10.6	12.9	NR	NR	5.58 / 33.2 *
AUC(0-t)(h*µg equiv/g)	527	858	2130	3670	1200
AUC(0-inf)(h*µg equiv/g)	560	990	NR	NR	1780
Cmax/D	0.42	0.48	0.45	0.45	0.73
AUC(0-t)/D	5.26	8.59	7.08	12.2	12.0
Gender ratios (male : female):
Cmax	0.88	-	0.99	-	-
AUC(0-t)	0.61	-	0.58	-	-
Dose proportionality ratios (group B : group A)
Cmax/ D	-	-	1.07	0.95	-
AUC(0-24)/ D	-	-	1.35	1.42	-
Accumulation ratios (group C : group A):
Cmax	-	-	-	-	1.74
AUC(0-t)	-	-	-	-	2.28

NR – Not reported (no clear terminal elimination phase).

* Half- life estimates for distribution phase (using 6, 8, 12 hour data) and terminal phase respectively.

Following single oral administration of [14C]-BMTP to male and female rats and fourteen daily oral administrations of [14C]-BMTP to male rats:

·        There was an indication of a gender difference after single administration at both the 100 and 300 mg/kg dose levels. Maximal concentrations occurred later in females and exposure to test substance related radioactivity (as determined by AUC) was also somewhat higher in female animals.

·        Maximal concentrations at the higher dose level were later relative to those at the low dose, indicating a possible delay in absorption.

·        After single doses, for both genders, Cmaxand exposure were essentially dose-proportional.

·        Concentrations of radiolabelled test substance related material following repeat administration were slightly higher, with both Cmaxand especially AUC(0-t)elevated after 14 doses, relative to single administration.

[14C]-BMTP was quickly and significantly absorbed. The residual radioactivity in blood 48 hours after single and repeated administrations were between 5 and 16% of the Cmax, indicating no tendency for bioaccumulation.

 

DISTRIBUTION and METABOLISM

According to the REACH Guidance, as a small molecule a wide distribution of 2,2 -bis(methylthio) propane is expected. This assumption is confirmed by the changes in spleen, liver and kidneys shown in the repeated dose toxicity studies in rats following oral application (Chevalier, 2008; Bentz, 2012 and 2015). Especially, the increase in P450 enzyme proteins (CYP2B1/2 and CYP3A1/2) in the liver of rats treated with 2,2 -bis(methylthio) propane for 28 days (Chevalier, 2008) is the indication of an activation of its own metabolisation.

The resting pH of the stomach of healthy people is around 1.8 but can be low as 1.0. An in vitro test performed according to OECD TG 111 (Gancet, 2009) shown that 2,2 -bis(methylthio) propane was readily hydrolysed at the pH of the stomach content (pH 1.2 at 37°C) with a half-life of 1.21h. Acetone and methyl mercaptan were identified as decomposition products. This data suggests that the toxicity of 2,2 -bis(methylthio) propane could be driven by the release in the stomach and the absorption of the toxic substance methyl mercaptan, the other decomposition product, acetone, being of low acute oral toxicity (Freeman and Hayes, 1985). However this hypothesis doesn’t fit with the experimental toxicological data. When administered orally as sodium methanethiolate to rats, methyl mercaptan is acutely toxic (LD50 = 116 mg/kg; REACH registration dossier, CAS no. 5188-07-8), whereas with 2,2-bis(methylthio) propane, no mortality but clinical signs of central nervous system depression were observed in rats at a dose level of 2000 mg/kg (Rokh, 2007). Therefore, the hydrolysis of 2,2-bis(methylthio) propane to acetone and methyl mercaptan in the stomach doesn’t seem a pre-required condition for its absorption.

Acetone and methyl mercaptan were identified as hydrolytic decomposition products of 2,2 -bis(methylthio) propane (Gancet, 2009) but can be also the result of the hepatic metabolism.. 

