Kenneth A. Palmer

Cytogenetic studies in rats of cyclohexylamine, a metabolite of cyclamate.

Cyclohexylamine, the major known metabolite of cyclamate, was tested in vivo for possible cytogenetic effects. In rats injected with this metabolite, there was a direct relation between dose concentration and percentage of spermatogonial and bone marrow cells showing chromosomal breaks. Single chromatid breaks predominated with infrequent exchange figures.

Cytogenetic effects of DDT and derivatives of DDT in a cultured mammalian cell line.

Cytogenetic effects of p,p′-DDT, o,p′-DDT, p,p′-DDD, o,p′-DDD, p,p′-DDE, o,p′-DDE and p,p′-DDA were investigated in a mammalian cell line (rat kangaroo). In this cell line, p,p′-DDA was toxic at 200 μg/ml, and the other compounds caused toxic effects at concentrations of 20–50 μg/ml. Mitotic inhibition was moderate in cultures treated with p,p′- or o,p′-DDT and slight in those treated with p,p′- and o,p′-DDD or DDE; none was observed with p,pt-DDA. The p,p′-isomers of DDT, DDD and DDE produced a 2-fold increase in chromosome abnormalities as compared with their corresponding o,p′-isomers. At a concentration of 10 μg/ml, the p,p′-isomers of DDT, DDD and DDE caused chromosome damage in 22.4, 15.5 and 13.7% of the cells, respectively. Of these cells, approximately 12% of the chromosome damage caused by p,p′-DDT and p,p′-DDE was exchange figures; only 1.2% of the abnormalities caused by p,p′-DDD was exchange figures. The o,p′-isomers and control cells exhibited no exchange figures.

A collaborative study of in vivo cytogenetic analysis: I. Interpretation of slide preparations

Abstract A collaborative study was conducted to determine the validity and reproducibility of an in vivo cytogenetic technique, which consisted of examining bone marrow cells from rats for chromosome abnormalities. Four laboratories participated: government, industry, a contract laboratory and an academic institution. The rats were treated and sacrificed, and slides were prepared in the laboratories of the Food and Drug Administration with all of the collaborators participating. Three dose levels of the unknown test compound ( p,′p -DDT), a positive control compound (trimethylphosphate), or the solvent control (corn oil) were given either ip or po and as either a single treatment or daily for 5 days. The rats were sacrificed at 18, 24 or 48 hr after the single treatment or at 6 hr after the last of the multiple treatments. Results showed that agreement between laboratories was close for the criterion of breaks, and even closer for the categories of rearrangement figures and cells with more than 10 aberrations. The number of aberrations per cell appears to be the most accurate, since this criterion showed the closest agreement between laboratories at the various time intervals.

In vitro cytogenetic investigation of calcium cyclamate, cyclohexylamine and triflupromazine

Abstract Cytogenetic effects after varied times of exposure to calcium cyclamate, cyclohexylamine (CHA) or triflupromazine HCl (TFP) were determined in a cell line derived from the kidney of the rat kangaroo ( Potorous tridactylis apicalis ). A preliminary study, using aflatoxin and mitomycin C, which are known to produce abnormalities in cell systems, established the suitability of this cell line for the detection of chromosomal aberrations. In the present study, mitomycin C produced frequent chromatid gaps and breaks in the arms of the chromosomes. Aberrations of the chromosome-type as well as exchanges were noted with aflatoxin. The X-chromosome was extremely sensitive to these agents. CHA produced single chromatid breaks and TFP produced single chromatid breaks and exchange figures. The X-chromosome was not exceptionally sensitive to either of these agents. Calcium cyclamate at concentrations up to 200 μg/ml produced no chromosomal aberrations.

Dominant lethal study of p,p′-DDT in rats

Abstract p , p ′-DDT was tested by the dominant lethal system in rats. Male rats were given DDT either ip or orally and were mated with two untreated females in each of the following 3 wk. The pregnant females were sacrificed at approximately mid-term and were scored for corporalutea, total implants, dead implants and pre-implantation losses. A statistically significant effect was found in the proportion of females having one or more dead implants after being mated during wk 3 with males given DDT orally in a dose of 100 mg/kg. No significant effects were found in females mated with ip-treated males. It is concluded that DDT is only marginally positive with respect to the dominant lethal test.

