Disulfiram

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted in 2010

Principles of method if other than guideline:

35S disulfiram (DSF), 7 mg/kg bw, was administered as a single dose to rats both orally (p.o.) or intraperitoneally (i.p.).

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

35S-disulfiram

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Madison, Wisc., USA
- Age at study initiation: young adult
- Weight at study initiation: 150 - 250 g
- Fasting period before study: 24 h prior to drug administration and until 4 h after drug administration
- Housing: groups of five in stainless steel wire-bottomed cages
- Individual metabolism cages: yes
- Diet: Purina Chow, ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: both, orally or i.p.

Vehicle:

other: The 35S-disulfiram was solubilized in polysorbate 80 and suspended in 0.5% methyl cellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 35S-DSF was solubilized in polysorbate 80 and suspended in 0.5% methyl cellulose.

Duration and frequency of treatment / exposure:

single dose

Dose / conc.:

7 mg/kg bw (total dose)

Remarks:

either i.p. or p.o.

No. of animals per sex per dose / concentration:

3 animals per timepoint

Control animals:

no

Positive control reference chemical:

No

Details on dosing and sampling:

Tissue analysis
Rats were given 35S-DSF in a dose of 7 mg/kg bw either i.p. or p.o., and then sacrificed by decapitation at 0.5, 2, 4, 6, 12, 24, and 48 hr following drug dosing. Exsanguinated blood was collected in citrate solution to prevent clotting. Tissue samples were removed, rinsed, blotted dry, und weighed. Tissues analyzed were liver, kidney, muscle, spleen, lung, stomach, testes, pancreas, thyroid, adrenals, brain, heart, skin, intestinal tract, and adipose layer. Three animals were used at each time period studied for each route of 35S-DSF administration. Approximately 100-300 mg of tissue sample was digested in a mixture of 0.8 mL of 70% perchloric acid and 0.4 mL of 30% hydrogen peroxide at 70ºC for 1 hr. Samples were allowed to cool to room temperature and then 20 mL of a triton X-100 scintillation cocktail was added. Samples were counted on a Beckman Scintillation counter (Model 1650). Appropriate corrections for quenching were made.

Gastrointestinal Tract Analysis
In a separate study 35S-DSF was given in a dose of 7 mg/kg bw either i.p. or p.o. and the animals then were immediately placed into stainless steel metabolism cages. The rats then were sacrificed by decapitation at 0.5, 1, 2, 4, 6, 12, 24, and 48 hr following drug administration. The intestinal tract plus contents (minus stomach) was removed and homogenized in a polytron homogenizer with 24 mL of 0.01 g EDTA/1% sodium chloride buffer, pH 8,5, and 6 mL of dimethyl sulfoxide (DMSO).


Breath, Urine, and Faeces Analysis
For the simultaneous collection of breath, urine, and faeces, animals were housed in a modified Roth-Delmar metabolism cage. The chamber was continuously vented with CO2-free air at a constant rate. Carbon disulfide in the expired air was collected by bubbling the air through a double trapping system containing a modified Viles reagent. Nonradioactive CS2 was determined spectrophotometrically. Radioactive CS2, was determined by taking 1 mL aliquots of the trapping solution and placing it in 15 mL of the triton X-100 scintillation cocktail and counted. Urine and faeces were collected in the metabolism cages separately. Aliquots of urine (50-100 mL) and faeces (200-300 mg) were treated similarly to the tissue samples and total radioactivity determined.

Disulfiram Metabolite Studies
Tissue analysis.
The time period at which the greatest radioactivity was found for each route of 35S-DSF administration was determined. One hour after i.p. and 5 hr after p.o. administration were the time periods selected. The 35S-DSF then was administered, and the rats sacrificed by decapitation 1 and 5 hr after dosing. Exsanguinated blood was collected in a citrate solution to prevent clotting. Tissues were excised and 35S-DSF metabolites determined as described previously.
Urine analysis.
A group of rats were given 35S-DSF i.p. and p.o., and immediately placed in stainless steel cages. Urine was collected for 48 hr and 35S-DSF and 35S metabolites determined.

Type:

absorption

Results:

The 35S DSF was rapidly absorbed (at least 80%) by either route.

