Ziram

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

Study period:

11 Apr 1988 - 04 Dec 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Blood kinetics and metabolism not investigated

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

not specified

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

not specified

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-labelled (CS2 moiety)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Massachusetts, USA
- Age at study initiation: 44 days (m); 51 days (f)
- Weight at study initiation: 149 - 169 g (m); 145 - 162 g (f)
- Housing: Individually in stainless steel, screen-bottom cages suspended on racks, with absorbent liners in the urine- and feces collecting trays. On the final day of acclimation, rats were housed in glass metabolism chambers overnight. After dosing rats were individually housed in glass metabolism chambers for the collection of expired air and the separation and collection of urine and feces.
- Diet: Certified Rodent Chow #5002 (Ralston Purina Company, Missouri, USA), ad libitum
- Water: Fresh water, ad libitum
- Acclimation period: 7 - 9 days (group-dependent)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved or suspended in 0.3 - 0.7% carboxymethyl cellulose.

Duration and frequency of treatment / exposure:

Single and multiple (15 days daily) application

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

352 mg/kg bw/day

No. of animals per sex per dose / concentration:

Control: 2/sex
Treatment: 5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

No

Details on study design:

Group 1: 2 male and 2 female rats received the vehicle control (0.5% carboxymethyl cellulose (CMC) in water) by gavage.
Group 2: 5 male and 5 female rats received a single dose of 15 mg/kg bw of the test substance by gavage.
Group 3: 5 male and 5 female rats received a repeated dose of 15 mg/kg bw/day of the non-labelled compound for 14 days followed by a single dose of 15 mg/kg bw/day of the labelled compound on Day 15 by gavage.
Group 4: 5 male and 5 female rats received a single dose of 352 mg/kg bw of the test substance by gavage.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: air, urine, faeces, blood, several organs
The CO2 trapping solution and the volatile traps were collected at 0 - 4, 4 - 8, 8 - 12, and 12 - 24 h following administration of the radiolabeled test substance and daily thereafter for a total of 4 days.
Urine and faeces samples were collected at 0 - 6, 6 - 12, and 12 - 24 h after the radiolabeled dose and daily thereafter for a total of 7 days. Urine and faeces were collected in plastic containers surrounded by ice. At the end of the collection period, the animals were anesthetized with halothane and exsanguinated by cardiac puncture.
Blood (2 to 5 mL) was collected and weighed in heparinized tubes and saved for radioanalysis.
After sacrifice the cages were washed with a 1.0% trisodium phosphate solution which was saved for analysis.

ANALYTICAL METHOD
- Sample preparation:
Organs/tissues: Fifteen matrices (including the carcass) were prepared for radio-analysis to determine the distribution of the radiolabelled material. Sample size permitting tissues were homogenized and duplicate aliquots were weighed for combustion.
Carcass: Carcasses were ground and homogenized and duplicate aliquots were weighed for combustion.
Fat: Fat samples were digested with Carbo Sorb for approximately 2 days. Duplicate aliquots were weighed and analyzed.
Blood: Whole blood samples were shaken to ensure homogeneity. Duplicate aliquots (0.2 g) were weighed for combustion.
Feces: Fecal samples were frozen using liquid N2 and then ground. Duplicate aliquots of the homogenates were weighed for combustion.
Samples of CO2 trap, urine, cagewash, and cage wipe (cut to four individual samples) were directly analyzed.
Volatile Trap: Sample was split into four aliquots, and each was combusted.
- Radio-analysis (measurement of radioactivity):
Samples were analysed by liquid scintillation counter (LSC) for 5 minutes or 100,000 counts. Scintillation counting data (cpm) were automatically converted to dpm using the external standardization technique and an instrument-stored quenchcurve generated from a series of sealed quenched standards.

Statistics:

Statistical analyses were limited to simple expressions of variation, such as mean and standard deviation.

Type:

absorption

Results:

Oral absorption was rapid and extensive, with the initial estimation of absorption ranging from 65 - 80% for male and female rats.

Type:

distribution

Results:

Radioactivity was further distributed to the organs/tissues, with the highest levels found in blood, liver, kidney, heart, lungs, spleen and thyroid. The mean total radioactivity retained in tissue was low.

Type:

excretion

Results:

The rate of elimination was relatively fast; the majority of the radioactivity was eliminated within 48 h after dosing via urine (17% to 35%), faeces (9% to 18%), and expired air (36% to 53%).

Details on absorption:

Recovery
The mean 14C recovery ranged from 79% to 92% of the total doses administered. For details, please refer to the attached background material 1 and 2.

