Thiram

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Reference 2

Reference Type:

study report

Title:

Unnamed

Materials and methods

Objective of study:

other: Absorption, distribution, metabolism and excretion

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted in 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Thiram

EC Number:

205-286-2

EC Name:

Thiram

Cas Number:

137-26-8

Molecular formula:

C6H12N2S4

IUPAC Name:

thiram

Radiolabelling:

yes

Remarks:

(14C)-test substance

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA, USA (Experiment A), Charles River, Portage, MI, USA (Experiment B), Taminco Farms, Germantown, NY, USA (Experiment C)
- Age at study initiation: 6 – 8 weeks
- Weight at study initiation: means were 207.3 -223.8 g (females), 217.9 – 227.3 g (males), Experiment A, 209.6 – 246.9 g (Experiment B) 200 -250 g (Experiment C)
- Housing: individually in suspended stainless steel, screen-bottom cages, in metabolism cages for sample collection (Experiment A, B, C)
- Diet: certified Rodent Chow #5002 (Purina Mills, Inc, Experiment A, B, C), Agway Pro Lab 1000 (Agway, Waltham, MA, Experiment C), all ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10-11 days (Experiment A, B)

ENVIRONMENTAL CONDITIONS (from Expeirment C)
- Temperature (°C): 20 - 30
- Humidity (%): 46 - 78
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 10 Aug – 17 Aug 1987 (Experiment A), 23 Jul 1990 – 2 Oct 1990 (Experiment C)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on exposure:

Amount applied: 4.75 mL/kg bw, 1.7×107 dpm/mL

Duration and frequency of treatment / exposure:

Single application (Experiment A and B) or 14 unlabelled doses followed by one labelled dose (Experiment C)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.9 mg/kg bw/day

Remarks:

low dose, Experiment A

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day

Remarks:

high dose, Experiment A

Doses / concentrations 3

Dose / conc.:

2 mg/kg bw/day

Remarks:

Experiment B (single dose) and C (for 14 days unlabelled, followed by a single dose of labelled test material)

No. of animals per sex per dose / concentration:

5 (Experiment A)
3 (males only, Experiment B)
5 (Experiment C)

Control animals:

yes

Positive control reference chemical:

No

Details on dosing and sampling:

Experiment A:
Sampling time
Urine and faeces: 4, 8, 12, 24 h, 2, 3, 4, 5, 6 and 7 days
Blood: at sacrifice
Tissues: bone (femur), brain, fat (peritoneal), gonads, heart, intestine w/contents, kidney, liver, lung, muscle (thigh), spleen, stomach w/ contents
Method: fecel samples and tissues were homogenized in distilled water, bone samples were ground. All samples were oxidized. Radioactivity was analysed with liquid scintillation counting



Experiment B:
Sampling time for expired air (trapping solution): 0, 2, 4, 6, 8, 11, 14, 16, 24, 28, 32, 48, 52, 72, 76, 80 and 96 h
Method: liquid scintillation counting, Beckman LS 9800 liquid scintillation system
Counting time: 10 min

In experiment B, the exit gases from the metabolism cages were passed through a scrubbing tower containing Harvey Carbon-14 Cocktail, then through a solution of diethylamine/ethanol (1:1) and finally through a solution of Viles CS2 reagent. The Harvey Cocktail is a very efficient trapping agent for CO2. The chemical reactivity of the cocktail would also make it an effective trapping agent for CS2 and COS, since it is capable of forming carbamates and/or thiocarbamates with CO2, CS2 or COS.

Experiment C:
Sampling time
Urine and faeces: 4, 8, 12, 24, 32, 48, 72 and 96 h
Blood and tissues: at sacrifice (96 h after administration)
Tissues: bone (femur), brain, adipose tissues, gonads, heart, kidney, liver, lung, skeletal muscle, spleen
Expired air (trapping solution): 4, 8, 12, 24, 32, 48, 72 and 96 h


Faeces were homogenized prior to measurement. Liver, kidney, heart, lung, brain, testes and spleen samples were finely minced. Adipose tissue, skeletal muscle and ovaries were not pretreated for combustion, blood cells were vortexed. Bone marrow was flushed from the bone prior to combustion.
Method: Tracor Analytic Mark III scintillation system.

