A proposal to improve clarity and communication in the EU Classification process for chemicals for carcinogenicity and reproductive and developmental toxicity.

There is an issue in the EU classification of substances for carcinogenicity and for reproductive or developmental toxicity which has brought difficulties to those involved in the process. The issue lies in the inability of the classification system to distinguish between carcinogens and reproductive toxicants with different levels of concern. This has its origins in the early years of toxicology when it was thought that a relatively small number of chemicals would be either carcinogens or reproductive toxicants, but this has turned out not to be the case. This can cause problems in communicating to the users of chemicals, including the public, the nature of the hazard presented by chemicals. Processes have been developed within the classification system for setting specific concentration limits which assess the degree of hazard for carcinogens and reproductive toxicants as high, medium or low. However these categories are not otherwise used in classification. It is proposed that their wider use would bring the advantages of transparency, clarity of communication, certainty of the process and would allow chemicals with a high degree of hazard to be identified and managed in an appropriate way.

Prenatal toxicity of inhaled polymeric methylenediphenyl diisocyanate (MDI) aerosols in pregnant wistar rats.

Mated Wistar rats, 25/group, were exposed to polymeric methylenediphenyl diisocyanate (MDI) aerosol of respirable size for 6 h/day, on gestational days (gd) 6 through 15, at 0, 1, 4, and 12 mg/m3. Maternal clinical signs, body weights, and feed and water consumption were measured throughout gestation. At scheduled sacrifice on gd 20, maternal body, gravid uterine, liver, and paired lung weights were documented. Corpora lutea were counted, implantation sites were identified: resorptions, dead and live fetuses, and placentas were weighed. All live fetuses were counted, sexed, weighed, and examined for external alterations; approximately 50% of the live fetuses/litter were preserved in Bouin's fixative and examined for visceral alterations, and the remaining live fetuses/ litter were cleared and stained with alizarin red S and examined for ossified skeletal alterations. Maternal toxicity was observed at 12 mg/m3, including mortality (2 of 24 pregnant), damage to the respiratory tract, reduced body weights and weight gain, reduced liver and increased lung weights, and reduced gravid uterine weight (the last not statistically significantly different from the control value). Developmental toxicity was also observed at 12 mg/m3, including reduced placental and fetal body weights and an increased incidence of fetal skeletal variations and skeletal retardations. There was no evidence of maternal or developmental toxicity at 1 or 4 mg/m3. The no observed adverse effect concentration for maternal and developmental toxicity was therefore 4 mg/m3. There were no treatment-related teratogenic effects at any concentrations evaluated.

Toluene diisocyanate: an assessment of carcinogenic risk following oral and inhalation exposure.

Although respiratory sensitization and pulmonary irritation have been the subject of particular studies with toluene diisocyanate (TDI), in recent years the potential carcinogenicity of TDI has been a reason for concern and speculation. This has arisen from the expectation that following exposure to TDI the chemical would hydrolyze at aqueous tissue surfaces to give rise to toluene diamine (TDA), a mutagen and rodent carcinogen. The chemistry of TDI suggests that the reaction with biological NH2 groups such as those on proteins, and polymerization to oligoureas, will compete with the hydrolysis reaction. This has been shown with results of in vitro studies where conjugation to protein occurs without detectable formation of TDA when protein solutions in saline are exposed to TDI vapor. Lower pH levels leading to high protonation of biological NH2 groups facilitate hydrolysis of TDI to TDA and subsequent formation of polyureas. These observations are consistent with comparative toxicokinetic studies in rats, which demonstrate significant levels of TDA following oral dosing with TDI--due to the acidic environment in the stomach--but not after inhalation. These results provide an explanation for the tumors observed in rodents after oral dosing of TDI in corn oil, but not after inhalation. Inhalation is the relevant route of human exposure for TDI and the toxicokinetics of TDI exposure at occupational exposure limits have been studied. These data provide a means by which quantitative estimates of the risk of carcinogenicity possibly resulting from the intermediate formation of TDA during TDI exposure can be obtained. Several calculations have been made, all of which lead to the conclusion that TDI exposure by inhalation at the recommended occupational limits will not give rise to significant carcinogenic risk.

