Harmonisation of rat fetal skeletal terminology and classification. Report of the Third Workshop on the Terminology in Developmental Toxicology. Berlin, 14-16 September 2000.

The initial efforts of the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) and the Free University of Berlin to standardise terminology in the field of developmental toxicology began in 1995. Procedures were undertaken to harmonise the terminology used by the International Federation of Teratology Societies (IFTS) and the International Programme on Chemical Safety (IPCS). This article reflects these activities and is a report on the Third Workshop on the Terminology in Developmental Toxicology held in September 2000. This Workshop served as a forum to discuss the results of a survey on the classification of skeletal anomalies that had been previously sent to scientists active in the field. Although high agreement was reached among the evaluators for several terms, the use of a number of terms was rather variable. Therefore, the discussions at the workshop among the experts from research institutions, regulatory agencies, and industry were mainly focussed on those terms for which there was disagreement and/or uncertainties and the possible reasons. Pictures provided by the participants for the illustration of "grey zone" anomalies constituted the basis for detailed discussions. In many of the cases with lower agreement, decisions were facilitated by the provision of the corresponding picture. The main reasons for lower agreement were imprecise terms, insufficient knowledge on postnatal consequences, theoretical terms that are unlikely to occur in isolation, and the possibility of observing a range of severity that might be decisive for the classification of either a malformation or variation. The attendees concluded that "grey-zone" anomalies will never disappear completely and that for the assessment, the grade of severity and/or the frequency of the observation can be decisive for the terminology chosen. A Joint IPCS/IFTS Project was proposed to further consensus of terminology and classification and to link these anomalies to pictures at different skeletal sites. In order to support the harmonisation of regulatory decisions, it was proposed to establish a "Clearinghouse" System under the umbrella of the IPCS. The Clearinghouse could be contacted either by the regulatory authorities or by any company to clarify their queries, particularly with regard to registration or authorisation processes. Finally, it was recommended to also carry out a similar survey on "soft tissue anomalies" and "external findings." The results of this survey will be discussed at a Joint IPCS/IFTS Workshop in Berlin in 2002.

Toxicological Evaluation of Acetolactate Decarboxylase.

The enzyme acetolactate decarboxylase (ALDC), which is used in the fermentation of beer, is produced by Bacillus subtilis containing the structural gene for ALDC production originating from a Bacillus brevis . ALDC and the glutaraldehyde-stabilized ALDC were subjected to a series of toxicological tests to investigate their safety. None of the ALDC preparations were mutagenic either in bacterial cultures (Ames test) or in mammalian cell cultures (mouse lymphoma assay) nor did they cause chromosomal damage (human lymphocyte assay). The test materials possessed no antimicrobial activity. To investigate the toxicological properties of ALDC, dietary concentrations of 200, 1,400, 10,000 ppm ALDC, or 10,000 ppm stabilized ALDC were given to rats continuously for 13 weeks. When given to pregnant rats at similar dose levels, no effect on the outcome of pregnancy was observed. The dietary concentration of 10,000 ppm corresponds to an intake of some 760 mg/kg/day, which represents approximately 120,000 times the estimated human intake. The toxicological investigations have proven ALDC to be a safe enzyme for use in fermentation of beer.

[Miporamicin teratogenicity study in rabbits].

A teratogenicity study on miporamicin (MPM) was performed in New Zealand White rabbits. Following suitable dose range finding experiments, 60 mated rabbits were randomised into 4 treatment groups, each containing 15 animals. These animals were dosed once daily, by oral gavage, over days 6-18 inclusive of gestation, where the day of mating was designated as day 0. The dose levels applied were 0, 50, 100 and 200 mg/kg/day. A standard dose volume of 5 ml dosing suspension per kilogram body weight was applied, the vehicle being an 0.5% solution of carboxymethylcellulose in distilled water. There was an effect on maternal food consumption at 200 mg/kg/day: cessation of consumption by over one quarter of the animals in the group. Under the conditions of this study, MPM had no effect on the outcome of pregnancy at dose levels of up to 200 mg/kg/day. There was generally no significant effect on either the mother or the conceptus at 100 mg/kg/day.

Evaluation of 60 chemicals in a preliminary developmental toxicity test.