Absorption:
After single oral treatment with the low dose of the test substance, the initial estimation of absorption (achieved by the total recovery of radioactivity excluding the percentage of dose in the faeces) was approximately 65% in males and females, while after single oral treatment with the high dose of the test substance, the absorption was approximately 77% in males and 66% in females. After repeated administration of the low dose, absorption was approximately 75% in males and 80% in females.

Details on distribution in tissues:

The mean total radioactivity retained in the tissues and carcasses ranged from 1.11% to 1.92% of the total dose administered. For the low dose groups, the residue levels in the blood and tissues ranged from 0.05 to 2.5 ppm (µg 14C-labelled test substance equivalents/g sample). The highest levels were found in blood, liver, kidney, heart, lungs, spleen and thyroid gland. For details, please refer to the attached background material 1 and 2.

Key result

Test no.:

#1

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was distributed to the peripheral tissues mainly liver, kidney, heart, lungs, spleen and thyroid gland.

Details on excretion:

The majority of the radioactivity was found in urine (17% to 35%), faeces (9% to 18%), and expired air (36% to 53%). The rate of elimination was relatively fast; the majority of the radioactivity was eliminated within 48 h after dosing. No apparent sex-related differences were observed for 14C elimination or distribution for any of the treated groups.
For details, please refer to the attached background material 1 and 2.

Metabolites identified:

not measured

Details on metabolites:

not applicable

Enzymatic activity measured:

not measured

Clinical observations:

Single oral low-dose group: One male had dark urine at the 96 to 120-h collection.

Multiple oral low-dose group: One male was found dead at the 96-hour collection. It appeared that the animal had suffocated because the Tygon tubing was found disconnected from the CO2 collection tower at the 96-h collection. This animal also had a rough hair coat on Days 15 and 16 (Days 1 and 2 after receiving the radiolabeled dose). One female was noted as having excessive salivation on Day 13 and rough haircoat on Day 15. Two females were noted as having rough haircoat on Days 14, 15, and 16. The last one female was noted as having rough haircoat on Days 14 and 15.

Single oral high-dose group: One male was noted as having excessive salivation on Day 0 and had dark urine at the 48 - 72-h collection. Another male was noted as having excessive salivation and lacrimation of the right eye on Day 0 and also had white material in its feces sample at the 0 - 6-h collection. Two other males had white material in their feces samples as well in the glass housing unit near the urine separator at the 0 - 6-h collection. The last male had white material in the glass housing unit near the urine separator at the 0 - 6-h collection. Three females were noted as having rough haircoat on Day 0, Three females had white material in their feces samples at the 0 - 6-h collection (two females with rough haircoat belonged to these animals).

Conclusions:

The biokinetic behaviour of the test compound was investigated in rats in a GLP-compliant study on rats according to EPA OPP 85-1 (Metabolism and Pharmacokinetics). Oral absorption was rapid and extensive, with the initial estimation of absorption ranging from 65 - 80% for male and female rats.
The mean 14C recovery ranged from 79% to 92% of the total doses administered. The majority of the radioactivity was found in urine (17% to 35%), feces (9% to 18%) and expired air (36% to 53%). The rate of elimination was relatively fast and the majority of the radioactivity was eliminated within 48 h after dosing. Radioactivity was further distributed to the organs/tissues, with the highest levels found in blood, liver, kidney, heart, lungs, spleen and thyroid. The mean total radioactivity retained in tissue was low with a mean total radioactivity retained in the tissues and carcasses ranging from 1.11% to 1.92% of the total dose administered. Based on the study results, no apparent sex-related differences were noted for 14C elimination or distribution for any of the treated groups and no bioaccumulation potential was observed.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun 1997 - 10 Nov 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

not specified

Deviations:

yes

Remarks:

Weight variation exceeds ± 20%. For the biliary excretion study only 1 male was used

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-labelled (CS2 moiety)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd, Margate, Kent, UK
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: 138 - 230 g
- Housing: Rats were housed in groups of up to five per cage according to sex in wire floor polypropylene cages suspended over polypropylene dirt trays containing soft white sawdust.
- Diet: SQC Rat and Mouse Maintenance Diet No. 1 (Special Diet Services, Essex, UK), ad libitum
- Water: Mains water, ad libitum
- Acclimation period: >3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 41 - 67
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose groups B, C, G and H: The radiolabelled test material was admixed with an appropriate amount of non-radiolabelled test substance and 0.3% aqueous carboxymethylcellulose (CMC) added to provide a nominal dose volume of 4 mL/kg bw.
Dose groups D, E, F and I: An appropriate amount of non-radiolabelled test substance was suspended in 0.3% aqueous CMC. Radiolabelled test material was then ground using an agate pestle and mortar with 1% aqueous ammonium lignosulphonate and transferred quantitatively into the non-radiolabelled test substance suspended in 0.3% aqueous CMC. Further 0.3% aqueous CMC was added to give a nominal dose volume of 4 mL/kg bw.

