Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

The ovine Booroola fecundity gene (FecB) is linked to markers from a region of human chromosome 4q.

The autosomal Booroola fecundity gene (FecB) mutation in sheep increases ovulation rate and litter size, with associated effects on ovarian physiology and hormone profiles. Analysis of segregation in twelve families (379 female progeny) identified linkage between the mutation, two microsatellite markers (OarAE101 and OarHH55, Zmax > 9.0) and epidermal growth factor (EGF) from human chromosome 4q25 (Zmax > 3.0). The marker OarAE101 was linked to secreted phosphoprotein 1 (SPP1, which maps to chromosome 4q21-23 in man) in the test pedigrees and independent families (Zmax > 9.7). The identification of linkage between the FecB mutation and markers from human chromosome 4q is an important step towards further understanding the control of ovulation rates in mammals.

Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner.

Multiple ovulations are uncommon in humans, cattle and many breeds of sheep. Pituitary gonadotrophins and as yet unidentified ovarian factors precisely regulate follicular development so that, normally, only one follicle is selected to ovulate. The Inverdale (FecXI) sheep, however, carries a naturally occurring X-linked mutation that causes increased ovulation rate and twin and triplet births in heterozygotes (FecXI/FecX+; ref. 1), but primary ovarian failure in homozygotes (FecXI/FecXI; ref. 2). Germ-cell development, formation of the follicle and the earliest stages of follicular growth are normal in FecXI/FecXI sheep, but follicular development beyond the primary stage is impaired. A second family unrelated to the Inverdale sheep also has the same X-linked phenotype (Hanna, FecXH). Crossing FecXI with FecXH animals produces FecXI/FecXH infertile females phenotypically indistinguishable from FecXI/FecXI females. We report here that the FecXI locus maps to an orthologous chromosomal region syntenic to human Xp11.2-11.4, which contains BMP15, encoding bone morphogenetic protein 15 (also known as growth differentiation factor 9B (GDF9B)). Whereas BMP15 is a member of the transforming growth factor beta (TGFbeta) superfamily and is specifically expressed in oocytes, its function is unknown. We show that independent germline point mutations exist in FecXI and FecXH carriers. These findings establish that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.

A linkage map of sheep chromosome X (OARX) aligned to human chromosome X (HSAX).

We have constructed a genetic linkage map of the sheep X chromosome (OARX) containing 22 new gene loci from across the human X chromosome (HSAX). The female OARX linkage map has a total length of 152.6 cM with average gene spacing of 5.5 cM. Comparison with HSAX confirms one previously reported major breakpoint and inversion, and other minor rearrangements between OARX and HSAX. Comparison of the linkage map with sheep sequence data OAR 1.0 reveals a different arrangement of markers on the q arm, which may more accurately reflect the genuine arrangement of this region.

Antibacterial agents that inhibit lipid A biosynthesis.

Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.

Regulatory aspects of genotoxicity testing: from hazard identification to risk assessment.

This symposium focused on the use of tests for chromosomal damage, and other genotoxicity measures, for detection of potentially harmful chemicals. The speakers discussed the information that has been gained over the last three decades about the use of "short-term tests" for genotoxicity in cultured cells and in animals (mainly rodents), and the ongoing debates about the rational use of data from such experimental systems in trying to extrapolate to an understanding of potential human risk. The overall theme was that the field of regulatory toxicology currently is over-reliant on qualitative outcomes of in vitro hazard-screening tests, generally conducted at the maximum achievable exposures, and needs a more realistic approach that incorporates in vivo exposure levels and dose-response information.

Comparative mapping of sheep chromosome 2q.

Sheep chromosome 2q (OAR2q), which is homologous with human chromosome 2q (HSA2q), and cattle chromosome 2 (BTA2), is known to contain several loci contributing to carcass traits. However, the chromosomal rearrangements differentiating these chromosomes among the three species have not yet been determined and thus precise correspondences between the locations of sheep and human genes are not known. Twenty-six genes from HSA2q (2q21.1-->2q36) have been assigned to OAR2q by genetic linkage mapping to refine this area of the sheep genome. Seventy-six genes were initially selected from HSA2q. Sixty-eight percent of the PCR primer sets designed for these genes amplified successfully in sheep, and 34% amplified polymorphic products. Part of the proximal arm of OAR2q was found to be inverted compared with HSA2q. The breakpoint has been localised near the growth differentiation factor 8 gene (GDF8), spanning 380 kb between the positions of the hypothetical protein (FLJ20160) (HSA2:191008944-191075046) and glutaminase (GLS) (HSA2:191453847-191538510) (Build36.1).