Type:

distribution

Results:

Kidney, pancreas, liver, and the gastrointestinal tract exhibited the greatest uptake of radioactivity, while the least was found in brain. Preferential tissue uptake was similar with both routes of administration.

Type:

metabolism

Results:

The 35S-DSF was rapidly metabolized to the 35S-diethyldithiocarbamate-glucuronide and 35S inorganic sulfate.

Type:

excretion

Results:

7% of the dose was excreted in the feces. Approximately 12% of the dose was eliminated by the breath as CS2. Most of the radioactivity is eliminated after 48 hr.

Type:

other:

Results:

Approximately 93% of the radioactivity was accounted for 48 hr after p.o. or i.p. 35S administration.

Details on absorption:

48 h after i.p injection of the test material, 93.21% of the radioactivity could be recovered. 6.73% of the radioactivity were excretet via feces and 5.25% were found in the gastrointestinal tract (+ contents). 66.1% were found in the urine, 12.64% in the exhaled air and 2.47% in tissues. Therefore, the absorption was at least 81.2% (Table 3).

48 h after oral administration of the test material, 92.36% of the radioactivity were recovered. 6.52% of the radioactivity were excretet via feces and 5.10% were found in the gastrointestinal tract (+ contents). 66.82% were found in the urine, 11.00% in the exhaled air and 2.91% in tissues. Therefore, the absorption was at least 80.7% (Table 4).

Details on distribution in tissues:

Radioactivity in the various tissues investigated peaked between 0.5 and 1 hour after i.p. (Table 1) and between 4 and 6 hr following p.o.35S-DSF administration (Table 2). Greatest uptake after i.p. dosing was found in kidney > pancreas > liver > largo intestine > small intestine > fat, while tissues exhibiting the least uptake were brain < muscle < heart < adrenal gland < testes. Generally, the p.o. route led to an uptake pattern similar that observed after i.p.35S-DSF. Only in the stomach was uptake greater after p.o.35S-DSF as compared to the i.p. route, which is not surprising since the stomach may not have been completely emptied. Furthermore, if uptake into tissues was compared either 1 hr after i.p. or p.o35S-DSF, or 6 hr after i.p. or p.o.35S-DSF, the rank order For each tissue uptake was similar. The dpm/g for each tissue studied after p.o.35S-DSF was found to be approximately 1/2 that observed after i.p. administration, reflecting greater bioavailability after i.p. dosing. After 48 hrs, approximately 93% of the dose of35S-DSF administered could be accounted for regardless of the route of administration.

Total tissue radioactivity 0.5 hr after i.p. 35S-DSF corresponded to approximately 23% of the total dpm administered, whereas 6 hr after p.o. 35S-DSF the tissues accounted for only about 10% of the radioactivity. After p.o. 35S-DSF, radioactivity in the tissues gradually increased, reflecting stomach emptying and drug absorption, and then declined at the same rate as that observed after i.p. administration.

Details on excretion:

Approximately 12% of the dose was eliminated as CS2. After either i.p. or p.o. dosing, radioactive CS2in breath was found within 2 hr. No urine was excreted for the first 4 hr after each route of administration.
After i.p. administration, total radioactivity in the tissues as the percent of35S-DSF administered declined in a biexponential manner, After p.o.35S-DSF, tissue radioactivity gradually increased peaking within 6 hr, The plasma decline was similar after both routes of administration. Radioactivity found in urine during the first 6 hr was less after p.o. than after
i.p. administration. This is due to the i.p. route being absorbed more rapidly und metabolized to water soluble metabolites that are subsequently excreted in the urine.

Metabolites identified:

yes

Details on metabolites:

One and 6 hr after i.p. and p.o. dosing respectively, the greatest amount of35S in the tissues was due primarily to35S-DDTC-glucuronide and35S inorganic sulfate. These water soluble metabolites constituted approximately 80% of the radioactivity regardless of the route of administration. An exception to this was the pancreas and fat in which only approximately 19% of the radioactivity was due to the glucuronide and inorganic sulfate after i.p. dosing, while after p.o. administration approximately 50% was due to these water soluble metabolites. Also, more parent35S-DSF was found in fat tissue after i.p. than after oral administration. Greater amounts of35S-DSF also were found in the pancreas and fat after i.p. than after p.o. 35S-DSF.
lncreased35S in the kidney, liver, and intestinal tract appears to be due to water soluble metabolites. Even though the testes, adrenal gland, heart, muscle, and brain have a high blood flow, the rapid metabolism of 35S-DSF precludes the uptake of polar metabolites into tissue.