Duration and frequency of treatment / exposure:

Single and multiple (15 days daily) application

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

No. of animals per sex per dose / concentration:

- Pharmacokinetics study/excretion balance study: 5/sex/group
- Tissue distribution study: 12/sex/group
- Biliary excretion study: 1 male/group

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

No

Details on study design:

Group A: Dose group A represents the pilo study. For details on the study design of the pilot study, please refer to table 1 under "Any other information on material and methods incl. tables"
Group B - I: Dose groups B to I represent the main study. For details on the study design of the main study, please refer to table 2 under "Any other information on material and methods incl. tables"

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Blood, expired air, bile, urine, faeces, cage wash, tissues (heart, lungs, liver, pituitary, spleen, thyroid, adrenals, kidneys, gonads, muscle (quadriceps), bone (femur), brain, fat (perirenal) and residual carcass

- Time and frequency of sampling:
Pharmacokinetics (dose groups B and C): Blood (300 µL from a lateral caudal vein) 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192 and 216 h
Excretion and Balance (dose groups D, E and F): Expired air (24, 48 and 72 h), urine (6, 12, 24, 36, 48, 72, 96, 120, 144 and 168 h), faeces (12, 24, 36, 48, 72, 96, 120, 144 and 168 h), tissues at sacrifice (168 h; heart, lungs, liver, pituitary, spleen, thyroid, adrenals, kidneys, gonads, muscle (quadriceps), bone (femur), brain, fat (perirenal) and residual carcass) and cage wash
Tissue Distribution (dose groups G and H): Plasma 2, 8, 24 and 96 h, tissues (same as above) 2, 8, 24 and 96 h
Biliary Excretion (dose group I): Bile 0, 1, 4, 6, 12, 24 and 48 h, urine 24 and 48 h, faeces 24 and 48 h, cage wash

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and feces
- Time and frequency of sampling: Urine and faeces samples were pooled between 0 and 48 h by sex and dose group.

MEASUREMENT OF RADIOACTIVITY AND USED ANALYTICAL METHODS
The following techniques for measuring radioactivity and for identification of substances were used in the present study:
1) For the measurement of radioactivity liquid scintillation counting (LSC) was used.
2) For metabolite profiling high-performance liquid chromatography (HPLC) was used.
3) For identification of metabolites liquid chromatography mass spectrometry (LC-MS) was used.

1) LCS
Measurement of liquid samples:
Urine, plasma, bile, expired air traps and cage washings: Portions were added directly to scintillant.

Measurement of solid samples:
Solubilisation: Adrenals, pituitary and thyroid: A suitable volume of a solubilising agent was added. After an appropriate period at room temperature, liquid scintillant was added and the samples allowed to dark adapt prior to LSC.
Digestion: Tissues, other than adrenals, pituitary and thyroids (and carcasses) were treated with a suitable volume of methanolic potassium hydroxide (40% w/v) and digested at room temperature (carcasses under reflux). Aliquots of digest were added to liquid scintillant and the samples allowed to dark adapt prior to LSC.
Combustion: Samples of bone, faecal, carcass and cage debris homogenates were added to ashless floe and combusted in oxygen. The combusted products were absorbed in Carbo-SorbR, mixed with Permafluor E+, and the radioactivity determined by LSC.

2) HPLC
In contrast to urine samples, pooled faeces samples were vortex mixed and sonicated with 10 mL of acetonitrile. The samples were centrifuged and the supernatants removed. The residual pellets were then homogenised and vortex mixed with 5 mL of water, centrifuged and the supernatant removed. The two supernatants were combined and reduced to dryness under nitrogen convection at room temperature. The extracts were reconstituted in ca 0.5 mL of water and analysed by HPLC.

3) LC-MS
Urine from one animal of dose group E was investigated directly by LC-MS to provide structural confirmation of the major radiolabelled metabolites present. A
pool of urine from group E (150 mg/kg bw) 0 - 48 h was also produced.

Limit of Detection
The Iimit of detection for the analysis of each sample type was taken as twice the background disintegration rate obtained from the measurement of blank samples of the same type.

Statistics:

No statistical analysis was performed.