A putative autosomal gene increasing ovulation rate in Romney sheep.

Ovulation rates were measured in 547 progeny of 24 rams in a Romney flock with a long history of high prolificacy. These sheep were from the same family line and the distribution of ovulation rates suggests the presence of a segregating major gene (FecW) that increases prolificacy. The phenotype differs from those previously described for major genes affecting prolificacy in sheep. The putative gene shows autosomal inheritance and one copy increases ovulation rate by 0.8-1.0 eggs per ewe ovulating. To date, we have found no evidence of infertility among putative homozygous ewes, as described in some autosomal major genes for prolificacy.

Investigation of the Booroola (FecB) and Inverdale (FecX(I)) mutations in 21 prolific breeds and strains of sheep sampled in 13 countries.

Twenty-one of the world's prolific sheep breeds and strains were tested for the presence of the FecB mutation of BMPR1B and the FecX(I) mutation of BMP15. The breeds studied were Romanov (2 strains), Finn (2 strains), East Friesian, Teeswater, Blueface Leicester, Hu, Han, D'Man, Chios, Mountain Sheep (three breeds), German Whiteheaded Mutton, Lleyn, Loa, Galician, Barbados Blackbelly (pure and crossbred) and St. Croix. The FecB mutation was found in two breeds, Hu and Han from China, but not in any of the other breeds. The 12 Hu sheep sampled were all homozygous carriers of FecB (FecB(B)/FecB(B)) whereas the sample of 12 Han sheep included all three genotypes (FecB(B)/FecB(B), FecB(B)/FecB+, FecB+/FecB+) at frequencies of 0.33, 0.58 and 0.08, respectively. There was no evidence of FecX(I) in any of the breeds sampled.

Growth regulation by peroxisome proliferators: opposing activities in early and late G1.

Peroxisome proliferators (PPs) are a diverse group of nongenotoxic rodent liver carcinogens. One potential mechanism for the carcinogenicity of PPs is epigenetic modulation of growth-regulatory signal transduction pathways. We investigated the effects of PPs on growth-regulatory gene expression and cell proliferation in immortalized mouse liver cells, comparing PPs with other growth regulators and tumor promoters of known activity. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, and ciprofibrate ethyl-ester were found to be potent inducers of immediate-early gene expression (including c-fos, c-jun, junB, egr-1, NUP475, and to a lesser extent fosB, JE, and KC, with maximal expression seen 1 h after treatment of serum-deprived quiescent cells. The gene induction was potently inhibited by protein kinase inhibitor H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride] but not by H8 [N-¿2-(methyl-amino)ethyl¿-5-isoquinolinesulfonamide dihydrochloride], indomethacin, or nordihydroguaiaretic acid. Compared with other growth regulators, the profile of PP-induced gene expression was most similar to that induced by arachidonic acid and eicosatetraynoic acid. The induction of immediate-early gene expression by PPs was followed by enhanced progression into S phase (DNA synthesis) when quiescent cells were treated with the PPs for only 1 h, washed, and then incubated without PPs. However, no stimulation of DNA synthesis was seen when the PPs were continually present. Furthermore, the PPs inhibited serum-induced DNA synthesis, even when they were added 6 h after serum stimulation (in late G1). Dehydroepiandrosterone-sulfate, a unique PP in being a steroid, had no detectable effect on immediate-early gene expression, did not stimulate DNA synthesis when applied for only 1 h, but did inhibit serum-induced DNA synthesis. Thapsigargin and A23187 mimicked this mitoinhibitory activity of PPs, suggesting that calcium mobilization by PPs might be involved. Our results demonstrate that PPs can modulate cell proliferation either by a stimulatory activity that functions in early G1, associated with activation of immediate-early gene expression, or by an inhibitory activity that functions in late G1; both activities could potentially play a role in tumor promotion by PPs.

Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery.

Bmp15 mutations and ovarian function.

BMP15, also known as growth and differentiation factor 9B (GDF9B), is a member of the transforming growth factor beta superfamily (TGFbeta) which in humans, rodents and sheep is expressed exclusively in the oocyte. BMP15 is closely related to GDF9, another oocyte-specific member of this superfamily which has been shown to be essential for early ovarian folliculogenesis. Inactivation of the BMP15 gene in mice has shown only minor effects on fertility. However, Inverdale and Hanna lines of sheep carry naturally occurring mutations in BMP15 which highlight differences in the action of this gene between mice and other mammals. Sheep which are heterozygous show an increase in ovulation rate whereas homozygotes are infertile. The granulosa cell receptor which mediates the BMP15 response has not yet been identified, but the discovery that a point mutation in the BMP1B receptor in Booroola sheep is responsible for increased ovulation rate highlights the importance of the TGFbeta signalling molecules in early folliculogenesis.