The data on the metabolism of methyl mercaptan are relatively scarce. Rats injected intraperitoneally with methyl mercaptan excreted CO₂and volatile sulphur containing compounds in the expired breath (Canellakis and Tarver, 1953).³⁵S-Methyl mercaptan injected into rats resulted in approximately 94% of the sulfur in the urine as³⁵SO4^2-(Derr and Draves, 1983 and 1984). One mouse, injected with methyl mercaptan, expired methyl mercaptan and dimethyl sulfide in its breath (Susman et al., 1978). Erythrocytes were found to oxidize methyl mercaptan producing formic acid, sulfite ion, and sulfate ion (Blom and Tangerman, 1988).

The metabolism of acetone has been extensively examined in laboratory animals, while only few data are available on humans. The primary site of metabolism of acetone is the liver. The first step includes the oxidation to acetol by acetone monooxygenase, associated with cytochrome P450IIE1. This step is followed by two different pathways: (i) oxidation to methylglyoxal (also associated with P450IIE1) and (ii) probably extrahepatic conversion to L-1,2-propandiol. Methylglyoxal is converted via D-lactate or directly to pyruvate. 1,2-Propandiol is also converted to pyruvate via L-lactate. Pyruvate is a main product of interediary metabolism that may enter e. g. the citric acid cycle or the gluconeogenic pathway. Consequently, in studies with¹⁴C-labelled acetone,¹⁴C-activity was also detected in other products and substrates of the intermediary metabolism and in carbon dioxide. The pattern of acetone metabolism can be altered by variations in the physiological status (WHO 1998; ATSDR 1994; Kalapos 1999).

Alternatively, 2,2 -bis(methylthio) propane can be metabolised without hydrolysis. 2,2 -bis(methylthio) propane contains 2 monosulphide functions (thioethers, CH3-S-R) which can primarily undergo S-oxidation, catalyzed by cytochrome P450 and flavin-containing monoxygenases (FMO), leading to the formation of sulphoxides, which can be further oxidised, at least partially, to sulphones. Sulphoxides and sulphones are hydrophilic and usually chemically stable. Sulphoxides are the major urinary excretion products in mammals exposed to thioethers, whereas the amount of sulphones is generally low. The S-oxidation of sulphoxides to sulphones is an irreversible reaction, whereas reduction of the sulphoxides back to sulphides is a common route of metabolism (EFSA, 2012).

ELIMINATION

According to the REACH Guidance, the n-Octanol/water partition coefficient (log Pow of 3.3) and the hydrolysis are not suggestive of accumulation of unchanged 2,2 -bis(methylthio) propane in fatty tissues subsequent to absorption. As well, the hydrolytic products, acetone and methyl mercaptan, are molecules of small size and are not subject to bioaccumulation. Therefore, no potential for bioaccumulation is to be expected for 2,2 -bis(methylthio) propane.

REFERENCES

ATSDR. 1994. Toxicological profile for acetone. Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry.

Blom, H. J. and Tangerman, A. 1988. Methanethiol metabolism in whole blood. J. Lab. Clin. Med. 111: 606-610.

Canellakis, E. S. and Tarver, H. 1953. The metabolism of methyl mercaptan in the intact animal. Arch. Biochem. Biophys. 42: 446-455.

Derr, R. F. and Draves, K. 1984. The time course of methanethiol in the rat. Res. Commun. Chem. Pathol. Pharmacol. 46: 363-369.

EFSA. 2012. Scientific Opinion on Flavouring Group Evaluation 08, Revision 4 (FGE.08Rev4): Aliphatic and alicyclic mono-, di-, tri-, and polysulphides with or without additional oxygenated functional groups from chemical groups 20 and 30. EFSA Journal 2012;10(2):2455.

Freeman JJ, Hayes EP (1985) Acetone potentiation of acute acetonitrile toxicity. J Toxicol. Environ Health 15: 609-621.

Kalapos, M. P. 1999. Possible physiological roles of acetone metabolism in humans. Med. Hypotheses 53: 236-242.

Susman, J. L., Hornig, J. F., Thomae, S. C., et al. 1978. Pulmonary excretion of hydrogen sulfide, methanethiol, dimethyl sulfide and dimethyl disulfide in mice. Drug Chem. Toxicol. 1: 327-338.

WHO. 1998. Acetone. Environmental Health Criteria (EHC) 207. International Programme on Chemical Safety (IPCS); World Health Organization.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100