Preliminary studies:

Initial experiments resulted in low recoveries of approximately 45%. Following a series of additional experiments designed to optimise the trapping systems, this recovery was increased to approximately 80%. An optimised trapping scheme wasused for the main experiment.

Type:

absorption

Results:

The absorption was estimated to reach 59 - 81.2% for male and female rats.

Type:

distribution

Results:

Distribution and uptake of radioactivity by tissues was rapid, with the highest radioactivity found in organs of metabolism and excretion (liver, lung, kidney), vascularised tissues (spleen, thyroid, adrenals), fat, blood and plasma.

Type:

excretion

Results:

Excretion of radioactivity was rapid, the major proportion being excreted as volatiles in expired air within 24 h of administration.

Type:

metabolism

Results:

The principal route of metabolism was hydrolysis to form and exhale CS2, COS and CO2.

Details on absorption:

Recovery:
Total recoveries of radioactivity ranged, on average, from 63.27 – 64.62% for males and females of the single low dose group to 75.88 – 76.46% of the single high dose group and 74.09 – 84.93% of the repeated-dose group and were mainly exhaled. For detail, please refer to the attached background material 1 and 2.

Absorption:
Based on the results of exhaled volatiles as well as urinary excretion, absorption is estimated to reach 59 % within 72 h after a single low dose exposure. Absorption increases up to 81.2% after repeated low dose exposure. Absorption was relatively slow with maximum concentrations of radioactivity being reached within 10 h at the low dose level and 24 h at the high dose level.

Details on distribution in tissues:

Pharmacokinetics (dose groups B and C):
Absorption was relatively slow with maximum concentrations of radioactivity being reached within 10 h at the low dose Ievel and 24 h at the high dose Ievel. The pharmacokinetic parameters of the test substance were approximately proportional, with a 10 fold increase in the dose Ievel resulting in a ca 10 fold increase in maximum concentrations of radioactivity and between a ca 13 - 18 fold increase in the respective values for the areas under the concentration/time curves. The half-life, in plasma, was approximately doubled for a 10 fold increase in dose Ievel. The pharmacokinetic parameters therefore indicated that both absorption and elimination of radioactivity were unlikely to be saturated at the dose Ievels used on this study. There was a ca 3 fold increase in the blood elimination half-life compared to plasma for the 150 mg/kg bw dose and about a 5 fold difference for the 15 mg/kg bw dose. This suggests that radioactivity was binding to the blood cells but binding was probably saturated between doses of 15 and 150 mg/kg bw. Overall, blood elimination half-life was higher than 24 h suggesting that accumulation may occur after repeated exposure.
For details on the toxicokinetic parameters, please refer to table 3 under "Any other information on results incl. tables".

Single Oral Administration at the Low Dose Level in Both Sexes (Group G):
Distribution and uptake of radioactivity by tissues was rapid. All tissues had been exposed to radiolabelled material within 2 h of administration. Levels of radioactivity absorbed into the carcass and tissues were 39/41% (♂/♀) at 2 h, 12/18% at 8 h, 2.7/2.1% at 24 h and 1.1% (both) at 96 h.
There were no sex-differences in concentration of radioactivity except for thyroids.
Greatest concentrations of radioactivity at all time points were found in organs of metabolism and excretion (liver, lung, kidney), vascularised tissues (spleen, thyroid, adrenals), fat, blood and plasma.

Single Oral Administration at the High Dose Level in Both Sexes (Group H):
All tissues had been exposed to radiolabelled material within 2 h of administration. Levels of radioactivity absorbed into the carcass and tissues were 74/76% (♂/♀) at 2 h, 45/54% at 8 h, 6.1/12.5% at 24 h and 1.1/1.3% at 96 h.
There were no sex-differences in concentration of radioactivity.
As in the low dose group the greatest concentrations of radioactivity at all time points were found in organs of metabolism and excretion (liver, lung, kidney), vascularised tissues (spleen, thyroid, adrenals), fat, blood and plasma.
For detail, please refer to the attached background material 1 and 2.

Key result

Test no.:

#1

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was distributed rapidly to organs of metabolism and excretion (liver, lung, kidney), vascularised tissues (spleen, thyroid, adrenals) and fat.

