A review of nonclinical toxicology studies of becaplermin (rhPDGF-BB).

Becaplermin (recombinant human platelet-derived growth factor-BB [BB homodimer, rhPDGF-BB]) has demonstrated a favorable safety profile in a series of nonclinical studies designed to assess its systemic toxicity, sensitization, local irritation, and genotoxic potential. No significant local or systemic toxicity directly attributable to becaplermin was observed following single and multiple intravenous or subcutaneous administration at doses up to 3 mg/kg in monkeys. Administration of single large intravenous doses (up to 100 mg/kg) and repeated dosing at 1 or 3 mg/kg in mice resulted in rapidly reversible vasodilation and central nervous system depression. In a bone-toxicity study, becaplermin produced histomorphologic changes suggestive of accelerated bone remodeling, which were judged to be potentially reversible. Similar findings have not been observed in humans. Although becaplermin was not considered a dermal or ocular irritant, some skin-sensitizing effects were observed in animals; this finding was not unexpected for a recombinant human-derived protein. Becaplermin was not genotoxic in a variety of in vitro assays and in one in vivo assay.

Genetic toxicity of a pharmacologically active group of ortho-(arylalkynyl)phenoxypropanolamines.

A series of ortho-(arylalkynyl)phenoxypropanolamines with antihypertensive activity in laboratory animals was screened in vitro for mutagenicity using the Ames test and the mouse lymphoma assay, and for DNA damaging potential in the primary rat hepatocyte/DNA repair assay. Those compounds with a dialkylamino group on the para position of the arylalkynyl function were shown to be genotoxic in both mutagenicity assays when tested in the presence of an Aroclor 1254-induced rat liver S-9 mix. They were also active in the DNA repair assay. Removal of the para-dialkylamino group or changing the position of this group on the aryl ring eliminated the genotoxic effect in these test systems. This collaborative effort between chemists, pharmacologists, and toxicologists successfully identified the structural feature responsible for the genotoxic activity and indicated structural alterations that would yield a pharmacologically active compound with no genotoxicity in these in vitro assays.

Genetic toxicity evaluation of McN-5195: a novel analgesic.

McN-5195, (+/-)-trans-3-(2-bromophenyl)octahydroindolizine, a novel analgesic, was tested for genotoxic potential in a battery of tests with endpoints of mutagenicity, chromosomal alterations and DNA damage/repair. McN-5195 was not mutagenic when tested in the Ames test using strains TA98, TA100, TA1535, TA1537 and TA1538, in the absence of metabolic activation and in the presence of Aroclor 1254-induced rat or hamster S-9. Negative results were also obtained in the mouse lymphoma assay in the absence of activation, but reproducible mutagenic responses were seen in this mammalian cell assay in the presence of rat S-9 at high levels of induced toxicity (reduced cell growth). Testing of the enantiomers of McN-5195 in this assay supported these findings. A predominance of small mutant colonies in the mouse lymphoma assay suggested a potential chromosomal effect of McN-5195. This was confirmed with positive findings in an in vitro cytogenetics assay using CHO cells, again at toxic exposure levels and only in the presence of S-9. McN-5195 did not induce DNA repair in the primary rat hepatocyte/DNA repair assay, nor did it induce alterations in vivo of chromosome structure or number when tested in a rat bone marrow cytogenetics assay. The findings from this battery of tests indicate that McN-5195 has modest genotoxic activity when tested in the presence of rat liver S-9 in in vitro systems sensitive to cytogenetic change. The absence of genotoxicity in vitro in Salmonella and intact liver cells and in vivo in rat bone marrow suggests that McN-5195 is unlikely to present a genotoxic risk to whole animals.

Cocarcinogenicity of saccharin and N-alkylnitrosoureas in cultured human diploid fibroblasts.