Table 1. Distribution of³⁵S after i-p.³⁵S-Disulfiram (% of³⁵S-Disulfiram administered)

Tissue	Time (hr) after administration
	0.5	1.0	2.0	4.0	6.0	12.0	24.0	48.0
Thyroid	0.0024	0.0028	0.0022	0.0022	0.0019	0.0018	0.0014	0.0017
Adrenals	0.0156	0.0054	0.0060	0.0041	0.0041	0.0025	0.0020	0.0014
Stomach	1.2259	0.3688	0.2521	0.2738	0.2841	0.3237	0.1826	0.1436
Muscle*	8.2965	4.5871	4.1079	3.7431	3.3109	2.1308	1.4491	1.0880
Brain	0.0627	0.0459	0.0459	0.0437	0.0442	0.0296	0.0222	0.0190
Liver	6.4349	2.8972	2.2768	2.3615	1.6110	1.0060	0.5057	0.3260
Testes	0.5389	0.4996	0.3162	0.3095	0.2646	0.1765	0.0972	0.0693
Kidney	1.6317	0.9493	0.6855	0.6102	0.5015	0.3160	0.1705	0.1131
Lung	0.4634	0.3053	0.3542	0.2506	0.2056	0.1227	0.0759	0.0704
Spleen	0.3851	0.1894	0.0979	0.0726	0.0606	0.0456	0.0284	0.0161
Heart	0.0972	0.0744	0.0637	0.0564	0.0479	0.0320	0.0179	0.0144
Blood**	3.8653	2.9395	3.5082	3.3053	2.7393	1.5999	0.9502	0.6005
Total	23.0296	13.0363	11.7166	11.0330	9.0724	5.7771	3.503	2.4658

Rats were given 7 mg/kg³⁵S-disulfiram. i.p. and sacrified at the times indicated. Tissues were excised and radioactivity determined. Each value is the average of three rats.

* based on 40% of body weight

** based on 7% of body weight

 

 

 

Table 2: Distribution of³⁵S after p.o.³⁵S-Disulfiram (% of³⁵S administered)

Tissue	Time (hr) after administration
	0.5	1.0	2.0	4.0	6.0	12.0	24.0	48.0
Thyroid	0.0005	0.0005	0.0011	0.0015	0.0013	0.0010	0.0007	-
Adrenals	0.0008	0.0014	0.0029	0.0035	0.0017	0.0015	0.0010	0.0015
Pancreas	0.0537	0.0480	0.0465	0.056 1	0.1214	0.0463	0.0384	0.0268
Stomach	2.2113	0.9214	0.9082	1.1354	0.3621	0.2813	0.1616	0.1446
Muscle*	0.5244	1.3940	2.2860	2.8859	3.2715	1.8280	1.4048	1.0915
Brain	0.0058	0.0114	0.0150	0.0273	0.0405	0.0244	0.0251	0.0196
Liver	0.9807	1.8321	1.4673	2.2578	2.2330	0.9998	0.6483	0.4160
Testes	0.0172	0.0696	0.0989	0.1709	0.2614	0.1268	0.1030	0.0739
Kidney	0.2639	0.4171	0.4626	0.6616	0.7426	0.3501	0.1879	0.1331
Lung	0.0467	3.1392	0.1233	0.1782	0.2264	0.0987	0.0790	0.0539
Spleen	0.0217	0.0301	0.0291	0.0462	0.0670	0.0401	0.0400	0.0232
Heart	0.0224	0.0224	0.0278	0.0427	0.0562	0.0357	0.0226	0.0154
Blood**	0.8803	1.0310	1.4400	2.0520	2.9300	1.7698	1.2116	0.9135
Total	5.0771	5.9182	6.8963	9.5184	10.3169	5.6038	3.9246	2.9123

Rats were given 7 mg/kg³⁵S-disulfiram. i.p. and sacrified at the times indicated. Tissues were excised and radioactivity determined. Each value is the average of three rats.