Details on excretion:

Following oral administration, the majority of radioactivity was excreted as volatiles (CS2, CO2) in expired air (36 - 50%) within 24 h, reaching 42 - 60% of the dose within 168 h. Excretion via urine and faeces reaches 16% and 3% respectively. Increasing the dose 10 times decreased slightly urinary excretion but increased exhalation via CS2. After repeated exposure, urinary excretion was slightly increased (21%) as well as CS2 exhalation (42 - 50%). Overall, within 168 h, 64 - 85% of the administered dose was excreted. The overall recovery in these experiments was lower than would normally be expected due to difficulty in trapping the high levels of volatiles.
For detail, please refer to the attached background material 1.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 15 mg/kg bw (males): 1.286 µg equiv./g (blood) and 0.859 µg equiv./g (plasma); 150 mg/kg bw (males): 12.88 µg equiv./g (blood) and 6.548 µg equiv./g (plasma)

Remarks:

15 mg/kg bw (females): 1.304 µg equiv./g (blood) and 0.804 µg equiv./g (plasma); 150 mg/kg bw (females): 15 µg equiv./g (blood) and 8.373 µg equiv./g (plasma)

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 15 mg/kg bw (males): 14.4 h (blood) and 6.8 h (plasma); 150 mg/kg bw (males): 33.6 h (blood) and 24 h (plasma)

Remarks:

15 mg/kg bw (females): 21.6 h (blood) and 10.4 h (plasma); 150 mg/kg bw (females): 43.2 h (blood) and 24 h (plasma)

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 15 mg/kg bw (males): 157.0 µg equiv.h/g (blood) and 21.97 µg equiv.h/g (plasma); 150 mg/kg bw (males): 1821 µg equiv.h/g (blood) and 296.1 µg equiv.h/g (plasma)

Remarks:

15 mg/kg bw (females): 194.2 µg equiv.h/g (blood) and 22.24 µg equiv.h/g (plasma); 150 mg/kg bw (females): 2348 µg equiv.h/g (blood) and 401.9 µg equiv.h/g (plasma)

Metabolites identified:

yes

Details on metabolites:

Approximately 52 - 65% of the administered dose was metabolised from which 43 - 59% were identified (see table below). The most important fraction of the test material was metabolised into volatile compounds excreted in air. These volatile metabolites, collectively accounted for ca 51% of the administered dose, and consisted of CS2, (COS) and CO2. Increasing the dose enhances slightly formation of these metabolites. In urine, independently of the dose, 3 main metabolites were detected, metabolites 4, 6 and 8. Two minor urinary metabolites were identified: dimethylamine-thiazolidine carboxylic acid (M1) and S-glucuronide (5) of dimethyldithiocarbamicacid. In faeces, low levels of Thiram were detected (0.08 - 0.18 %).

Enzymatic activity measured:

not measured

Clinical observations

At the dose level of 352 mg/kg bw rats were lethargic and suffered from acute hypothermia for approximately 12 h post dose. At the lower dose levels of 150 and 15 mg/kg bw similar, but less severe, toxicological signs were noted.

 Table 3:   Plasma and blood toxicokinetics of¹⁴C-labelled test material after single oral administration at two dose levels to male and female rat

Kinetic Parameters	Group B (5/sex; 15 mg/kg; single)				Group C (5/sex; 150 mg/kg; single)
	Plasma		Blood		Plasma		Blood
	♂	♀	♂	♀	♂	♀	♂	♀
Cmax[µg equiv./g]	0.859	0.804	1.286	1.304	6.548	8.373	12.88	15.00
Tmax[h]	6.8	10.4	14.4	21.6	24.0	24.0	33.6	43.2
T1/2[h]	33.44	38.90	170.5	196.2	57.59	65.40	170.6	234.2
AUC(0-t)[µg equiv. x h/g]	21.97	22.24	157.0	194.2	296.1	401.9	1821	2348
AUC(0-∞)[µg equiv. x h/g]	29.95	31.31	273.8	357.5	419.4	558.1	3215	5177

Conclusions:

The biokinetic behaviour of the test compound was investigated in rats in a GLP-compliant study on rats similar to OECD 417. Based on the results of exhaled volatiles as well as urinary excretion, absorption is estimated to reach 59 % within 72 h after a single low dose exposure. Absorption increases up to 81.2% after repeated low dose exposure. Oral absorption was slow and the total recoveries of radioactivity ranged, on average, from 63.27 – 64.62% for males and females of the single low dose group to 75.88 – 76.46% of the single high dose group and 74.09 – 84.93% of the repeated-dose. Radioactivity was rapidly cleared from plasma although there was evidence of cell binding. Distribution and uptake of radioactivity by tissues was rapid, with the highest radioactivity found in organs of metabolism and excretion (liver, lung, kidney), vascularised tissues (spleen, thyroid, adrenals), fat, blood and plasma. Excretion of radioactivity was rapid, the major proportion being excreted as volatiles in expired air. The remaining dose was excreted in urine and faeces and none via bile. Excretion was essentially complete within 24 h. Based on the study results, no apparent sex-related differences were noted for 14C elimination or distribution for any of the treated groups and no bioaccumulation potential was observed. The principal route of metabolism was hydrolysis to form and exhale CS2, COS and CO2 (ca 51%). Urine contained 2-dimethylamine-thiazolidine carboxylic acid, and the S-glucuronide of dimethyldithiocarbamic acid. Faeces contained thiram.