Cytotoxicity and chromosome aberrations in vitro: experience in industry and the case for an upper limit on toxicity in the aberration assay.

The chromosome aberration assay in vitro is a useful and sensitive test for detection of genotoxins. However, aberrations can occur secondary to toxicity, with compounds that do not react with DNA and are not genotoxic in vivo. Thus, some positive results in the in vitro aberration assay are not relevant to human risk. To help evaluate the influence of toxicity, data were collected from 27 pharmaceutical and chemical companies and contract laboratories. When cytotoxicity was measured by cell counts or confluence, compounds expected to damage DNA (Category 1) generally induced aberrations without severe concomitant cytotoxicity, i.e., at cell growth 60% or more of control. The more toxic nucleoside analogues, topoisomerase inhibitors, fluoroquinolone antibiotics, antifolates, and producers of reactive oxygen were still positive with cell growth 50% or more of control. In contrast, when there was evidence that the compounds were not DNA damaging (Category 2), there was a higher proportion of toxicity-associated clastogens, with positive results at less than 50% of control cell growth. When mitotic index (MI) was used as an indicator of cytotoxicity, the pattern was less clear, although there was a tendency to more mitotic suppression with the Category 2 compounds. Overall the data indicate that a limit on toxicity, and a more accurate way of estimating it, would increase the accuracy of the assay by reducing the frequency of nonrelevant positive results with a threshold-type of dose relation. The rationale for evaluating positive results in the in vitro aberration assay, especially those associated with toxicity, is discussed, as is the need for a harmonized regulatory approach.

Chromosome aberrations induced in vitro: mechanisms, delayed expression, and intriguing questions.

Chromosome aberrations, including breakage and rearrangement and numerical changes, are important in carcinogenesis, heritable mutations, embryonic loss, and developmental abnormalities. We can detect DNA reactive agents in in-vitro chromosome aberrations assays, but aberrations are also induced by chemical that do not directly interact with DNA. This article discusses briefly some important aspects of using aberrations in genetic toxicology testing but concentrates on highlights of recent research on aberrations, in particular two areas: (1) persistence through multiple cell cycles of changes that lead to chromosome aberrations, and (2) the relations among DNA synthesis inhibition, DNA damage, cell cycle regulation, and genomic instability, expressed as chromosome breakage, gene amplification, and aneuploidy. An understanding of these mechanisms not only may lead to insights into carcinogenesis but ultimately may help us to interpret results of chromosome aberration tests and to develop a rational assessment of the degree of human risk implied by a positive aberration test.

Human liver S-9 metabolic activation: proficiency in cytogenetic assays and comparison with phenobarbital/beta-naphthoflavone or aroclor 1254 induced rat S-9.

Induced rat liver S-9 is routinely used for metabolic activation in cytogenetic assays. When a compound gives a positive test result only with rat S-9, the significance for humans should be assessed. To evaluate the use of human S-9, we used sister-chromatid exchanges (SCEs) and chromosome aberrations (Abs) in Chinese hamster ovary cells to test five pro-mutagens, each preferentially activated by a different family of P-450: benzo(a)pyrene (BP), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), aflatoxin B1 (AFB), and 2-acetylaminofluorene (2-AAF). We tested two human S-9 preparations, one from a single liver and a second pooled from two livers known to have good activity for several P-450s. Concentrations and ratios of NADP and isocitrate were adjusted to optimize NADPH generation by the S-9. Abs were scored 20 hr, and SCEs 29-45 hr, after the beginning of a 3 hr treatment. P-450 enzyme activities were generally higher in rat than human S-9. With the single-liver human S-9, increase in SCEs were seen with all chemicals; with both human S-9s, increases in Abs were seen with all chemicals except BP. (The level of P-450 1A1, required for BP activation, is very low in human liver.) Compared with rat S-9, generally higher concentrations of human S-9 and of promutagens were required to see positive results. However, human S-9 effectively activated 2-AAF, whereas neither of the two types of rat S-9 produced Abs with 2-AAF. We also compared rat S-9s induced with Aroclor 1254 or phenobarbital/ beta-naphthoflavone (PB/beta NF). Although there were some differences in P-450 enzyme activities, these did not translate into differences in Abs induction. At low doses of AFB and of BP, PB/beta NF induced S-9 appeared more effective than Aroclor 1254 induced S-9.

Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals.