Previous attempts to transform human foreskin fibroblasts in vitro with N-methylnitrosourea (MNU) or N-ethylnitrosourea (ENU) have been unsuccessful, and concurrent treatment with cocarcinogens or tumor promotors and either MNU or ENU have also failed to produce a neoplastic response. The present study was undertaken to test the effect of sodium saccharin on MNU- or ENU-induced cell transformation. Saccharin alone was not effective in inducing the growth of colonies in soft agar (anchorage-independent growth). However, concurrent treatment with saccharin (50 micrograms/ml, nontoxic dose) and MNU or ENU (29 micrograms/ml or 44 micrograms/ml, respectively) was effective in inducing transformation (greater than 300 colonies/10(5) cells), but only when the cells were treated with saccharin after being released from a G1 block (amino acid deprivation) and followed by MNU or ENU treatment in early S phase. In contrast to results obtained with other chemical carcinogens, transformation frequencies induced by saccharin and MNU or ENU were only slightly decreased in the absence of insulin, which is normally required for growth in this system. Saccharin-MNU- or saccharin-ENU-treated cells that exhibited growth in soft agar also exhibited cellular invasiveness in 9-d-old embryonic chick skin in vitro. In addition, these cells reacted with a monoclonal antibody prepared against a molecular weight 115,000 sarcoma-cell surface-associated glycoprotein and also developed tumors in nude mice. These data demonstrate the cell-cycle-dependent cocarcinogenic potential of saccharin and MNU or ENU in cultured human skin fibroblasts.

Mutagenicity testing of selected analgesics in Ames Salmonella strains.

Acetaminophen (APAP), aspirin (ASA), phenacetin (PA) and ibuprofen (IB) were tested for mutagenic activity in the Ames Salmonella plate incorporation assay using strains TA98, TA100, TA1535, TA1537 and TA1538. These analgesics were tested in four separate tests: without metabolic activation, and in the presence of a rat, hamster or mouse liver post-mitochondrial supernatant (S-9, Aroclor 1254-induced). Treatment of all five strains of Salmonella with APAP, ASA or IB under all four metabolic conditions did not induce any appreciable increases in revertant colony counts, as compared to the negative controls. A dose-related increase in revertant colony counts, reaching levels twice the negative control values, were seen with PA at doses greater than or equal to 500 micrograms per plate. This response was only seen in strain TA100 in the presence of hamster S-9. Therefore, these findings constitute a positive result for PA in the Ames test. APAP, ASA and IB did not show any mutagenic potential under these conditions of testing. These findings are discussed along with previously published results concerning the genotoxicity of these analgesics.

Stability of Aroclor 1254-induced rat liver postmitochondrial supernatant (S-9) following long-term storage (-75 degrees C).

The stability of Aroclor 1254-induced rat liver postmitochondrial supernatant (S-9) over a 5-year period was investigated in a retrospective study. S-9 was uniformly prepared at 6-month intervals, and aliquots were stored at -75 degrees C. The protein and cytochrome P-450 content of these lots of S-9 were very similar, and no differences attributable to duration of storage were observed in the activities of ethoxycoumarin O-deethylase, aniline hydroxylase, cytochrome P-450 reductase or aryl hydrocarbon hydroxylase. There was no decrease following 5 years of storage in the ability of S-9 to activate 2-aminoanthracene, as measured in the Ames test (TA98), but there was a notable reduction following more than 1 year of storage in the ability of the S-9 to generate Ames test activity with benzo(a)pyrene. Based on the results of these studies, S-9 prepared and stored under these conditions appears to be suitable for use in vitro genotoxicity assays for at least 1 year.

Lack of genotoxicity of the cancer chemopreventive agent N-(4-hydroxyphenyl)retinamide.

As part of the preclinical drug safety evaluation of the cancer chemopreventive agent N-(4-hydroxyphenyl)retinamide (HPR) in vitro and in vivo tests were conducted to assess its genotoxic activity. Negative findings from HPR testing were demonstrated in the Ames Salmonella/microsomal activation test, the L5178Y mouse lymphoma assay, and a rat bone marrow cytogenetics study. These data imply that HPR lacks the ability to induce point mutations or chromosomal aberrations, and is therefore not genotoxic. Limited testing of retinyl acetate in the Ames test, the L5178Y mouse lymphoma assay, and the primary rat hepatocyte/DNA repair assay yielded consistently negative results. These findings and previously published results concerning retinoid genotoxicity are discussed.

Alterations of drug-induced toxicity in the mouse lymphoma assay by a rat hepatic microsomal metabolizing system (S-9).