The evaluation of carcinogenic risk to humans: occupational exposures in the spraying and application of insecticides.

The International Agency for Research on Cancer (IARC) has reviewed the carcinogenic risk to man of the occupation of spraying or applying insecticides. IARC was unable to conclude that a causal relationship had been established (category 1), but there was some evidence which led them to consider the occupation to be probably carcinogenic (in category 2A). These conclusions have been reviewed by a working group of the International Group of National Associations of Manufacturers of Agrochemical Products (GIFAP) to determine what steps needed to be taken which might improve safety for those working with insecticides. The working group reinforced the view that a causal relationship could not be established. In addition, the usage rate of insecticides has decreased since the time of the studies that IARC considered, their general toxicity has decreased, and the products themselves have been the subject of close regulatory scrutiny. It is considered that these changes are addressing any concerns that may be raised by the IARC report.

Olfactory and hepatic changes following a single inhalation exposure of 3-trifluoromethyl pyridine in rats: concentration and temporal aspects.

The effects of a single exposure to 3-trifluoromethyl pyridine (3FMP), were investigated in two studies. In the first study, rats were exposed nose only to 0, 50 or 800 ppm 3FMP for periods of 15 min to 4 h. Half were sacrificed on day 3 and the remainder on day 10. In the second study, rats were exposed whole body to 0, 0.1, 1.0, 10 or 50 ppm 3FMP for 6 h, with sacrifices immediately after exposure (6 h), 24 h and on days 3 (48 h after exposure started) 5, 8, 11, 35, 70 and 157. Effects were seen in the olfactory epithelium at concentrations of 1 ppm and above and in the liver at concentrations of 50 ppm and above. In the olfactory epithelium the earliest changes were seen immediately after exposure and by 24 h this progressed to extensive necrosis with sloughing of the epithelium. By day 3, the epithelium was replaced by undifferentiated basophilic cells, considered to reflect early regeneration. Regeneration progressed to complete recovery between days 70 and 157, no changes were seen in the nasal respiratory epithelium, an olfactory function test on rats exposed for 6 h to 50 or 10 ppm 3FMP showed a reduced sense of olfaction at days 3 and 5 with complete recovery on subsequent days, indicating functional recovery in advance of histological normality. Single cell necrosis was seen in the liver at day 3 after 30 min exposure and immediately after 6 h exposure to 50 ppm 3FMP. At 24 h after a 6 h exposure to 50 ppm this had progressed to necrosis, haemorrhage and moderate cytoplasmic hepatocyte vacuolation in centrilobular areas. The lesion had completely recovered by day 5.

Assessment of the reproductive toxicology of bromochlorodifluoromethane (BCF, halon 1211) in the rat.

The effect of bromochlorodifluoromethane (BCF) on reproduction in the rat has been investigated in two studies. Pregnant female rats were exposed by inhalation to 1000, 10,000, or 50,000 ppm BCF for six hours a day on days six to 15 of gestation (day of mating = day 0). Exposure to 50,000 ppm BCF caused a reduction in maternal weight gain over the exposure period but there was no evidence of either teratogenicity or embryo/fetotoxicity at any concentration. In a study designed to assess the potential effect of BCF during a complete reproductive cycle male and female rats were exposed to 5000 ppm or 25,000 ppm BCF for six hours a day for five days a week for 10 weeks (males) or three weeks (females) before mating. Exposure to BCF continued during mating and up to day 20 of gestation for half the females which were subsequently allowed to litter and the development of their offspring monitored. The remaining females were removed from exposure to BCF after mating and killed on day 20 of gestation for examination of their uterine contents. There were no effects on adult fertility, pup numbers, survival, or pup development. It was concluded that BCF had no reproductive toxicity potential in the rat.

Inhalation hazard test. Interlaboratory trial with OECD method 403.

Six industrial laboratories validated the Inhalation Hazard Test (OECD Method 403) using eight volatile chemicals. The test gave similar results in all laboratories, despite variation in inhalation exposure systems and strain of rat used. Detailed atmosphere analyses are not necessary, since nominal atmosphere concentrations were close to the analysed values. The method gives reproducible results directly applicable to hazard evaluation, and is quicker and cheaper and uses fewer animals than the conventional LC50 test.

