Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Reducing the risks of children living near the site of a former lead smeltery.

The sources of lead exposure, soil, household dust, diet and ambient air near a former lead smeltery were studied. The blood lead level of small children was also determined. The aim of the study was to define, based primarily on blood lead measurements, whether children living in the contaminated area may be at risk. Within 500 m from the site of the smeltery, there were several areas where the Finnish limit value for soil Pb, i.e. 300 mg/kg, was exceeded. In the recently built areas, the surface soil has been replaced and soil remediation has taken place in schoolyards and the playgrounds of children's day-care centres. Lead content in household dust was clearly elevated in the contaminated areas. In approximately 20 years, after the smeltery was closed in 1984, the lead concentrations of the fruits and berries in local gardens have decreased to one-tenth. In some samples, the limit values are still exceeded. The lead concentration in ambient air is now 50 times lower than in the 1970s. The blood lead level of the children living in the area is slightly but statistically significantly higher than that of the children in the control areas. The critical blood lead level, i.e. 10 microg/100 ml, was not exceeded in any of the children examined. The average and maximum lead concentrations of 63 analysed blood samples were 2.2 and 5 microg/100 ml, respectively. In contrast, the average and maximum blood lead levels of school children in 1981 were 6.7 and 13.0 microg/100 ml, respectively. The risk reduction measures undertaken during the past 20 years are described.

Intake of polychlorinated biphenyls (PCB) from the Finnish diet.

Concentrations of seven single PCB indicator congeners were measured in 99 food samples. These concentrations were converted into a total PCB content and used for estimating total PCB intake among Finns. The average intake from food is 15 micrograms/day or 0.25 mu/kg body weight per day, which showed no change during the late 1980s. Approximately half of the intake comes from fish. The composition of the PCB mixture varies a lot in different sample types, which emphasizes the importance of single compound determination.

Estimation of dietary intake of cadmium: reliability of methods.

The reliability of intake studies depends on representative sampling of foods, quality assurance of analytical methods, selection of adequate food consumption data and combining concentrations of heavy metals in foods with food consumption figures. The food balance sheet is usually unsuitable for intake estimations of cadmium, since it covers mainly unprocessed food and does not include individual food consumption data. The reliability of household surveys could be improved by taking food wastage into account, more detailed description of meals eaten outside the home, and calculating per capita consumption figures. The duplicate portions method seems to be the most reliable of the available techniques, but does not provide realistic information on variations in long-term intake, for which dietary surveys, food recall studies or food frequency questionnaires should be used. The duplicate portions technique is suitable for studies of risk groups. The average intake of cadmium in Finland is about 10 micrograms per day, but 5-10% of the population ingests more than 20 micrograms of cadmium per day. Additional exposure from smoking, ambient air and drinking-water considerably increases the variation in exposure.

Estimated dietary intake of lead and cadmium and their concentration in blood.

Dietary intake of lead and cadmium and the concentration of Pb and Cd in blood were determined for 42 non-smoking subjects not occupationally exposed to Pb or Cd. The aim of the study was to analyze: (i) the relation between calculated dietary intake of Pb and Cd and the concentration of these metals in blood; and (ii) the methodological problems associated with these two measurements of exposure. The mean dietary intakes of Cd and Pb were 14.5 (SD, 3.1) and 52.9 (SD, 17.9) micrograms day-1, respectively. The concentrations of Pb and Cd in blood were 0.28 mumol l-1 (SD, 0.12) and less than 0.1 microgram l-1, respectively. The relation between dietary intake and concentration in blood was similar to that found in other countries. However, the distributions of these two variables were quite different. This suggests that dietary intake does not accurately reflect the concentration of lead in blood. The methodological problems associated with estimating the dietary intake of toxic metals were: mistakes in keeping food diaries; errors in transferring data from diaries to the computer; invalid food composition or recipe data in the data base used in the calculations. The concentration of Pb and Cd in blood is not necessarily a good indicator of exposure, since only a small proportion of the total body burden is in the blood, and interactions of Pb and Cd with other food constituents during absorption are possible.

Variation and modifying factors of the exposure to lead and cadmium based on an epidemiological study.