Large differences in induced cellular toxicity were observed in the presence or absence of a rat liver microsomal metabolizing system (S-9) during drug testing in the mouse lymphoma assay. After studying the fate of three drugs in this test system, several mechanisms were demonstrated whereby S-9 reduced cellular toxicity. For N-(4-hydroxyphenyl)retinamide (HPR), fenoctimine sulfate and methyl palmoxirate, the drug concentrations (EC50) in the presence of S-9 were, respectively, 11.5, 14.3 and 4.1 times the concentrations required to achieve comparable levels of toxicity in the absence of S-9. HPR was metabolized by the S-9 and sequestered in the microsomal membranes. This was associated with a marked reduction in the cellular accumulation of the drug. The reduced toxicity of fenoctimine sulfate in the presence of S-9 was associated with extensive biotransformation to polar metabolites. This was accompanied by a reduction of radioactivity associated with the cells from 5.7% to 0.4% of the administered drug. Methyl palmoxirate was rapidly converted to its acid, palmoxirate, by horse serum enzymes present in the treatment medium. This provides an example of metabolism by a test system component other than the S-9 or lymphoma cells. The reduced toxicity of this drug in the presence of S-9 was attributed to further metabolism of palmoxirate and a reduction of the proportion of total radioactivity associated with the cells from 3.1% to 0.4%. These results emphasize the need for pilot toxicity studies, especially when components of the test system are varied, to assess the effect of drug concentration on the toxic response.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiparametric evaluation of the toxic responses of normal human cells treated in vitro with different classes of environmental toxicants.

Eight compounds representing three classes of chemicals were evaluated for their toxic effects on normal neonatal human foreskin fibroblasts in vitro. A battery of toxicity assays was employed to measure the effects of the chemicals on cell viability, DNA synthesis, protein synthesis, DNA repair synthesis, cell ultrastructure, membrane-bound and soluble cytoplasmic proteins, and the activities of six enzymes: beta-glucuronidase, acid phosphatase, gamma-glutamyl transpeptidase, alkaline phosphatase, 5'mononucleotidase, and calcium-magnesium activated (Na+,K+)-dependent ATPase. The compounds evaluated included two antibiotics, each with a metabolic derivative-sulfamethazine (SMZ) and acetylsulfamethazine (ASZ), and carbadox (CBX) and desoxycarbadox (DCX); two anthelmintics-haloxon (HAL) and sansalid (SAN); and a steroid with a metabolic derivative, 17 alpha-estradiol (17-AE) and 17 alpha-estradiol-17-beta-D-glucoside (AE-G). Compounds with similar biological functions often elicited different patterns of response in the normal fibroblasts. For example, the two anthelmintics, HAL and SAN, were similar to each other in that they induced 50% relative cloning efficiencies (EC50) at approximately the same concentrations (HAL = 52 microgram/ml, SAN = 58 microgram/ml), and neither inhibited protein synthesis. They differed, however, in their effects of DNA synthesis. SAN did not inhibit DAN synthesis, while HAL was a profound inhibitor of DNA synthesis (98% inhibition after 4 h at 100 microgram/ml). Because the various toxicants elicited such a variety of response patterns as measured by a multiplicity of parameters, we conclude that similarities in survival responses of cells to closely related toxicants may arise frequently through toxic action at different sites within the cells.

Characterization of human cells transformed by chemical and physical carcinogens in vitro.

Several different classes of chemical carcinogens induced the transformation of human fibroblasts grown in vitro. Characteristics of the events that occur from time of treatment through the expression of neoplastic transformation are presented. The S-phase appeared to be the portion of the cell cycle most vulnerable to insult. Staging of the cells by blocking them in G1 before releasing them to proceed through scheduled DNA synthesis (S) was required to induce reproducible transformation. Compounds such as insulin were added to the cells upon release from the block to sensitize the cells to the carcinogen that was added during S. Growth of the transformed cells as distinct from nontransformed cells was promoted by growth in medium supplemented with 8X nonessential amino acids. Carcinogen-treated cells in the early stage of transformation exhibited abnormal colony morphology and were able to grow at 41 degrees C, in air atmosphere, and in medium supplemented with only 1% serum. In addition, the transformed cells were insensitive to KB cell lysate and exhibited density independent, as well as anchorage independent, growth (i.e., growth in 0.33% agar). Cells that grew in soft agar also produced undifferentiated mesenchymal tumors in preirradiated nude mice.