The number of chemicals in commerce which have not been evaluated for potential developmental toxicity is large. Because of the time and expense required by conventional developmental toxicity tests, an abbreviated assay is needed that will preliminarily evaluate otherwise untested chemicals to help prioritize them for conventional testing. A proposed short-term in vivo assay has been used in a series of studies in which a total of 60 chemicals were tested. Some were independently tested two or four times each. In this preliminary test, pregnant mice were dosed during mid-pregnancy and were then allowed to deliver litters. Litter size, birth weight, and neonatal growth and survival to postnatal day 3 were recorded as indices of potential developmental toxicity. Results in this assay and conventional mouse teratology tests were generally concordant. Conventional data were available for 14 chemicals (ten teratogens, one fetotoxin, three nonteratogens), of which 11 (nine teratogens, one fetotoxin, one nonteratogen) produced evidence of developmental toxicity. This included conventional data for three chemicals (ethylene glycol, diethylene glycol dimethyl ether, and triethylene glycol dimethyl ether) that were untested before the present study. As high priority candidates for conventional testing on the basis of results here, all were subsequently studied in a standard teratology assay and were confirmed to be teratogenic in mice. Additionally, one of them (ethylene glycol) plus a fourth high priority candidate for conventional study (diethylene glycol monomethyl ether) were subsequently tested in rats and were found to be teratogenic in that species.

Toxicological Safety Evaluation of a Bacillus acidopullulyticus Pullulanase.

Promozyme®, an amylopectin debranching enzyme produced by Bacillus acidopullulyticus , was studied to evaluate its safety in the food industry. A dietary subchronic toxicity study incorporating fertility and teratogenicity studies was performed in 1-month-old rats at concentrations of 0.5, 1.5 and 5% Promozyme. No adverse effects were seen at the 0.5 and 1.5% dose levels, and at the 5% dose level only minor or equivocal signs of toxicity were recorded. With the exception of a moderate reduction in body weight gain the FIA litters at the 5% dose level, no effects were found in the fertility study, and Promozyme was not teratogenic. In a 13-wk oral toxicity study in dogs, no adverse effects resulted from 0.5 g/kg/d, whereas mild gastrointestinal disturbances were seen clinically at 1.5 and 5.0 g/kg/d. In dogs given 5.0 g/kg/d, terminal investigations showed increased kidney weights and mineralized casts in renal cortical tubules. This was probably due to the high content of ash (phosphorus) in the test material. Lack of mutagenic potential was confirmed in bacterial mutagenic assays with Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and in an in vivo cytogenetic study in rat bone marrow cells after a single dose and daily dosing for 5 d of up to 8 g/kg/d. In an acute inhalation study with 4 h of exposure of rats, no death occurred at the highest dose level used, i.e., 2 mg/L. The test material was non-irritating to skin and did not produce eye injury in rabbits. A skin sensitization study in guinea pigs revealed no indication that the enzyme is a sensitizer. The pathogenic potential of the enzyme-producing B. acidopullulyticus was investigated by single intraperitoneal and subcutaneous administrations to rats and mice; the microorganism was found to be nonpathogenic (LD50>1010 cells/kg). Tests of culture broths revealed that the microorganism does not produce antibiotics. Results indicated that production and the intended use of Promozyme can be regarded as safe for plant workers and consumers.

Results of testing fifteen glycol ethers in a short-term in vivo reproductive toxicity assay.

Fifteen glycol ethers were investigated for their potential to cause adverse reproductive toxic effects using an in vivo mouse screening bioassay. Pregnant mice were orally dosed once per day on days 7 through 14 of gestation at concentrations causing 0 to 41% maternal mortality. Reproductive endpoints included pup survival in utero (percent of live litters/pregnant survivors), pup perinatal and postnatal survival (number of live pups per litter, number of dead pups per litter, and pup survival to 2.5 days of age), and pup body weight statistics (weight at birth and weight at 2.5 days of age). The study was conducted in two phases: a dose range-finding phase using nonpregnant female mice, and a definitive reproductive phase using time-mated mice. The range-finding phase sought to identify, for each chemical, the maternal LD10 as the target dose. However, based upon reproductive phase results, such an exact dose was impractical to achieve. Thus, a range from the LD5 to the LD20 was considered a sufficient challenge dose that would not affect results due to high mortality, i.e., greater than the LD20. Glycol ethers were assigned to groups having different priorities for further testing based upon whether a sufficient challenge dose was administered and the degree of effects recorded for each chemical.(ABSTRACT TRUNCATED AT 250 WORDS)