Protocols for the dominant lethal test, host-mediated assay, and in vivo cytogenetic test used in the food and drug administration's review of substances in the gras (generally recognized as safe) list.

Protocols are described for the dominant lethal and in vivo cytogenetics test in rats and the host-mediated assay, using Salmonella typhimurium and Saccharomyces cerevisiae in mice, as used by the Food and Drug Administration in its mutagenicity review of substances from the generally recognized as safe (GRAS) list. In addition proctolols are described for in vitro mutagenicity tests with S. typhimurium and S. cerevisiae and for statistical treatment for evaluation of data from dominant lethal tests.

Effects of cyclohexylamine on the fertility of male rats

Abstract Cyclohexylamine (CHA) was tested for possible dominant lethal mutations in male rats. Female rats were mated with CHA-treated males and were then examined for the number of corpora lutea, total number of implantations and the number of implantations regarded as early deaths. The results showed significant pre-implantation loss in females mated during wk 1 and 2 after intraperitoneal treatment of the males with 100 or 300 mg CHA/kg. The number of early deaths was not significant. An average of 35% of the ova flushed 48 hr after insemination from the oviducts of females mated with CHA-treated males showed no cleavage and did not exhibit two pronuclei. This indicated that fertilization had not occurred. It is concluded that the pre-implantation loss in females mated with CHA-treated males results from some mechanism other than that of dominant lethal mutation.

Characterization of Thymidine Kinase in L5178y mOuse Lymphoma Cells

Thymidine kinase (TK) was isolated from four different cell lines (TK +/+ P4, TK +/- P4.3.4, TK+/- 3.7.2C, and TK -/- P4.3) that represent the genotypes of L5178 Y mouse lymphoma cells. TK isolated from each of the different cell lines was characterized with respect to the estimated isoelectric point (pI), temperature sensitivity, estimated substrate dissociation constants (Km), estimated inhibitor constant (Ki), and response to the activator deoxycytidine diphosphate. The characteristics of TK from the different cell lines were compared to determine whether the product of the TK gene was changed by the mutation that produced the TK-/- genotype and reverse mutation to the TK +/- genotype. The results indicate that the TK enzymes isolated from the TK+/+ P4, TK +/P4.3.4, and TK +/- 3.7.2C cells have similar, if not the same, characteristics. The small amounts of TK associated with TK -/P4.3 and the mitochondria from TK +/+ P4 had similar characteristics, and both were different in many respects from the TK associated with the TK +/+ P4, TK +/- P4.3.4, and TK +/-3.7.2C. These results raise a question about whether a structural gene is the target for chemical mutagens in the L5178Y TK +/assay, however, this can only be answered by the isolation of the TK gene from each of the L5178Y genotypes and determination of the nucleotide sequence.

Mutagenicity studies of R-amino salt, a metabolite of amaranth (FD & C Red No. 2), in mouse lymphoma cells heterozygous at the thymidine kinase locus and in the rat dominant lethal test

Abstract The R-amino salt of amaranth (FD & C Red No. 2) was tested for mutagenicity in the thymidine kinase heterozygous (TK+−) mouse lymphoma assay and in the dominant lethal test. The results from the mouse lymphoma assay showed a dose-related increase in mutation frequency compared with values for the negative controls. Giant cells were present in the cultures after treatment and a distinct change of colour was observed when the R-amino salt was dissolved in the tissue-culture medium. In the dominant lethal test. the effect of R-amino salt was not statistically significant.

In vitro toxicity screening tests: A comparison of activation systems and treatment protocols among different laboratories

A survey conducted to determine methods of choice for providing procarcinogen activation and treatment protocols for mammalian cell tests in various genetic toxicology testing laboratories showed that one-third of respondents were performing assays without exogenous activation systems, one-third used liver homogenate preparations with short exposure to the test compound, and one-third used cocultivation with metabolically competent cells with longer exposure times. Treatment protocols were variable even among similar assays, indicating that development of standard operating procedures is still in progress, or may need to be individually defined for different test systems.