"Materials

Hexachloro-1,3-butadiene (at least 98%) was obtained from Aldrich-Chemie (Steinheim, Germany) and E. Merck (Darmstadt, Germany), d-limonene from Sigma Chemical Co. (St Louis, MO, USA), mercuric chloride (at least 99.5%) from E. Merck, tetrachloroethylene (99.9 + %, HPLC grade) from Aldrich Chemical Co. Ltd (Dorset, UK), trichloroethylene (99 + %, spectrophotometric grade) from Aldrich Chemical Company, Inc. (Milwaukee, USA) and l,l,2-trichloro-3,3,3-trifluoro-1-propene from ABCR GmbH & Co. (Kalsruhe, Germany). Free synthetic lysinoalanine.2HCl was synthesized in the former Organic Synthesis section of our Institute, following the directions of Okuda and Zahn (1965). Corn oil was obtained from Remia (Den Dolder, The Netherlands)."

"Second study

Administration route. The test compounds were dissolved in corn oil, either alone or in combination, and administered by oral gavage in a volume of 10 ml/kg body weight, once daily for 32 consecutive days. Concurrent controls were dosed with corn oil only. The dose volumes were adjusted twice per wk for changes in body weight. The dosing solutions were prepared shortly before the start of the study, divided over daily portions, and stored at 4°C until use. Stability of the test compounds under these conditions was confirmed by analysis.

Study design.

On arrival the animals were randomly allocated to one of 15 groups of 10 (vehicle controls) or five female rats each. After an acclimatization period of 16 days, treatment was started. The dose levels (selected on the basis of range-finding studies with the individual compounds) are given in Table 2. The four nephrotoxicants were given either alone at their NONEL or LONEL, or in combinations of four at their NONEL or one-half of their LONEL, or in the four possible combinations of three at one-third of their LONEL. For each compound, the NONEL and LONEL were selected such that the NONEL was one-fourth of the LONEL, to enable comparison of the effect of combined exposure to four different compounds at their respective NONEL values with the effect of exposure to four times the NONEL ( = the LONEL) of a single compound. When full additivity is assumed, the effects of the combinations at the NONEL or 1/3 LONEL and those of the single compounds at their LONEL are expected to be the same."

"Table 2. Treatments and dose levels in a 32-day oral toxicity study testing the additivity assumption for combinations of similarly acting nephrotoxicants

Treatment

Control: corn oil 10 ml/kA

Individual compounds at NONEL

TETRA 600 mg/kg

TRI 500 mg/kg

TCTFP 1.5 mg/kg

HCBD 1 mg/kg

Individual compounds at LONEL

TETRA 2400 mg/kg

TRI 2000 mg/kg

TCTFP 6 mg/kg

HCBD 4 mg/kg"

Female Crl:(WI)WU BR Wistar Rats