Effect of pH on the neoplastic transformation of normal human skin fibroblasts by N-hydroxy-1-naphthylamine and N-hydroxy-2-naphthylamine.

The conversion of urinary N-hydroxy arylamines to carcinogenic electrophiles under mildly acidic conditions in the bladder lumen has been proposed as an essential step in arylamine-induced urinary bladder carcinogenesis. To test the hypothesis that extracellular generation of an ultimate carcinogenic species can initiate a neoplastic event normal human fibroblasts were exposed to N-hydroxy-1- and 2-naphthylamine (N-HO-1-NA and N-HO-2-NA) at pH 5 and pH 7. With both compounds, anchorage independent growth of transformed cells in soft agar were enhanced 3- to 7-fold in the pH 5 incubations. Injection of the N-HO-1-NA and N-HO-2-NA-transformed cells into nude mice resulted in tumors in 1/8 and 2/7 animals, respectively. In a control experiment, no differences in transformation of these cells by aflatoxin B1 were observed between pH 5 and pH 7 exposures. Thus, the results are consistent with the hypothesis that ultimate carcinogenic electrophiles, generated extracellularly, can enter an intact cell and induce neoplasia. Alternatively, the possibility of a local intracellular acidic environment near the cell surface and its role in the generation of a reactive electrophile leading to urinary bladder carcinogenesis is discussed.

Comparative induction of unscheduled DNA synthesis by physical and chemical agents in non-proliferating primary cultures of rat hepatocytes.

The incorporation of [3H]thymidine into DNA due to unscheduled DNA synthesis (UDS) induced by N-OH-2-acetylaminofluorene (N-OH-AAF), aflatoxin B1 (AFB1), ethyl methanesulfonate (EMS) and ultra-violet light was quantitated by autoradiography and by scintillation spectrometry on acid precipitable macromolecules or DNA insolated by isopycnic banding in cesium chloride (CsCl). Dose-dependent increases in UDS due to N-OH-AAF and AFB1 treatment were found. Only 2-fold increases at the highest dose levels were found, however, when incorporated [3H]thymidine was quantitated by scintillation spectrometry. Seven, 11, and 25-fold increases in UDS induced by AFB1, N-OH-AAF and ultra-violet light, respectively, were found when incorporated [3H]thymidine was quantitated by autoradiography, indicating a high sensitivity for detecting 'long patch' repair by this technique. Scintillation spectrometry was completely ineffective in detecting EMS-induced UDS, whereas autoradiography demonstrated a small, but significant induction in [3H]thymidine incorporation at high dose levels. The non-proliferative nature of the primary hepatocyte prohibits the uniform radioactive prelabeling of DNA, necessary in other techniques, for the detection of 'short patch' repair induced by compounds such as EMS. Therefore, the sensitivity of the primary cultured rat hepatocyte in conjunction with UDS for detecting DNA damage caused by mutagens and carcinogens which induce 'short patch' repair may be limited to the autoradiographic analysis of the unscheduled incorporation of [3H]thymidine.

2-acetylaminofluorene-induced unscheduled DNA synthesis in hepatocytes isolated from 3-methylcholanthrene treated rats.

The dose-dependent induction of unscheduled DNA synthesis (UDS) by 2-acetylaminofluorene (AAF) and N-OH-2-acetylaminofluorene (N-OH AAF) in primary rat hepatocytes isolated from untreated and 3-methylcholanthrene (3-MC) treated rats was investigated. 3-MC treatment was not necessary for the dose-dependent induction of UDS induced by N-OH AAF, suggesting the presence of enzyme levels adequate for its esterification to an active form in isolated primary hepatocytes. Although all doses of AAF increased the level of [3H]thymidine incorporation above that of the control, a dose-dependent response could not be demonstrated, suggesting inadequate constitutive levels of N-hydroxylating enzymes. Treatment of rats with 3-MC 24 h prior to hepatocyte isolation resulted in a dose-dependent induction of UDS by AAF. 3-MC treatment increased the amount of UDS per cell, as well as the percentage of cells induced to repair their DNA.