Metabolism and environmental fate

Radiolabels

Three different ¹⁴C compounds were used. One labelled in the ring carbons of the 4-ethylphenyl ring, referred to as "A-ring labelled", the second labelled in the ring atoms of the 3,5-dimethylphenyl ring and referred to as "B-ring labelled" and the third with the central carbon of the tert-butyl group labelled and referred to as "t-butyl labelled".

In studies where it was possible or likely that the entire molecule would be metabolized all three labelled compounds were used to allow for the isolation and identification of fragments of the parent compound. In other studies only one label was used.

Animal Metabolism

The metabolism of tebufenozide has been studied in rats, lactating goats, and laying hens. In each case the test material was administered orally. All the studies included test material labelled in all three positions.

Rats. The metabolism of tebufenozide in male and female rats was studied using a single high dose (250 mg/kg), a single low dose (3 mg/kg), or a pulse dose (3 mg/kg) given after dosing for two weeks with unlabelled test material. The low-dose study was with all three radiolabels, the high-dose study with the B-ring and t-butyl labels and the pulse dose study with only the t-butyl label. Each test group consisted of 5 male or 5 female rats. Pooled samples of urine and faeces from each group, each containing >95% of the excreted radioactivity, were extracted and analysed (Hawkins et al., 1992b).

For the determination of metabolites samples from the 5 animals in each test group were combined for analysis. After dosing, urine and faeces were collected at approximately 24-hour intervals for 7 days.

Almost all the radioactivity (94-104%) in all test groups was eliminated in the faeces. Low levels (0.5-8.0%) were excreted in the urine and only traces of radioactivity were left in the carcase, blood and tissues or excreted as carbon dioxide or other volatile compounds.

The extent of metabolism in the high- and low-dose groups was found to be highly dependent on the dose. When fed a nominal dose of 250 mg/kg, <4% of the dose was metabolized. When fed a nominal dose of 3 mg/kg about 46% was metabolized.

No qualitative differences in metabolism between the different labelled compounds were seen in the faeces samples from either high or low doses where multiple labels were used. Most of the activity in the high-dose faeces was due to parent tebufenozide. In the low-dose samples the parent accounted for 35-43% of the dosed activity. In addition to the parent compound a total of eleven metabolites were identified in the high-dose faeces samples: RH-6595, RH-1788, RH-9886, RH-2631, RH-9871, RH-0126, RH-0282, RH-0897, RH-120898, RH-0875, and RH-0970. All of these metabolites except RH-0970 were also found in the low-dose faeces samples together with four others, RH-2703, RH-3065, RH-2777, and RH-2778, which were not identified in the high-dose samples. The structures of the metabolites are shown in Figure 1.

In the pulse dose study, male and female rats were dosed with 30 ppm of unlabelled compound in the diet for two weeks before administration of a single 3 mg/kg dose of labelled tebufenozide. The metabolism in these rats was qualitatively the same as in the rats receiving only a low single dose of the test material, but higher levels of the more highly oxidized metabolites were observed (Hawkins et al., 1993).

The faeces samples showed a similar metabolic profile to the low-dose samples, but they did not contain RH-9871 or RH-2703 and contained one additional metabolite, RH-122652. The parent compound accounted for 26-39% of the dose.

Figure 1. Proposed metabolic pathways of tebufenozide in rats.

No tebufenozide was found in the urine nor were several of the less polar faecal metabolites, but eleven of the metabolites identified in the faeces were also found in the urine, together with one additional whole-molecule metabolite, RH-2652. Although no differences were observed between the metabolites in the faeces produced from the three different labelled compounds the same was not true of the metabolites in the urine samples; small amounts of cleavage products containing only the A-ring or only the B-ring label (none greater than 1.5% of the dosed activity) were also seen but not identified. Some compounds with the B-ring label were similar in chromatographic behaviour to some of the whole-molecule metabolites.

As can be seen from Figure 1, all identified metabolites result from oxidation of the alkyl substituents of the aromatic rings of tebufenozide, particularly at the carbons adjacent to the rings. On the A-ring side of the molecule, this can result in either a secondary alcohol or a ketone, or an acid produced by oxidation of the terminal carbon of the ethyl group. On the B-ring side there are two positions which can be oxidized to combinations of alcohols acids.

Table 1. Tebufenozide and its metabolites in the combined urine and faeces of rats (Hawkins et al., 1992, 1993).

Compound (see Figure 1)	% of 14C in dose
	High-dose1		Low-dose1		Pulse dose2
	Male	Female	Male	Female	Male	Female
Tebufenozide	96.6	99.7	43.5	34.6	39.3	26.1
RH-6595	0.48	0.40	2.5	2.8	0.6	0.8
RH-2652	0.002	0.12
RH-1788	0.19	0.31	0.58	0.8	1.7	2.0
RH-2631	0.11	0.1	10.0	1.0	3.5	3.8
RH-9886	0.19	0.28	1.04	0.99	0.2	0.26
RH-2703			0.26		0.44	0.18
RH-0126	0.22	0.03	3.83	3.3	5.7	4.5
RH-0282	1.4	1.1	2.64	13.9	11.1	16.3
RH-0875	0.06	0.11	3.8	0.1	0.57
RH-9871	0.14	0.38	1.74	1.54	0.84	1.23
RH-0897	0.07	0.03	1.81	0.12	3.35	0.34
RH-120898	0.04		2.2	3.1	6.1	15.2
RH-2778		0.003	0.11	8.3	3.5	2.7
RH-0970	0.4	0.5
RH-2777	0.006		0.41	0.34	0.39	10.9
RH-3065	0.02		1.63	1.05	2.49	0.41
RH-122652					1.24	4.41
Unknowns3	0.19	0.14	1.1	6.6	3.0	4.7
TOTAL	100.1	103.2	77.1	78.5	84.0	93.8

¹ Hawkins et al., 1992
² Hawkins et al., 1993
³ Only whole-molecule compounds