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The Health Council of The Netherlands advises the Dutch government on scientific issues concerning health and environmental protection. In 1978 a committee of the Council established mechanistic criteria for quantitative risk assessment of carcinogenic substances. For this purpose it adopted the multistage model of carcinogenesis. Based on this model two categories of carcinogens were distinguished. The first category includes complete carcinogens and tumor initiators: these agents induce irreversible modification of DNA and act by a stochastic mechanism. The second category includes promotors and other substances acting as cocarcinogens; these agents act by nonstochastic mechanisms. Stochastic action implies that there is no threshold for the contribution to the carcinogenic effect, whereas nonstochastic action implies a threshold dose. Recently, reevaluation of the method for carcinogenic risk assessment was considered due because of remarkable scientific progress since 1978, such as the identification of cellular oncogenes and tumor suppressor genes, and the partial clarification of mechanisms at the cellular and/or molecular level by which tumor promotors stimulate tumor development. The present paper discusses the Dutch method for carcinogenic risk assessment in the light of the recent scientific developments. It concludes that this method for risk assessment is still a valid and appropriate one.

Bacteriochlorin a (BCA) is an effective second-generation photosensitizer both in vitro and in vivo. BCA has a high molecular absorption coefficient (32,000 M-1 cm-1) at 760 nm. At this wavelength tissue penetration of light is almost optimal and melanin absorption is relatively low. BCA is preferentially retained in a number of tumour model systems and is rapidly cleared from non-cancerous tissues, thus inducing no or minor skin photosensitivity. Mutagenicity of BCA has been tested using the Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104. In all tester strains used, BCA induces, in the dark, a minute increase in the number of revertants. No linear correlation between the number of revertants and the BCA dose is observed. Incubation of isolated rat hepatocytes with BCA, in the dark, does not result in increased cell death as measured by leakage of cytosolic lactate dehydrogenase. A convenient bioassay to test possible genotoxicity in vivo is the established Somatic Mutation and Recombination Test (SMART) on Drosophila melanogaster. Bacteriochlorin was tested for induction of loss of heterozygosity in the white/white+ eye mosaic assay, which predominantly measures homologous mitotic recombination in somatic cells of Drosophila after treatment of larval stages. BCA did not induce loss of heterozygosity above the level of the incorporated controls, with or without illumination. Based on these results, obtained in prokaryotic and eukaryotic cells and in vivo, we are inclined to conclude that the dark toxicity and mutagenic properties of BCA, as measured by the applied bioassays, are negligible.

The effect of storage on MMS-induced recessive lethals in the zeste-white (3A1-3C2) and the maroon-like (18F4-20F) regions was studied by complementation analysis. 1. (1) Without any exception, all 52 mutants (from unstored spermatozoa) mapped in the zeste-white region were restricted to single complementation units. Furthermore, none of an additional 15 lethals, sampled from sperm that had been stored in females for 9–12 days, was associated with a deletion. 2. (2) Of 34 mutations induced by 8.5 × 10−2 mM MMS in the maroon-like (mal) region, 4 spanned 2 or more complementation units, and thus are considered to be deletions. A high dose of 2.5 mM MMS provided 55 lethals for analysis of which 4 were deletions. There was no evidence for any difference in the frequency of deletions as the MMS concentration was enhanced from 8.5 × 10−2 mM to 2.5 mM. However, with storage, 47.1% lethals (16 of 34 mutants induced by 2.5 mM MMS) mapped in the mal region were found to involve large structural changes. 3. (3) A high proportion of double mutants in both the zeste-white (z w) and the maroon-like regions was found among the chromosomes analyzed. These double mutants have one lethal positioned within the region studied and the other outside it. Clearly, the proportion of double mutants increased with dose, from 6.3 to 41.7% in z w and from 14.7 to 61.8% in the mal section. Apurinic sites in DNA reacted with MMS are considered as the likely primary lesions responsible for the storage effect on MMS-induced recessive lethals in the mal region. Thus, the ability of MMS to produce delayed deletion lethals seems to correlate with preference for alkylation of base nitrogens. An interesting aspect for further analysis is the apparent infrequency in the zeste-white region of alkylation-induced chromosomal breakage, as observed by various investigators for MMS, EMS and MNNG.