Experiment D
Urine samples analysed
From Experiment B: 24 h samples from the low (1.9 mg/kg bw) and high (125 mg/kg bw) dose
From Experiment C: 4, 8, 12 and 24 h
For analyzation, samples from animals from the same sex and dose were pooled.

Method: HPLC with a LDC/Milton Roy CM4000 pump, LDC spectroMonitor variable wavelength UV detector and a FLO-ONE radioactivity detector. The analytical column was a Baxter Burdick & Jackson C-18. (5 µm, 4.6 x 250 mm) with a Brownlee precolumn (C-18, 7 µm). Initial mobile phase was 100% phosphoric acid buffer, increased to 30% acetonitrile , increased to 100% acetonitrile.

The amount of radioactivity in the HPLC fractions was analysed with Beckman LS 9800 liquid scintillation system.

Results and discussion

Preliminary studies:

Total recoveries of radioactivity were 32.1 and 27.0% for males and females of the single low dose group, respectively, not accounting for volatile residues. In the single high dose group, the recovery (without volatiles) was 35.7% (♂) and 32.0% (♀). In the repeated-dose study (Experiment C), volatile residues were accounted for. Accordingly, the recoveries were much higher with 85.4% (♂) and 92.9% (♀) (see Table A6_2-1 in the attached background material).

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Based on the the high excretion via air and urine (total 83 - 87%), high absorption is assumed, but no specific experiments were conducted.

Main ADME results 2

Type:

excretion

Results:

[14C]-test substance-derived radioactivity was excreted in the urine (ca. 35—40% of the dose within 96 hr.), faeces (Ca. 2-5%) and expired air (ca. 47-48%). Most of the radioactivity was excreted in the first 12 hours after dosing.

Main ADME results 3

Type:

distribution

Results:

Distributions in tissues were low, the highest percentage of the dose was seen in blood (0.9-1.99 %), liver (0.4-1.8%), muscle (0.05-0.6%), bone (± 0.5%) and kidney (0.07- 0.16%).

Main ADME results 4

Type:

metabolism

Results:

The test substance is extensively and almost completly metabolized to more polar products, diethylthiocarbamic acid being formed as the principal metabolite. Other metabolic products include carbon disulfide, methyldiethyldithiocarbamate, and sulfate.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

No designated experiments were conducted to examine the absorption of the test susbtance. However, the majority of the applied radioactivity is either excreted in urine or exhaled (total 83-87%, see Table A6_2-1 in the attached background material). This demonstrates a high degree of absorption.

Details on distribution in tissues:

Four to seven days after oral exposure to single or multiple doses, trace levels of test-substance-derived radioactivity were detected in all tissues. Total recovery was low (from 1.8 to 4.2%): the highest percentage of the dose was seen in blood (0.9-1.99 %), liver (0.4-1.8%), muscle (0.05-0.6%), bone (± 0.5%) and kidney (0.07- 0.16%). All other tissues, in both sexes, contained less than 0.1% of the dose. (Table A6_2-2 under "Any other informations on results incl. tables"). This would indicate that the test substance did not accumulate in the rat upon repeated dose administration.

Details on excretion:

The majority of the dose (at least 83.7% for males and 89.6% for females) was eliminated from the body within four days following dosing (Table A6_2-1 under "Any other informations on results incl. tables").
As far as urinary and faecal excretion is concerned, the following data were obtained.

After a single dose, maximal urinary excretion occurred within 4 to 24 h and accounted for 6% and 14 % for a low or high dose respectively. Faecal excretion was low, occurring within 48 and 96 h and representing approximately 3.7% of the dose. Seven days after exposure, total urinary and faecal excretion ranged from 24 to 34% of the administered dose, mostly present in urine. There were no significant differences in total recovery between dose levels or between sexes. In these experiments, a majority of the material was unaccounted for (Table A6_2-3 under "Any other informations on results incl. tables").