Results from the testing of 108 coded chemicals in Chinese hamster ovary (CHO) cells for the induction of chromosome aberrations and sister chromatid exchanges (SCEs) are presented. All chemicals were tested with and without exogenous metabolic activation, using protocols designed to allow testing up to toxic doses. Cell harvest times could also be extended if chemical-induced cell cycle delay was seen. Chromosome aberrations were induced by 43 of the chemicals, and 66 induced SCEs; 37 of the chemicals were positive for both endpoints.

Chromosome aberrations in vitro related to cytotoxicity of nonmutagenic chemicals and metabolic poisons.

Chromosome aberrations can occur by secondary mechanism(s) associated with cytotoxicity, induced by chemicals that do not attack DNA. Aberrations are formed from DNA double-strand breaks, and DSBs are known to be induced by nonmutagenic (Ames test negative) noncarcinogens at toxic levels [Storer et al. (1996): Mutat Res 368:59-101]. Here, 8 of 12 of these chemicals caused aberrations in CHO cells at cytotoxic doses, and often only when cell counts (survival) at 20 hr approached < or =50% of controls. Five of eight noncarcinogens (2,4,-dichlorophenol, dithiocarb, menthol, phthalic anhydride, and ethionamide) and one of two equivocal carcinogens (bisphenol A) caused aberrations, usually over a narrow dose range with steeply increasing cytotoxicity. Phthalic anhydride and ethionamide were positive only at doses with precipitate. Phenformin was negative even at toxic doses and ephedrine and phenylephrine were negative and gave little toxicity. Aberrations were also induced by metabolic poisons, 2,4-dinitrophenol, (uncouples oxidative phosphorylation), and sodium iodoacetate, (Nal; blocks ATP production). Five of the chemicals that induced aberrations in CHO cells were tested in human TK6 cells and four were positive, the fifth being equivocal. Stable aberrations (translocations) were induced in human cells by Nal. Clearly, chemicals can give "false-positive" results in the chromosome aberration assay at cytotoxic levels, though cytotoxicity does not always produce aberrations, so that further information (e.g., DNA reactivity) is needed to determine whether a result is a "false-positive." Primary DNA-damaging chemicals such as alkylators are also cytotoxic, but give strong increases in aberrations without marked initial toxicity by the measures used here, although the aberrations they induce do reduce long-term survival in colony-forming assays.

Multilaboratory comparison of in vitro tests for chromosome aberrations in CHO and CHL cells tested under the same protocols.

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.

Placebo controlled double-blind trial of fluvoxamine maleate in the obese.

Obesity and depression are common disorders which may co-exist. The management of the combination is complicated because some antidepressants cause weight gain fenfluramine, an effective antiobesity agent, may cause depression. Fluvoxamine is an antidepressant which, like fenfluramine, inhibits serotonin re-uptake within the brain. Forty obese female subjects with refractory obesity participated in a double-blind placebo controlled trial. During the twelve week study, those subjects receiving fluvoxamine achieved a mean weight loss greater than, but not significantly different from, that of the placebo group. The result suggests that fluvoxamine may be particularly useful in the management of obese patients requiring treatment with an antidepressant.

The micronucleus test and NTP rodent carcinogens: not so many false negatives.

In the study by Shelby et al. (1993) on 49 chemicals, the results of the micronucleus (MN) test in mouse bone marrow were compared with the results of the 2 year rodent carcinogenicity assays. Seven of the 25 rodent carcinogens were considered positive in the MN test, 5 following a protocol in which chemicals were given in three daily doses, and a further 2 when the chemical was administered only once. This low rate of positive results has led to disappointment in the MN test as a screen for carcinogens, but a careful examination of the data and of its analysis by Shelby et al. (1993) shows that many of the negative results are appropriate because: of the 18 carcinogens that were negative in the MN test, 1 has been retested and found to be non-carcinogenic, 9 were non-genotoxic and at least 2 were site-of-contact carcinogens not expected to be detected in the bone marrow. Two others were clearly positive in the MN test in other labs. Thus, the MN test 'missed' not 18 carcinogens, but 4 genotoxic carcinogens. The significance of these 4 needs further assessment, since three were liver specific carcinogens and the fourth was a very weak inducer of hemangiosarcomas in female mice only. Overall, the results of Shelby et al. (1993) do not cast such a shadow on the micronucleus test as many feared, and must be examined in the context of all the information available on each chemical. As Ashby and Tinwell emphasize in the accompanying article and in Tinwell and Ashby (1994), the data show that the MN test is capable of identifying human carcinogens and rodent germ cell mutagens, and remains a useful part of genotoxicity evaluation of chemicals.