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Mar 1988 - 03 Aug 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Recovery was not in the required range.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

not specified

Deviations:

yes

Remarks:

Recovery was not in the required range. Volatile 14C-compounds were not collected.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

(14C)-labelled

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Wilmington, Massachusetts, US
- Weight: 237 - 267 g (Individual data of control group not reported)

Type of coverage:

open

Vehicle:

water

Duration of exposure:

0.5, 1, 2, 4, 10 and 24 h

Doses:

1.1, 12 and 91 mg/animal corresponding to 0.086, 0.95 and 7.25 mg/cm², respectively

No. of animals per group:

4/dose/time point

Control animals:

yes

Signs and symptoms of toxicity:

no effects

Dermal irritation:

not examined

Absorption in different matrices:

The mean absorption values (sum of dose in urine, carcass, skin at the application site and nonaccountable) at 24 h were 28.5, 30.7 and 4.89% for the 1.1, 12 and 91 mg/animal doses respectively. For details, please refer to Table 1 under "Any other information in results incl. tables".

Total recovery:

The mean total recoveries were 81.28, 74.63 and 101.1%, for the 1.1, 12 and 91 mg/animal doses respectively. For details, please refer to Table 1 under "Any other information in results incl. tables".

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.086 mg/cm²

Parameter:

percentage

Absorption:

28.5 %

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

0.95 mg/cm²

Parameter:

percentage

Absorption:

30.7 %

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

7.25 mg/cm²

Parameter:

percentage

Absorption:

4.89 %

Table 1:    Mean recovery of radioactivity from male rats following a single dermal application of (^14C)-labelled test substance
Parameter / Tissue	1.07 mg/rat						11.9 mg/rat						90.6 mg/rat
	[%] of administered dose
	0.5 h	1 h	2 h	4 h	10 h	24 h	0.5 h	1 h	2 h	4 h	10 h	24 h	0.5 h	1 h	2 h	4 h	10 h	24 h
Skin rinse	75.22	85.35	78.90	74.64	70.07	70.53	72.39	69.03	67.03	60.71	72.33	68.79	101.96	99.79	99.73	97.20	100.26	93.49
Skin cell cover	0.06	0.08	0.12	0.12	0.26	0.60	0.02	0.04	0.05	0.06	0.14	0.16	0.04	0.06	0.18	0.26	0.15	0.35
Skin enclosure	0.10	0.14	0.33	0.20	0.07	0.35	0.42	0.17	0.31	0.17	0.09	0.43	1.02	1.44	1.02	0.22	1.03	1.26
Total nonabsorbed dose	75.38	85.57	79.35	74.96	70.40	71.48	72.83	69.24	67.39	60.94	72.56	69.38	103.02	101.29	100.93	97.68	101.44	95.10
Urine	ND	ND	0.01^a	0.02	0.06^a	0.16	ND	ND	0.01	0.01	0.02	0.05	ND	ND	ND	0.01	0.01	0.01
Faeces	ND	ND/NS	ND	ND/NS	ND	0.01	ND/NS	ND/NS	ND/NS	ND/NS	ND/NS	0.01^a	ND/NS	0.01^c	ND/NS	ND/NS	ND/NS	ND
Cage wash	ND	ND	ND	ND	ND	0.02^b	ND	ND	0.01^c	ND	ND	ND	ND	ND	ND	ND	ND	ND
Cage wipe	ND	ND	0.01^a	0.01	0.01	0.01	ND	0.01^c	0.01^c	0.01^b	0.01	0.01	0.01^c	0.01^b	ND	0.01^b	ND	0.01^b
Total excreted	ND	ND	0.01	0.02	0.06	0.18	ND	0.01	0.01	0.01	0.02	0.05	0.01	0.01	ND	0.01	0.01	0.01
Carcass	ND	ND	ND	ND	0.08	0.14	ND	ND	0.01	ND	ND	0.03	ND	ND	ND	ND	ND	ND
Skin	5.63	3.18	4.57	5.05	7.88	3.79	3.04	3.48	5.67	9.93	8.86	4.46	0.56	0.93	1.11	1.48	1.44	1.52
Total absorbed dose	5.63	3.18	4.57	5.07	8.02	4.11	3.04	3.48	5.68	9.93	8.88	4.54	0.56	0.93	1.11	1.48	1.44	1.52
Total recovery	81.01	88.75	83.92	80.03	78.42	75.59	75.87	72.72	73.07	70.87	81.44	73.92	103.58	102.22	102.04	99.16	102.88	96.62
NS   Not sampled ND  Not detectable ^a      Mean of three animals ^b      Mean of two animals ^c      Mean of one animal