After repeated oral administration of a low dose, urinary excretion became more important, approximately 33-35%. Faecal excretion was low, only 0.5% of the dose. No significant differences were found between male and female. (Table A6_2-4 under "Any other informations on results incl. tables"). While urinary excretion trends to increase with the dose, faecal excretion decreased. These effects are in agreement with the quantitative differences occurring in the metabolism of thiram suggesting saturation of a metabolic route.

Total 14C test substance-derived exhaled radioactivity over 96 h was 47.0 and 48.2% for male and female rats respectively. Exhaled radioactivity was collected in KOH traps (likely as CO2 and CS2), mainly within the first 12 hours, and in Viles CS2 traps (likely CS2), mainly excreted within the first 8 hours. The extent and rate of exhalation of 14C test substance-derived radioactivity were similar in male and female rats. These data also indicate that the test substance is rapidly metabolized by the rat. After multiple doses, exhalation is slightly lower (Table A6_2-5 under "Any other informations on results incl. tables").

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

In general, the metabolism of dialkyldithiocarbamates such as the test substance in mammals is straightforward, diethylthiocarbamic acid being formed as the principal metabolite. Other metabolic products include carbon disulfide, methyldiethyldithiocarbamate, and sulfate.
The metabolic decomposition of ethylene bisdithiocarbamates such as nabam, maneb and zineb is complex and results in the formation of carbon disulfide, hydrogen sulfide, ethylene diamine, ethylene bisthiuram disulfide, 5,6-dihydro-3H-imidazo(2,1-C)-1,2,4-dithiazole-3-thione, ethylene diisocyanate, ethylene thiourea, ethylene urea and 2-imidazoline (Table A6_2-6 under "Any other informations on results incl. tables").
The test substance is extensively metabolized in rats to more polar products. The metabolic pathway involved a reduction of the disulfide bond and subsequent reactions of the thiol moiety to form oxidative and conjugative polar products. No unchanged test substance was detected in the urine.
Among the five metabolites identified in the urine, the alanine conjugate of dimethyldithiocarbamate is one of the major metabolites, occurring both after single or repeated, and low or high dose administration.
Quantitative differences occurred for the other metabolites: dithiocarbamate thiosulfenic acid was the second major metabolite after subchronic or high dose, while in the low dose animals 2-thioxo¬thiazolidine-4-carboxylic acid was the second major metabolite.
Three additional urinary metabolites, P1, P2 and P3, were not identified. P1 and P2 constitute together less than 5% of the radioactivity present in the urine of subchronically treated rats. P3 constituted 12-21% of the radioactivity present in urine of low dose animals. None of these three were present in the urine of high dose rats.
Sexual difference occurred in the metabolism after low dose exposure: males had almost twice as much P3 as the females, whereas females had ten times more dithiocarbamate thiosulfenic acid than the males (Table A6_2-6 under "Any other informations on results incl. tables").
After subchronic exposure, a major portion of the dose (ca.47-48%) was exhaled in air. It is likely that radioactivity collected in the KOH trap was 14CO2 and/or 14CSO (carbamyl sulfide, carbon thioxide), since it has been reported that CSO is soluble in KOH. Radioactivity collected in the Viles CS2 traps is likely to be CS2, as it was reported earlier that CS2 is a metabolite of the test substance. (Table A6_2-4 under "Any other informations on results incl. tables").

Any other information on results incl. tables

Summarized results of Experiment A, B, C and D can be found in Attachment 1, 2, 3 and 4 in the attached background material, respectively. An overview of relevant results can be found below. 

 