Classification of dangerous substances and pesticides in the European economic community directives: a proposed revision of criteria for inhalation toxicity.

Criteria for the classification of dangerous substances with respect to their inhalation toxicity were originally proposed by the European Economic Community (EEC) on the basis of a 1-hr LC50, as were the similar United Nations (UN) "transport" criteria. Both sets of criteria have since been amended for a 4-hr LC50, but whereas the UN criteria limits have been decreased to compensate for the increased exposure time, the EEC limits have not. This has introduced an anomaly into the EEC classification scheme whereby substances are classified more severely than they were previously. The EEC scheme is now out of line with the UN criteria and other international guidelines and gives a much more stringent toxicity classification for individual substances by inhalation than by the oral route, as well as causing an unjustifiable duplication of animal tests. This anomaly has led to a proposal by the Federal Republic of Germany for revised criteria. This paper examines the scientific basis for the relationship between inhalation exposure duration and toxicity, and for a comparison of the LD50 and LC50 classifications for individual substances. It is concluded that the proposed German revised classification scheme is more in line with the UN transport criteria and international guidelines and provides a rational basis for a classification scheme for inhalation toxicity. The classification criteria therefore should be harmonized by a revision of the EEC classification limits for inhalation toxicity.

Further studies on the toxicology of the glycol ethers with emphasis on rapid screening and hazard assessment.

The discovery that ethylene glycol monomethyl ether (EGME) could affect the testis and the developing fetus in laboratory animals prompted further work to understand the effect of EGME and to examine additional glycol ethers to see if they showed EGME's reprotoxicity. Propylene glycol monomethyl ether (PGME) was shown not to cause testicular atrophy or to affect the development of rats at 600 ppm by inhalation, whereas EGME caused testicular atrophy at 300 ppm and showed teratogenic potential at 100 ppm. Diethylene glycol monomethyl ether (diEGME) was found to show no teratogenic potential when administered subcutaneously in rats at up to 1000 microL/kg, whereas EGME had effects at 40 microL/kg. EGME has been shown to cause effects on the testis in rats after a single exposure to 600 ppm or above for 4 hr. The effects can be seen as little as 24 hr after exposure. Ethylene glycol monoethyl ether (EGEE) also causes a reduction in testicular weight following a single exposure to saturated vapor for 3 hr (17 mg/l), but ethylene glycol monoisopropyl ether (EGPE) at 15 mg/l and ethylene glycol monobutyl ether (EGBE) at 4 mg/l showed no effect on the testis.

Ethylene glycol monoethyl ether and ethylene glycol monoethyl ether acetate teratology studies.

An inhalation teratology study was conducted in rats at 10, 50 and 250 ppm ethylene glycol monoethyl ether (EGEE) and in rabbits at 10, 50 and 175 ppm EGEE. This study was designed to supplement a study conducted for NIOSH which showed teratogenic effects in rats at 200 ppm EGEE and in rabbits at about 160 ppm EE. In this study, EGEE was found not to cause teratogenic effects at concentrations up to and including 250 ppm in rats and 50 ppm in rabbits, while 175 ppm EGEE was considered to be a marginal effect level for teratogenic effects in rabbits. Fetotoxicity was observed at 250 ppm EGEE and possibly at 50 ppm EGEE in rats. An inhalation teratology study using ethylene glycol monoethyl ether acetate (EGEEA) has been conducted in rabbits at 25, 100 and 400 ppm. There was evidence of teratogenicity (vertebral malformations) at 400 ppm EGEEA and slight fetotoxicity at 100 ppm; 25 ppm was a no effect level.

The effects on the rat testis of single inhalation exposures to ethylene glycol monoalkyl ethers, in particular ethylene glycol monomethyl ether.