Exposure to cadmium and lead was studied using a dietary survey. Data for 1348 individuals aged 25-64 years were collected using 3-day food records. In addition to the dietary intake of heavy metals, the effect of the following were also analyzed: smoking frequency, place of residence, occupation and age. Total cadmium and lead exposure was estimated by calculating intake from food plus that from inhalation, i.e. from urban air and smoking. Heavy smoking increased the cadmium exposure three-fold. The distribution curve of total cadmium exposure was bimodal due to the additional exposure caused by smoking. The distribution curve of the lead and cadmium exposure from food was also skewed, being mostly influenced by food intake. Contaminated urban air slightly increased the total exposure to lead. Occupation had a minor effect on the exposure to both metals. Housewives and retired persons were the least exposed groups. The distribution pattern and wide range of heavy metal exposure should be taken into account in risk assessment for contaminants in food. The additional exposure to cadmium and lead from smoking and from air and water should be considered.

Exposure to cadmium from foods, estimated by analysis and calculation--comparison of the methods.

Cadmium intake of 40 middle-aged Finnish men was determined both by calculation based on computer files and by analysis of duplicate portions. A significant difference was observed between intake estimates of the two methods. The average calculated cadmium intake was 15.8 micrograms/day compared to the 8.2 micrograms/day obtained by analysis. Factors causing the difference between the methods and affecting the comparability are: (i) expression of concentrations below the detection limit; (ii) the calculation is based on the analysis of foods being only washed and peeled whereas duplicate portions are normally processed; (iii) when duplicate portions are prepared using non-representative lots or brands of food, their cadmium content differs from average values used in calculation; (iv) the level of cadmium content in duplicate portion samples is very low affecting the accuracy of the results. The analytical level of cadmium during the analysis of duplicate portions was somewhat lower as compared to the analysis on which the food composition file and the calculation is based. It seems that the complicating factors identified in the present study potentially affect the reliability and comparability of studies of trace element intakes. Therefore one has to be careful when comparing intake estimates derived independently or by different methods.

Intake of heavy metals from foods in Finland, West Germany and Japan.

Comparison of heavy metal intakes between Finland, West Germany and Japan suggests that the average oral exposure to lead and cadmium in Finland is 30-40% of that in the other two countries. Environmental contamination is the obvious cause of high heavy metal content of vegetables and liver in West Germany and Japan and this explains a part of the difference observed in the intake rates. For example cabbage and lettuce in West Germany contain on average over 10 times more lead than the corresponding Finnish products. On the other hand a part of the difference is explained by food consumption habits. For example there is a high consumption of fish and seaweed in Japan, and beer in West Germany, and these products also significantly contribute to intake of heavy metals. Since international comparisons of intakes of food contaminants are at present inaccurate and difficult to conduct, a co-ordinated international study should be implemented. The analytical work should be centralized or alternatively an intercalibration of participating laboratories could precede the actual study. The sampling should cover polluted areas in proportion to their yield of crops.

Validity of toxicity documents of pesticides and good laboratory practice.

Attention has been centered on the validity of the original toxicity reports supporting the registration of pesticides. In Finland, the present standard of the toxicity reports on pesticides was studied. Of the 299 pesticide formulations on the Finnish market only 96 were supported by original toxicity reports. Altogether 16 US formulations and 121 reports, as well as 11 control formulations with 170 reports were selected for the study. Reports from the questionable laboratories proved to be invalid according to the OECD guide-lines more frequently (61%) than those from control laboratories (20%). The frequency of invalid reports among long-term studies was higher (38%) than among acute studies (26.4%). The percentage of valid toxicity studies for the registration of pesticides in Finland was increased from about 50% in the fifties and sixties to 93% in 1980. The lack of reports on old pesticide formulations rather than the validity of the toxicity reports creates a problem in pesticide registration in Finland.

Status of the toxicity reports of pesticides for their registration in Finland.

Recently, attention has centered on the validity of toxicity reports for the registration of pesticides. In Finland, a study was conducted to evaluate the validity of the toxicity reports of pesticides done in U. S. laboratories. Twenty-seven pesticide formulations of the 230 on the market in Finland were selected for the study. The material contained 61 toxicity reports of 16 pesticide formulations done in U. S. laboratories considered questionable, 64 reports from U. S. laboratories considered reliable, and 142 reports originating from European laboratories, totally 267 reports. These reports were divided into valid according to the Organization for Economic Cooperation and Development (OECD) guidelines, and included those with minor discrepancies but in compliance with the good laboratory practice at the time of the study (group I), and and invalid according to the OECD guidelines (group II). After a closer evaluation of the 125 U. S. reports, 85 reports were considered valid and 40 invalid. As a function of time, the proportion of valid studies of all studies on pesticides for the registration purposes in Finland increased during 1961-1981 from 47 to 93%, being 100% at the present time. The lack of information of pesticide formulations registered earlier creates a problem to be solved in the future in Finland, rather than the lack of the validity of toxicity reports.