Table A6_2-1:   Recovery of radioactivity [%] in rats following single oral administration of (^14C)-test substance*
Sample [hour]	50 ppm		500 ppm		1000 ppm
	Male	Female	Male	Female	Male	Female
Urine
0-6	8.09	17.26	11.60	25.69	12.03	15.54
6-12	0.00	3.01	2.14	7.65	4.37	0.79
12-24	5.44	2.06	2.94	3.93	3.45	4.65
24-48	0.73	0.79	0.48	1.01	0.66	0.43
48-72	0.37	0.46	0.18	0.36	0.28	0.31
72-96	0.24	0.30	0.17	0.25	0.17	0.20
Total Urine	14.87	23.88	17.51	38.89	20.96	21.93
Faeces
0-6	0.00	0.01	0.01	0.00	0.02	0.00
6-12	0.02	0.00	0.01	0.00	0.00	0.00
12-24	5.80	3.82	2.14	0.77	0.93	0.12
24-48	2.31	2.45	1.52	1.30	1.16	0.82
48-72	0.28	0.56	0.27	0.31	0.66	0.46
72-96	0.07	0.18	0.06	0.07	0.22	0.17
Total Faeces	8.47	7.00	3.99	2.45	2.99	1.57
Cage
Wipes	0.05	0.14	0.02	0.08	0.01	0.06
Ringe	1.21	3.24	1.83	1.23	2.27	5.85
Wash	0.07	0.16	0.05	0.10	0.06	0.13
Total Cage	1.33	3.54	1.89	1.40	2.34	6.04
Total Elimination	24.66	34.42	23.38	42.74	26.28	29.54
Tissue
Muscle	0.28	0.56	0.10	0.37	0.24	0.25
Fat	0.04	0.08	0.03	0.04	0.03	0.03
Lung	0.03	0.05	0.02	0.04	0.02	0.02
Spleen	0.01	0.01	0.01	0.02	0.01	0.01
Kidney	0.11	0.13	0.06	0.08	0.06	0.06
Liver	0.97	0.87	0.55	0.50	0.55	0.62
Stomach	0.02	0.03	0.02	0.02	0.01	0.00
Stomach content	0.03	0.06	0.03	0.05	0.03	0.04
Intestine	0.01	0.01	0.00	0.00	0.00	0.00
Intestine content	0.02	0.03	0.02	0.03	0.08	0.07
Blood	0.85	1.35	0.73	1.55	0.74	0.91
Total Tissue	2.35	3.15	1.54	2.68	1.76	2.01
Total Recovery**	27.01	37.56	24.92	45.42	28.04	31.55
*   Data are presented as group mean values with n = 2 for each group except females - 1000 ppm where n =1 ** Total recovery does not include volatile^14C-residues in expired air which was not collected in this study.

 

Table A6_2-2:    Tissue distribution of radioactivity from ^14C-test substance
dose - mg/kg bw (frequency)	1.9 (1 x)		2 (15 x)		125 (1x)
day of sacrifice after treatment	7	7	4	4	7	7
sex	♂	♀	♂	♀	♂	♀
bone	0.55	0.48	-	-	0.476	0.42
blood	0.975	1.632	-	-	1.236	1.99
plasma + blood cells	-	-	0.04+0.5	0.04+1.43	-	-
brain	0.0075	0.0076	0.01	0.01	0.01	0.01
fat	0.015	0.0084	0.06	0.08	0.014	0.022
gonads	0.01	0	0.01	0.00	0.01	0
heart	0.03	0.045	0.02	0.03	0.016	0.014
intestine + content	0.097	0.127	-	-	0.072	0.078
kidney	0.127	0.165	0.07	0.10	0.048	0.068
liver	1.862	1.479	0.69	0.94	0.44	0.376
lung	0.065	0.077	0.03	0.04	0.03	0.038
muscle	0.087	0.136	0.39	0.58	0.05	0.11
spleen	0.01	0.01	0.01	0.03	0.008	0.018
stomach + content	0.032	0.065	-	-	0.016	0.024
total	3.867	4.23	1.83	3.27	2.42	3.168

 

Table A6_2-3:    Mean urinary and faecal excretion as a function of time after a single low or high dose
Dose [mg/kg bw]	Urine				Faeces
	1.9		125		1.9		125
Sex	♂	♀	♂	♀	♂	♀	♂	♀
Time [h]
0	0	0	0	0	0	0	0	0
4	5.46	6.48	0.95	1.07	0.13	0	0	0.188
8	3.6	3	0.58	0.95	0.022	0	0	0
12	3.8	2.7	2.21	2.27	0.19	0.465	0.16	0.168
24	2.4	2.8	10.8	8.65	1.18	1.01	0.204	0.28
48	2.2	1.19	10.4	8.12	1.56	1.62	0.77	0.492
72	0.84	0.79	2.31	3.4	0.54	0.49	1.08	0.244
96	0.66	0.42	0.86	1.11	0.25	0.482	0.56	0.606
120	0.48	0.5	0.7	0.62	0.18	0.288	0.39	0.3
144	0.26	0.47	0.85	0.33	0.114	0.354	0.35	0.238
168	0.17	0.44	0.59	0.43	0.128	0.242	0.29	0.278
total	19.87	18.79	30.25	26.95	4.3	4.95	3.8	2.79