* based on 40% of body weight

** based on 7% of body weight

Table 3: Distribution and Excretion of Radioactivity after i.p.³⁵S-Disulfiram (% of³⁵S-Disulfiram administered, mean values)

 

	Time (hr) after administration
	12	24	48
Diverse tissues	5.78	3.5	2.47
Gastrointestinal tract	10.38 (5)	4.74 (5)	5.25 (4)
Urine	32.6 (5)	48.64 (5)	66.10 (4)
Faeces	-	-	6.75 (3)
Breath	10.34 (5)	10.97 (5)	12.64 (3)
Total			93.21

Number of rats in parenthesis

 

Table 4: Distribution and Excretion of Radioactivity after p.o.³⁵S-Disulfiram (% of³⁵S-Disulfiram administered, mean values)

 

	Time (hr) after administration
	12	24	48
Diverse tissues	5.60	3.92	2.91
Gastrointestinal tract	12.30 (5)	8.86 (5)	5.10 (4)
Urine	45.24 (5)	59.48 (5)	66.82 (4)
Faeces	-	-	6.52 (4)
Breath	8.82 (2)	10.70 (4)	11.00 (4)
Total			92.36

Number of rats in parenthesis

 

 

Conclusions:

The studies were conducted similar to guidelines and under GLP conditions. The test substance was excreted mainly via urine and expired air. Kidney, pancreas and liver were the tissues which contained the highest concentration of 14C-test substance residues. The 35S-DSF was rapidly metabolized to the 35S-diethyldithiocarbamate-glucuronide and 35S inorganic sulfate. No bioaccumulation potential was assumed based on the study results, as most of the radioactivity was excreeted after 48 h.

Description of key information

Disulfiram is well and rapidly absorbed from the gastro intestinal tract. Based on physico-chemical data and information from the analogue substance thiram dermal absorption is rather low (≤10%). Disulfiram and its metabolites is widely distributed throughout the body in the lipids of the various tissues and is rapidly degraded by the rats to more polar products. The majority of the dose is eliminated from the body within 3 days after dosing either via urine or the expired air, and does not accumulate in the organism.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

80

Additional information

Disulfiram (tetraethylthiuramdisulfide, TETD) has been used extensively in the treatment of alcohol abuse for several decades.

It causes a blockade of the enzyme aldehyde dehydrogenase with consequently increasing levels of acetaldehyde upon ethanol consumption. The unpleasant clinical signs are known as Disulfiram-ethanol reaction (DER).

Therefore, relatively detailed toxicokinetic data for disulfiram in both, rat and man has been published as peer-reviewed literature.

Absorption

Disulfiram was found to be absorbed in a proportion of 70-90% of the administered dose upon oral administration in rats (Saint Blanquat, 1976). In rats,³⁵S-disulfiram was rapidly absorbed by either route, upon oral or intraperitoneal administration (Faiman, 1980).

 

Regarding dermal absorption no data is available for disulfiram. On the basis of the following considerations, the dermal absorption of disulfiram is considered to be rather low. Regarding the molecular weight of 296.4 g/mol, the octanol/water partition coefficient of 3.6 in combination with the low water solubility of 4.09 mg/L, a low dermal absorption rate is anticipated. Based on the molecular weight absorption may occur (ECHA, 2012). Log Pow values between 1 and 4 (optimal 2-3) favour dermal absorption, particularly if water solubility is high (ECHA, 2012), which is not the case for disulfiram. A QSAR calculation with DERMWIN (v2.01) supports this assumption. Based on the physico-chemical properties, low dermal absorption was predicted (10% absorption). This is in concordance with the decision for thiram, which is similar to disulfiram in structure and physico-chemical properties, whereas default value of 10% has been set by the European Commission as result of the evaluation of thiram by RMS Belgium according to Directive 91/414/EEC. This is further supported from an acute dermal toxicity study with disulfiram, where no systemic effects and no mortality were observed up to the limit dose of 2000 mg/kg bw, which is in contrast to the results after oral application (LD50_oral= 500 mg/kg bw).