Table 2:    Concentations of radioactivity in blood from male rats following a single dermal application of (^14C)-labelled test substance
Dose level	µg test substance/g
	0.5 h	1 h	2 h	4 h	10 h	24 h
1.07 mg	0.002	ND	ND	ND	0.002	0.006 .006
11.9 mg	ND	ND	ND	ND	ND	ND
90.6 mg	ND	ND	ND	ND	ND	ND
ND   Not detectable

Conclusions:

The study was conduct according to GLP and similar to OECD guideline 427. Following topical application of 0.086, 0.95 and 7.25 mg/cm² of the 14C-labelled test material formulated as wettable powder to male rats, the total absorbed dose was 28.5, 30.7 and 4.89% of the applied dose, respectively after 24 h.

Reference 4

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Dec 2005 - 04 Jan 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

adopted in 2004

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-labelled (methyl moiety)

Species:

other: human skin

Sex:

male/female

Type of coverage:

unoccluded

Vehicle:

water

Duration of exposure:

6 h

Doses:

1.55 mg/mL: 9.95 µg/cells equivalent to 15.5 µg/cm²
625 mg/mL: 4114 µg/cell equivalent to 6429 µg/cm²

Control animals:

no

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Full-thickness human skin samples (3 abdomen) were obtained from human donors post-mortem from two females and one male aged 24 to 64 years old. These skin samples were supplied by the International Institute for the Advancement of Medicine. Prior to use, the skin samples were thawed to room temperature. The resulting full thickness skin membrane was swabbed with 70% v/v ethanol/water to remove residual fat and blood, wiped dry and re-hydrated with distilled water prior to dermatoming.
- Thickness of skin (in mm): 200-400 µm
- Membrane integrity check: Yes

PRINCIPLES OF ASSAY
- Diffusion cell: at least 7 diffusion cells per dose
- Receptor fluid: 0.01 M phosphate-buffered saline, supplemented with 5% w/v bovine serum albumin, adjusted to pH 7.4 was used as the receptor fluid for the barrier integrity assessment and permeability assessment of the test substance.
- Solubility of test substance in receptor fluid: yes
- Test temperature: 32°C
- Occlusion: no
- Specific activity of test substance: 11.3 MBq/mg (= 305.4 µCi/mg)
- Volume applied: 6.4 µL of each preparation (per 0.64 cm²)
- Sampling time: Receptor fluid: in hourly fractions for the duration of the experiment (24 h).
Tape strips, remaining receptor fluid and washings: at study termination (24 h).

Absorption in different matrices:

A total amount of applied material absorbed after 24 h at the high dose level was 0.02% and at the low dose level was 0.39%. For details, please refer to Table 1 und "Any other information on results incl. tables".

Total recovery:

The total recovery was 91.74% of the applied test material concentration for the low dose applied and 94.11% of the applied test material concentration for the high dose. For details, please refer to Table 1 und "Any other information on results incl. tables"

Key result

Time point:

24 h

Concentrate / Dilution:

concentrate

Dose:

15.5 µg/cm²

Parameter:

percentage

Absorption:

0.39 %

Key result

Time point:

24 h

Concentrate / Dilution:

concentrate

Dose:

6429 µg/cm²

Parameter:

percentage

Absorption:

0.02 %

Table 1       Table for in vitro percutaneous absorption study in human skin (mean of 4-6 values)
	Distribution of Radioactivity (% Applied Dose)
Samples	1.55 mg/mL	643 mg/mL
1.         Receptor fluid (0-24 h)	0.38	0.02
2.         Receptor fluid at termination	0.01	nd
3.         Skin	nd	nd
4.         Receptor chamber	nd	nd
5.         Tape (stratum corneum)	1.84	0.04
6.         Total absorbed (1+2+3+4+5)	2.23	0.06
7.         Skin swab (after 6 h)	87.82	93.95
8.         Tape (surface)	1.49	0.11
9.         Donor chamber	0.21	nd
10.      Total non-absorbed (7+8+9)	89.52	94.06
11.      Total recovery (6+10)	91.75	94.12

Conclusions:

The study was conduct according to GLP and OECD guideline 428. Following topical application of a low (1.55 mg/mL) and a high concentration (643 mg/mL) of the 14C-labelled test material to human skin, the total absorbed dose was 0.39% and 0.02% of the applied dose, respectively after 24 h.