 

 

Table A6_2-4:          Excretion of radioactivity after 2 doses: comparison with excretion after 1 dose
Dose [mg/kg bw]	Urine						Faeces
	1.9			125			1.9		125
Sex		♂	♀		♂	♀	♂	♀	♂	♀
Time [h]
0		0	0				0	0
4		5.46	6.48		16.3	16.4	0.13	0	0.1	0
8		3.6	3		6.4	6.4	0.022	0	0	0
12		3.8	2.7		4.2	4.4	0.19	0.465	0	0.3
24		2.4	2.8		4.2	4.4	1.18	1.01	0.3	0.2
48		2.2	1.19		0.6	0.6	1.56	1.62	0	0.1
72		0.84	0.79		0.9	1.4	0.54	0.49	0.9	1.8
96		0.66	0.42		0.4	0.9	0.25	0.482	0.3	0.8
120		0.48	0.5		0.3	0.6	0.18	0.288	0.9	2.1
144		0.26	0.47				0.114	0.354
168		0.17	0.44				0.128	0.242
total		19.87	18.79		33.3	35.2	4.3	4.95	2.6	5.3

 

Table A6_2-5:          Exhalation of volatile ^14C [% of administered dose]
Dose [mg/kg bw]	1.9 (2 rats)	2.0
frequency	1 x	15 x
Time [h]
2	9.08
4	16.31	24.3
6	9.79
8	6.13	15.6
11	7.98
14	3.78
16	1.01
24	4.10	1.7
28	1.105
32	0.605	0.3
48	1.165
52	0.25
56	0.095
72	0.395	0.3
76	0.115
80	0.085
96	0.337	0.2
total	62.86	47.0

 

Table A6_2-6:          Metabolites of ^14C-test substance in rat urine [% of radioactivity in urine]
Dose [mg/kg bw]	1.9		125		2 (x 15)
Sex	♂	♀	♂	♀	♂	♀
Metabolites
2-thioxothiazolidine-4-carboxylic acid	33.6	30.2	12.6	7.8	2.4	2.1
dimethyldithiocarbamate glucuronide	3.2	4.2	5.6	8.7	6.6	6.9
dimethyldithiocarbamate thiosulfenic acid derivative	0.9	9.1	37.3	42.4	44.4	48.1
dimethyldithiocarbamate ethyl ester	ND	ND	3.4	2.4	0.8	0.8
dimethyldithiocarbamate alanine	34.8	42.1	33.9	36.5	41.8	38.3
P1 unknown	ND	ND	ND	ND	1.3	1.8
P2 unknown	ND	ND	ND	ND	2.8	2.1
P3 unknown	20.8	12.2	ND	ND	ND	ND

Applicant's summary and conclusion

Conclusions:

The test substance, studied at doses between 2 and 125 mg/kg bw in the rat, is well absorbed from the GI tract. It is eliminated, for most part within 24 hours, mainly via urine (33%) and exhaled air (47%), and does not accumulate in the organism. 96 hours after dosing, the highest tissue levels are found in blood, liver, muscle, bone and kidney, representing about 3% of the dose administered. It is extensively metabolised, ~85% of the administered dose was detected as metabolites in urine, faeces (5%) and expired air. The metabolic pathway involves a reduction of the disulfide bond and subsequent reactions of the thiol moiety to form oxidative and conjugative polar products. No unchanged test substance was detected in the urine.
In general, the metabolism of dialkyldithiocarbamates such as the test substance in mammals is straightforward, diethylthiocarbamic acid being formed as the principal metabolite. Other metabolic products include carbon disulfide, methyldiethyldithiocarbamate, and sulfate.