Case examples of an evaluation of the human relevance of the pyrethroids/pyrethrins-induced liver tumours in rodents based on the mode of action

The CAR-mediated MOA for liver tumorigenesis is of no carcinogenic risk for humans.

Abstract

Rodent carcinogenicity studies are useful for screening for human carcinogens but they are not perfect. Some modes of action (MOAs) lead to cancers in both experimental rodents and humans, but others that lead to cancers in rodents do not do so in humans. Therefore, analysing the MOAs by which chemicals produce tumours in rodents and determining the relevance of such tumour data for human risk are critical. Recently, experimental data were obtained as case examples of an evaluation of the human relevance of pyrethroid (metofluthrin and momfluorothrin)- and pyrethrins-induced liver tumours in rats based on MOA. The MOA analysis, based on the International Programme on Chemical Safety (IPCS) framework, concluded that experimental data strongly support that the postulated MOA for metofluthrin-, momfluorothrin- and pyrethrins-produced rat hepatocellular tumours is mediated by constitutive androstane receptor (CAR) activation. Since metofluthrin and momfluorothrin are close structural analogues, reproducible outcomes for both chemicals provide confidence in the MOA findings. Furthermore, cultured human hepatocyte studies and humanized chimeric mouse liver studies demonstrated species difference between human hepatocytes (refractory to the mitogenic effects of these compounds) and rat hepatocytes (sensitive to their mitogenic effects). These data strongly support the hypothesis that the CAR-mediated MOA for liver tumorigenesis is of low carcinogenic risk for humans. In this research, in addition to cultured human hepatocyte studies, the usefulness of the humanized chimeric liver mouse models was clearly demonstrated. These data substantially influenced decisions in regulatory toxicology. In this review I comprehensively discuss the human relevance of the CAR-mediated MOA for rodent liver tumorigenesis based on published information, including our recent molecular research on CAR-mediated MOA.

Prediction of human carcinogenic risk and carcinogenicity assays in animals

In the 1950s and 1960s, several human carcinogenic chemicals were identified as rodent carcinogens. Thus, the two-year bioassay was developed to provide a standardized screening procedure for evaluating chemicals with the assumption that these were predictive of human carcinogenic risk. In utilizing experimental animals as a bioassay screening model, two fundamental assumptions are made: (1) the results observed in the animal model are relevant to humans (species extrapolation); and (2) the dose administered to the animals is relevant to the exposure levels in humans (dose extrapolation).1 The validity of these assumptions for the two-year bioassay had been based on the tumour response for potent DNA reactive carcinogens. However, for non-genotoxic chemicals, one or both of these assumptions have been demonstrated to be incorrect.1

Five decades after the performance of two-year bioassays, it was revealed that about half of all the chemicals tested were positive in the standard rodent high-dose cancer bioassay, no matter whether natural or synthetic chemicals were used.2,3 Not only were numerous food ingredients, natural and synthetic shown to be positive in the two-year bioassay, but also a large number of pharmaceutical agents, consumer products and environmental chemicals were positive. Nevertheless, many of these chemicals have continued in commercial use because of significant differences between the mode of action (MOA) in the animals versus humans and/or differences in exposure.1 A survey showed a compendium of information retrieved on carcinogenicity, in animals and humans, of 535 marketed pharmaceuticals whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time.3 Of these 535 drugs, 530 have the results of at least one carcinogenicity assay in animals, and 279 (52.1%) of them gave a positive response in at least one assay. With respect to the correlation between the results obtained in animals and epidemiological findings, 58 drugs (31.2%) displayed at least one positive result in animals and also to a variable extent showed epidemiological findings of potential carcinogenicity in humans.3 However, 53 drugs (28.5%) gave at least one positive result in animals in the absence of positive findings in humans.3 Some of these positive results have been for agents that are widely used by humans, such as statins (liver tumours) and proton pump inhibitors (stomach tumours), but lack carcinogenicity in humans as confirmed in multiple large-scale epidemiology studies.1

Evaluation of the modes of action by which chemicals produce tumours in rodents

Different carcinogens may have different MOAs. Some MOAs lead to cancers in both experimental rodents and humans, but others that lead to cancers in rodents do not do so in humans, at least under realistic circumstances of human exposure. A particular concern was the increased incidence of tumours in certain tissues, such as the liver and lung in mice, and the liver, mammary gland, and various endocrine organs in rats.1 However, several of the tumours identified in the rodent models were demonstrated to occur by MOAs that were not relevant to humans.1 Such examples of non-relevant MOAs include the production of calcium phosphate-containing urinary precipitate by sodium saccharin and other sodium salts leading to the induction of bladder tumours in rats, d-limonene and the induction of kidney tumours in male rats by binding to α2u-globulin, ethyl acrylate induction of forestomach tumours in rodents, and a variety of others.4,5 Therefore, without additional data on how a chemical causes cancer, the interpretation of a positive result in a rodent bioassay is highly uncertain.2

To improve the process of carcinogen hazard identification and to avoid misidentification of harmless substances as possible human carcinogens, it has become imperative that a MOA analysis be undertaken, and that data to support such analyses be collected in a thorough and scientifically rigorous manner. Determining the relevance of tumours in the bioassay to humans had to be determined in follow-up mechanistic research.1,4 In recent years, frameworks for analysing the MOAs by which chemicals produce tumours in rodents and the relevance of such tumour data for human risk have been developed through the International Life Sciences Institute (ILSI)6–8 and the International Programme on Chemical Safety (IPCS).9–11 In terms of the human relevance of an animal carcinogenic MOA there are three questions to consider before reaching a conclusion, as shown in the ILSI/IPCS framework (Table 1).