Description of key information

In rats, the test substance is absorbed from the gastrointestinal tract approximately 59 - 81% within 48 h after oral administration. Distribution is rapid and large. Partial inactivation, by first-pass hepatic elimination, is suggested. Based on the kinetic parameters (T1/2 >24 h), it was concluded that the test substance might accumulate. High levels of radioactivity were detected in carcass, organs of metabolism and excretion as well as in high vascularised tissues. Metabolism of absorbed doses is extensive. The principle route is hydrolysis to dimethyldithiocarbamate, which further decomposed to CS2, COS and CO2 exhaled as volatile metabolites or conjugated. Following oral administration, radioactivity was essentially excreted as volatiles (CS2 and CO2) in expired air within 24 h. Excretion via urine was less compared to expired air and excretion via faeces was minor.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

66

Absorption rate - dermal (%):

5

Additional information

Toxicokinetics

Rats

Toxicokinetic behavior of the test substance was investigated in two GLP-compliant study, which were both conducted similar to OECD 417 and considered as key (Report number: 6225-106 and 514/40-D1141). After single oral and repeated-dose administration of the radiolabelled test substance to rats, the test material was absorbed from the gastrointestinal tract approximately 59 - 81% within 48 h after oral administration. Distribution was rapid and large. High levels of radioactivity were detected in carcass, organs of metabolism and excretion (liver, lung, kidney), as well as in high vascularised tissues (spleen, thyroid, adrenals), fat, blood and plasma. Metabolism of absorbed doses is extensive. The most important fraction of the test material was metabolised into volatile compounds excreted in air. These volatile metabolites, collectively accounted for ca 51% of the administered dose, and consisted of CS2, (COS) and CO2. The principle route is hydrolysis to dimethyldithiocarbamate, which further decomposed to CS2, COS and CO2 exhaled as volatile metabolites or conjugated. Low levels of the metabolite thiram were detected in faeces (0.08 - 0.18%). In urine, S-glucuronide of dimethyldithiocarbamic acid and the 2-dimethylamine-thiazolindine carboxylic acid were detected. Other metabolic products included carbon disulfide, sulfate and dimethylamine. Following oral administration, radioactivity was essentially excreted as volatiles (CS2 and CO2) in expired air within 24 h. Excretion via urine was less compared to expired air and excretion via faeces was minor. Following oral administration, the majority of radioactivity was excreted as volatiles (CS2, CO2) in expired air (up to approximately 50%) within 24 h. Excretion via urine and faeces reaches a maximum of 35% and 18% respectively. Increasing the dose 10 times decreased slightly urinary excretion but increased exhalation via CS2. After repeated exposure, urinary excretion was slightly increased as well as CS2 exhalation. The rate of elimination was relatively fast; the majority of the radioactivity was eliminated within 48 h after dosing

Goat

Two further GLP-compliant toxicokinetic studies were performed in lactating goats (Report number: WIL-223005 and HLA 6225-01). As these studies were performed in goats, assessment of toxicokinetics and the extrapolation to humans is expected to be limited. Thus, these studies are considered as supporting. In lactating goats, orally administered test material was excreted via urine (~32%) and faeces (9%). Another 1.6% was eliminated in the milk. The test material was extensively metabolized in goats. Dimethylamine was the dominating metabolite in milk, urine and bile, no parent was detected in tissues, milk or excreta.

 

Dermal Absorption

Dermal absorption was investigated in vitro on human (Report number: PFX0009) and rat skin (Report number: HLA 6225-107). Both studies were conducted according to GLP and OECD guideline (428 or 427). Following topical application of a low (1.55 mg/mL) and a high concentration (643 mg/mL) of the 14C-labelled test material to human skin, the total absorbed dose was 0.39% and 0.02% of the applied dose, respectively after 24 h. After topical application of 0.086, 0.95 and 7.25 mg/cm² of the 14C-labelled test material formulated as wettable powder to male rats, the total absorbed dose was 28.5, 30.7 and 4.89% of the applied dose, respectively after 24 h. On the basis of these results a dermal absorption of 5% for the concentrate and 30% for the diluted formulation has been derived.