This study presents the analysis of chemically-induced somatic mutations and chromosomal damage in the eye imaginal discs of Drosophila larvae, assayed later as twin (TS) and single light (LS) mosaic spots in the adult eyes. Regarding the question as to what kind of DNA alterations contribute to somatic cell mutagenicity, the approach followed here has been to investigate the possible differences in response between male (hemizgous for an X) and female (homozygous) larvae, rod-X / rod-X versus ring-X / rod-X genotypes and inversion-heterozygotes versus genotypes not carrying an inversion. The systems chosen for analysis were the white-coral/white (wco / w) and the white (w+ / w) eye mosaic system. The principle findings with 12 mutagens of different modes of action are as follows: 1. (1) At least 98% of all TS and LS induced by cisplatin (DDP) in wco / w female larvae and about 95% of those by formaldehyde (FA) appear as the result of recombinogenic activity between the two homologous X-chromosomes. The corresponding estimates for MMS, EMS and ENU are 81%, 73% and 61%, respectively. 2. (2) The long scS1Lsc8R inversion, which also contains In(1)dl-49, suppresses induction of TS to 83–93%. There was also a sharp decline in the frequency of LS in inversion heterozygotes for DDP (91%), FA (86%), MMS (52%) and EMS (47%). 3. (3) Ethylnitrosourea (ENU) was the mutagen for which introduction of the inverted chromosome reduced only slightly (23%) the frequency of LS, indicating that the majority of them were somatic mutations (and deletions) at the white locus. 4. (4) In w/RX females heterozygous for a ring-X chromosome, the frequency of LS was only approximately one tenth of that of the control (w+ / w) group, after exposure to MMS or DDP. The explanation is that exchange processes involving the ring frequently lead to genetic imbalance with subsequent cell killing. 5. (5) Low frequencies of TS in wco males (one fifth to one tenth of the rates in females) after exposure to MMS, EMS, DEN, ENNG, ENU and DDP are indicative of unequal sister-strand recombination in the X-chromosome of males. Unequal mitotic recombination had been predicted by Auerbach (1945) to occur in males, and this event was later demonstrated after exposure to X-rays and triethylenemelamine (Becker, 1975). 6. (6) Frequencies of LS were clearly higher in wco / w females than in wco males for MMS (2 : 1) and DDP (7 : 1) but not for EMS and ENU. After correcting for that portion of LS due to recombinogenic activity, there was no significant departure from a 1 : 1 ratio for MMS and ENU. EMS seems to represent a mutagen which is more genotoxic in males than in females, both in germinal and somatic tissue. Part of this study also dealt with methodological aspects: 1. (7) Four different routes of application, i.e., chronic exposure, acute treatment, surface treatment and inhalation, were examined in order to devise suitable protocols for chemicals with different properties. Exposure doses as low as 1.4 ppm 1,2-dibromoethane were still mutagenic in inhalation experiments with wco / w and w+ / w female larvae. 2. (8) The white coral / white bioassay gave problems in the sense that small mosaic spots, i.e., size classes 1, 2 and 3–4, but also 5–8 (ommatidia), mostly escaped detection. This drawback could be overcome by replacing the wco marker by the wild-type allele w+. Thus, we recommend for mutagenicity testing the use of w+ / w crosses because this test enables the quantitative scoring of both small and large mosaic sectors (LS). 3. (9) Comparative experiments with MMS and ENU revealed that eye mosaics can be induced in first, second and third larval instar. However, the high number of small spots produced when treating third-instar larvae could only be detected in wild-type (w+ / w) phenotypes.

The ability of caprolactam (CAP) to induce somatic mutation/mitotic recombination (SMART) in heterozygous white/white-coral female larvae was examined in comparison with 2 assays measuring sex-linked recessive lethals (SLRL) in male and female larvae. The overall result is a positive response in both the SMART and SLRL assays after treatment of female genotypes, indicating that CAP acts as a weak genotoxin in Drosophila. The SLRL assay on male larvae was devoid of any activity. This finding is consistent with the view that an intrinsic limitation to assays measuring heritable genetic damage in premeiotic cell stages, particularly in hemizygous condition as in males, is that mutational events in those cells may be eliminated either by repair, by selection, or by both mechanisms. Thus the failure of CAP to elicit mutagenic activity in premeiotic male cells could be interpreted to mean that in Drosophila as in human lymphocytes, CAP acts as a clastogenic cell mutagen.

The origin of strains of Drosophila and mice susceptible to xray and chemical mutagenesis is discussed. The probable causes of the genetic differences are briefly reviewed and a possible solution to the problem is suggested. (ERB)

In July 1984 the Minister of Welfare, Public Health and Culture, representing the Dutch government, sent a request to the Health Council of the Netherlands to advise on the health risks presented by environmental exposure to several carcinogenic substances. One of these substances was vinyl chloride (VC). On the basis of a working document prepared by the National Institute of Public Health and Environmental Hygiene, a committee of the Health Council of The Netherlands prepared a report concerning a health risk assessment of VC which was published in May 1986. A short review is presented of the available data and the considerations that formed the basis for the risk assessment of the carcinogenicity of VC to humans. The advice was based mainly on human data from epidemiological studies of workers occupationally exposed to VC. The committee concludes that continuous exposure to 0.001 mg/m 3 VC corresponds to an additional cancer mortality risk of 10 −6 per lifetime. The Dutch government considers this additional risk to the general population to be acceptable.