 

Disulfiram has a very low vapour pressure of <0.0001 Pa at 20 °C thus being of low volatility. Therefore, under normal use and handling conditions, inhalation exposure and thus availability for respiratory absorption of the substance in the form of vapours is not significant.

However, the substance may be available for respiratory absorption in the lung after inhalation of aerosols or dusts. In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Among this inhalable fraction, only about 1.6% are ≤ 10 µm in diameter and belong to the thoracic fraction that reaches the tracheo-broncheal region. Only particles below 4 µm are likely to reach the alveolar region where absorption via lung can occur. Only about 0.02% of the airborne disulfiram particles belong to this fraction. Inhaled particles > 4 µm are eventually coughed out or swallowed which can then be treated as oral exposure.

Thus, absorption after inhalation of disulfiram is considered to be as high as after direct oral ingestion.

 

Distribution

Kidney, pancreas, liver, and the gastrointestinal tract exhibited the greatest uptake of radioactivity, while the least was found in brain. Preferential tissue uptake was similar with both routes of administration, oral and intraperitoneal (Faiman, 1980). Radioactivity in the various tissues investigated peaked between 0.5 and 1 hour after i.p. and between 4 and 6 hr following p.o.³⁵S-disulfiram administration. Disulfiram, diethyldithiocarbamate (DDTC), and CS₂are widely distributed throughout the body in the lipids of various tissues, and highest levels of these compounds are found in skeletal muscle (Peachey, 1981).

 

Metabolism

In rats³⁵S-disulfiram was rapidly metabolized to the³⁵S-diethyldithiocarbamate-glucuronide and³⁵S inorganic sulfate. The main metabolite DDTC appeared already in the gut (Saint Blanquat, 1976).

Upon absorption, disulfiram is immediately reduced to DDTC when it reacts with thiol groups. DDTC is metabolized to diethylamine, carbon disulfide (CS₂), DDTC methyl ester, DDTC glucuronide, and DDTC sulphate; a small amount of DDTC is reoxidized to disulfiram (Peachey, 1981). DDTC is a potent copper chelator, and it can thereby affect the activity of copper-dependent enzymes such as monooxygenases, amine oxidase, cytochrome oxidase, microsomal carboxylesterase, and plasma cholinesterase (Gaval-Cruz, 2009). In human blood, concentrations of zero to 0.6 µg carbon disulfide and 0.2 to 1.0 µg diethyldithiocarbamate per mL blood were found upon oral uptake of 200 mg disulfiram (Sauter, 1976).

It was suggested that the metabolite DDTC-methyl ester actually may be the metabolite of disulfiram which produces the disulfiram-ethanol reaction. It is proposed the reaction be more correctly identified as the DDTC-Me-Ethanol Reaction or D-MER (Yourick and Faiman, 1987).

 

Excretion

48 hours after oral administration of³⁵S-disulfiram, 7% of the dose was excreted in the faeces and 67% via urine and approximately 11% of the dose was eliminated by the breath as CS₂(Faiman, 1980).

In humans over 90% of orally administered disulfiram is eliminated within 3 days via the urine principally as DDTC and DDTC gluruconide and to a small extent as DDTC sulphate, and via the breath exclusively as CS₂(Peachey, 1981).

Considering the likeness of the toxicokinetic characteristics of disulfiram and thiram (CAS# 137 -26 -8) it is possible to use thiram as a surrogate substance in a read-across on several endpoints. (For detailed information on the justification of read-across, please refer to the analogue justification document attached in IUCLID section 13). Thiram is well and rapidly absorbed from the gastrointestinal tract. The majority of the dose is eliminated from the body within four days after dosing either via urine (~34%) or the expired air (~48%), and does not accumulate in the organism (Banijamali et al, 1990). The highest tissue levels of thiram are found in blood and liver, independent of the administered dose and the elapsed time. Thiram is rapidly degraded by the rats to more polar products. The urine, which held ca. 30% of the [¹⁴C]-thiram-derived radioactivity, contained virtually no unchanged [¹⁴C]-thiram. Five urinary metabolites were detected and isolated by HPLC and identified by mass spectrometry (Gay et al., 1991).The same metabolites were described for disulfiram by Strome, 1963.