The effects of a single inhalation exposure to the rat of the saturated vapours derived from four ethylene glycol monoalkyl ethers have been investigated. No effects on the testis were observed following exposure to ethylene glycol isopropyl ether (EG ISOPE) and ethylene glycol butyl ether (EGBE), but there were marked reductions in testicular weight 14 days after exposure to ethylene glycol monomethyl ether (EGME) and ethylene glycol monoethyl ether (EGEE). Further studies were designed to establish the effect of a single exposure to EGME. Mature male albino rats were exposed to various levels of EGME vapour for a single 4-h period and killed 14 days later. Following this single exposure a dose-related decrease in testis weight was observed in rats exposed to 5,000, 2,500 or 1,250 ppm EGME. Histopathological examination revealed disordered spermatogenesis and tubular atrophy in these animals. Minimal degenerative changes were seen in the testis of rats exposed to 625 ppm EGME. When rats were examined at various time intervals after exposure to EGME vapour for 4 h, testis weight was reduced in rats examined 2 days after exposure to 2,500 and 1,000 ppm EGME and remained depressed when compared with control values for up to 19 days following exposure. Histopathological examination of the testis revealed disordered spermatogenesis in exposed animals evident at 1 day following exposure to either 2,500 or 1,00 ppm EGME.

Comparative aspects of the reproductive toxicology by inhalation in rats of ethylene glycol monomethyl ether and propylene glycol monomethyl ether.

This study was designed to provide a rapid assessment of the effect of two glycol ethers on some aspects of reproduction in the rat. Exposure was by inhalation at 100 and 300 ppm (EGME) and 200 and 600 ppm (PGME) for 6 hr/day. The study was in two parts: (a) Pregnant females were exposed on Days 6 to 17 of gestation. Body weight gain was reduced in both EGME groups. No litters were delivered in the 300-ppm EGME group and only 9/20 rats in the 100-ppm EGME group produced litters where the number, weight, and viability of the pups were reduced, but the pups appeared normal externally. Exposure at 200 and 600 ppm PGME had no effect. (b) Male rats were exposed for 10 days. There was a reduction in testicular weight accompanied by semeniferous tubular atrophy in the 300-ppm EGME group. There were no effects at 100 ppm EGME or 200 and 600 ppm PGME. 300 ppm EGME caused significant reductions in white blood cell count, red blood cell count, hemoglobin concentration, hematocrit, and mean cell hemoglobin. The study design proved capable of demonstrating the effects of EGME on the testes and on fetal development and highlighted PGMEs lack of activity at the doses tested.

Specific inhibition of contact sensitivity in the guinea pig following tolylene diisocyanate inhalation.

Guinea pigs were exposed by head only inhalation for 5 h to concentrations of approximately 1 ppm of tolylene diisocyanate (TDI) and 14 days later pinnal application of TDI on 3 consecutive days was followed by a dermal application of TDI after a further 7 days. The degree of contact sensitivity, measured 24 h later, showed that prior inhalation inhibited the skin reaction to TDI but it did not change the skin reaction to chloro-dinitrobenzene (DNCB) in guinea pigs sensitised and challenged similarly with DNCB. The inhibition lasted up to 9 weeks. However, it did not occur in guinea pigs after intranasal instillation (instead of inhalation) and only occurred after oral dosage with TDI when the dose was relatively high. Treatment with cyclophosphamide before TDI inhalation reversed the inhibition of contact sensitivity, suggesting an involvement of suppressor cells.

A comparison of drug-induced responses on rat tracheal, bronchial and lung strip in vitro preparations.

1. A preparation of rat trachea and a new preparation of rat bronchi are described. Both preparations are fluid-filled and the intraluminal pressure is monitored. 2. A preparation of rat peripheral airways, the lung strip, is described. The preparation consists of a thin strip of lung parenchyma which is superfused, and contractions monitored isotonically. 3. The rat trachea and bronchi have no intrinsic tone but increases in pressure are elicited in response to methacholine. The preparations relax in response to isoprenaline and aminophylline in the presence of a methacholine-induced contraction. Both preparations respond weakly and show tachyphylaxis to 5-hydroxytryptamine (5HT). 4. The lung strip contracts with equal magnitude to methacholine and 5-HT. It exhibits intrinsic tone which is inhibited by indomethacin and relaxed by isoprenaline.