The potential of organoids in toxicologic pathology: role of toxicologic pathologists in in vitro chemical hepatotoxicity assessment.

The development of in vitro toxicity assessment methods using cultured cells has gained popularity for promoting animal welfare in animal experiments. Herein, we briefly discuss the current status of hepatoxicity assessment using human- and rat-derived hepatocytes; we focus on the liver organoid method, which has been extensively studied in recent years, and discuss how toxicologic pathologists can use their knowledge and experience to contribute to the development of in vitro chemical hepatotoxicity assessment methods for drugs, pesticides, and chemicals. We also propose how toxicological pathologists should assess toxicity regarding the putative distribution of undifferentiated and differentiated cells in the organoid when liver organoids are observed in hematoxylin and eosin-stained specimens. This was done while considering the usefulness and limitations of in vitro studies for toxicologic pathology assessment.

Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of food-flavouring substances uniquely used in Japan that belong to the class of aliphatic primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated functional groups.

We performed a safety evaluation using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) of the following four flavouring substances that belong to the class of 'aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups' and are uniquely used in Japan: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. Although no genotoxicity study data were found in the published literature, none of the four substances had chemical structural alerts predicting genotoxicity. All four substances were categorised as class I by using Cramer's classification. The estimated daily intake of each of the four substances was determined to be 0.007-2.9 µg/person/day by using the maximised survey-derived intake method and based on the annual production data in Japan in 2001, 2005 and 2010, and was determined to be 0.250-600.0 µg/person/day by using the single-portion exposure technique and based on average-use levels in standard portion sizes of flavoured foods. Both of these estimated daily intake ranges were below the threshold of toxicological concern for class I substances, which is 1800 µg/person/day. Although no information from in vitro and in vivo toxicity studies for the four substances was available, these substances were judged to raise no safety concerns at the current levels of intake.

The Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of acetal food flavouring substances uniquely used in Japan.

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on five acetal flavouring substances uniquely used in Japan: acetaldehyde 2,3-butanediol acetal, acetoin dimethyl acetal, hexanal dibutyl acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal. As no genotoxicity study data were available in the literature, all five substances had no chemical structural alerts predicting genotoxicity. Using Cramer's classification, acetoin dimethyl acetal and hexanal dibutyl acetal were categorised as class I, and acetaldehyde 2,3-butanediol acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal as class III. The estimated daily intakes for all five substances were within the range of 1.45-6.53 µg/person/day using the method of maximised survey-derived intake based on the annual production data in Japan from 2001, 2005, 2008 and 2010, and 156-720 µg/person/day using the single-portion exposure technique (SPET), based on the average use levels in standard portion sizes of flavoured foods. The daily intakes of the two class I substances were below the threshold of toxicological concern (TTC) - 1800 µg/person/day. The daily intakes of the three class III substances exceeded the TTC (90 µg/person/day). Two of these, acetaldehyde 2,3-butanediol acetal and hexanal glyceryl acetal, were expected to be metabolised into endogenous products after ingestion. For 4-methyl-2-pentanone propyleneglycol acetal, one of its metabolites was not expected to be metabolised into endogenous products. However, its daily intake level, based on the estimated intake calculated by the SPET method, was about 1/15 000th of the no observed effect level. It was thus concluded that all five substances raised no safety concerns when used for flavouring foods at the currently estimated intake levels. While no information on in vitro and in vivo toxicity for all five substances was available, their metabolites were judged as raising no safety concerns at the current levels of intake.

The JFFMA assessment of flavoring substances structurally related to menthol and uniquely used in Japan.

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on four flavoring substances structurally related to menthol (L-menthyl 2-methylbutyrate, DL-menthyl octanoate, DL-menthyl palmitate, and DL-menthyl stearate) uniquely used in Japan. While no genotoxicity study data were available in the literature, all four substances had no chemical structural alerts predictive of genotoxicity. Moreover, they all four are esters consisting of menthol and simple carboxylic acids that were assumed to be immediately hydrolyzed after ingestion and metabolized into innocuous substances for excretion. As menthol and carboxylic acids have no known genotoxicity, it was judged that the JECFA procedure could be applied to these four substances. According to Cramer's classification, these substances were categorized as class I based on their chemical structures. The estimated daily intakes for all four substances were within the range of 1.54-4.71 µg/person/day and 60-1250 µg/person/day, using the methods of Maximized Survey-Derived Intake and Single Portion Exposure Technique, respectively, based on the annual usage data of 2001, 2005, and 2010 in Japan. As the daily intakes of these substances were below the threshold of concern applied to class I substances viz., 1800 µg/person/day, it was concluded that all four substances raise no safety concerns when used for flavoring foods under the currently estimated intake levels.

Assessment of developmental effects of hypothyroidism in rats from in utero and lactation exposure to anti-thyroid agents.

To clarify the developmental effects of hypothyroidism and to establish a detection system of resultant brain retardation, pregnant rats were administered 3 or 12 ppm of 6-propyl-2-thiouracil (PTU) or 200 ppm of methimazole (MMI) in the drinking water from gestation day 10 to postnatal day 20 and maintained after weaning until 11 weeks of age (adult stage). Offspring displayed evidence of growth retardation lasting into the adult stage, which was particularly prominent in males. Except for hypothyroidism-related thyroid follicular cell hypertrophy, most histopathological changes that appeared at the end of chemical exposure were related to growth retardation and reversed by the adult stage. A delayed onset of puberty and an adult stage gonadal enlargement occurred by exposure to anti-thyroid agents, both being especially evident in males, and this effect might be related to gonadal growth suppression during exposure. At the adult stage, the distribution variability of hippocampal CA1 pyramidal neurons reflecting mismigration could be detected in animals receiving both thyrotoxins, with a dose-dependent effect by PTU. Similarly, a reduction in the area of the corpus callosum and oligodendroglial cell numbers in the cerebral deep cortex, both reflecting impaired oligodendroglial development, were detected in rats administered both chemicals. Thus, all effects, except for impaired brain development, might be linked to systemic growth retardation, and the brain morphometric methods employed in this study may be useful to evaluate the potency of chemicals to induce hypothyroidism-related brain retardation.

Pathological assessment of the nervous and male reproductive systems of rat offspring exposed maternally to acrylamide during the gestation and lactation periods - a preliminary study.

To evaluate the developmental effects of exposure to acrylamide (ACR) on the nervous and male reproductive systems, pregnant Sprague-Dawley rats were given ACR at 0, 50, 100 or 200 ppm in the drinking water from gestational day 10 to postnatal day 21 and histopathological assessment of offspring was performed at weaning and postnatal week 11. Neurotoxicity was quantitatively assessed with reference to nerve fiber density, percentages of degenerated and small caliber axons in the sciatic nerves, and numbers of aberrant dot-like structures immunoreactive for synaptophysin in the cerebellar molecular layer. Although maternal neurotoxicity was evident from 100 ppm, no changes suggestive of neurotoxicity or testicular toxicity were observed in offspring. However, lowering of body weights was dose-dependently observed from birth at the dose levels of > or =50 ppm in males and > or =100 ppm in females. Maternal malnutrition was apparent at >/=100 ppm during the lactation period. Therefore, poor lactational ACR-exposure due to maternal toxicity might account for the lack of ACR-induced offspring toxicity other than retarded body growth.