Table 1. Framework for analyzing the cancer mode of action.

1. Is the weight of evidence sufficient to establish a mode of action in animals?

A. Postulated mode of action (MOA) for the induction of tumor in experimental animals

B. Key events in experimental animals

C. Concordance of the dose–response relationship

D. Temporal association

E. Strength, consistency, and specificity of association of tumor response with key events

F. Biological plausibility and coherence

G. Other modes of action

H. Uncertainties, inconsistencies, and data gaps

I. Assessment of the postulated mode of action

J. Human applicability of the proposed mode of action

2. Can human relevance of the mode of action be reasonably excluded on the basis of fundamental, qualitative differences in key events between experimental animals and humans?

3. Can human relevance of the mode of action be reasonably excluded on the basis of quantitative differences in either kinetic or dynamic factors between experimental animals and humans?

Liver carcinogenesis

The liver is by far the most common target tissue affected in the rodent bioassay.4,12–14The various MOAs that have been specifically identified in rodents for hepatocellular carcinogenesis are listed in Table 2.15 For liver carcinogenesis, the MOAs can be divided into those that are DNA reactive and those that are non-DNA reactive. For non-DNA reactive MOAs, they can be divided into those that act through specific receptors versus those that do not. Furthermore, as for all non-DNA reactive MOAs, the increase in cell proliferation can either be due to an increase in cell births or a decrease in cell deaths, which results in the accumulation of more cells. Increased cell births can be produced by cytotoxicity and regeneration or by direct mitogenesis. Details of this were reviewed by Cohen and Arnold.4 Since essential key events for carcinogenesis should precede the appearance of the tumours, these MOAs can readily be identified in short-term evaluations ranging from a few days to a few months.4,16

Table 2. Modes of action for hepatocellular carcinogenesis.

I. DNA reactivity

II. Increased cell proliferation

A. Receptor mediated

1. PPARα (peroxisome proliferation)

2. Enzyme induction (CAR, PXR, AHR)

3. Estrogen

4. Statins

5. Cytotoxicity

6. Other

B. Non-receptor mediated

1. Cytotoxicity

2. Infectious

3. Iron (copper) overload

4. Increased apoptosis (e.g., fumonisin B1)

5. Other

Regarding human relevance of the MOAs identified for hepatocellular carcinogenesis, genotoxicity, a known MOA for certain liver carcinogens such as aflatoxin, is relevant to humans.4 In addition, it is known that estrogen is related to the rare development of hepatocellular carcinomas in humans, evolving in those circumstances from adenomas.4 In humans, the most common MOAs for induction of hepatocellular carcinomas involve cytotoxicity, inflammation, and regenerative hepatocellular proliferation, whether due to inherited disorders, iron overload, or viral infection.4 The MOAs such as constitutive androstane receptor (CAR) activation, peroxisome proliferator-activated receptor alpha (PPARα) activation, or exposure to statins are generally considered not relevant to humans.4,17–21

Pyrethroids/pyrethrins-produced hepatocellular tumour in rodents

Pyrethroids are a class of synthetic insecticides designed and optimized based on the structure of the pyrethrins found in natural pyrethrum extracted from chrysanthemum flowers. Pyrethroids are widely used to control insect pests in agriculture and public health because of their relative safety for humans and high insecticidal potency. Since pyrethroids, including pyrethrins, have been used for many years, it is important to evaluate their carcinogenicity in humans. The Agency for Toxic Substances and Disease Registry (ATSDR) has provided an excellent review entitled “Toxicological Profile for Pyrethrins and Pyrethroids”.22 According to this review, no reports were located regarding cancer in humans or domestic animals following inhalation or dermal exposure to pyrethrins or pyrethroids. However, in the case of oral exposure to these chemicals, while no reports were located regarding cancer in humans, pyrethrins and some pyrethroids have been shown to cause tumours in rodent models as shown in Table 3.23 Of these, the liver was the most common target tissue of the pyrethroids. This may be because the liver is the major site of metabolism of these chemicals, and furthermore, the liver is the first organ exposed to the chemical following absorption from the gastrointestinal tract if administered orally. These results are not specific to pyrethroids,12 and therefore, these data indicate that tumour induction does not appear to reflect a common carcinogenic endpoint for this particular subset of compounds. Instead, tumorigenic responses appear to be specific to the compound, dose levels and test organism employed.

Table 3. Possible tumorigenic target organs in carcinogenicity studies with pyrethroids and pyrethrins.

Possible tumorigenic target organs	Pyrethroids/pyrethrins
Kidney	Allethrin (R)
Liver	Bifenthrin (M), Metofluthrin (R), Momfluorothrin (R), Permethrin (M), Pyrethrins (R), Resmethrin (R&M), Tefluthrin (M), Tetramethrin (M), Transfluthrin (M)
Lung	Bifenthrin (M), Cypermethrin (M), Permethrin (M)
Leukaemia	Bifenthrin (M)
Testis	Fenvalerate (R), Tetramethrin (R)
Thyroid	Pyrethrins (R), Etofenprox (R)
Urinary bladder	Bifenthrin (M), Transfluthrin (R)
Uterin	Tefluthrin (R)
None (R&M)	Bioresmethrin, Cyfluthrin, Cyhalothrin, Cyphenothrin, Deltamethrin, Esfenvalerate, Fenprorathrin, d-Phenothrin, Prallethrin, Tau-fluvalinate, Tralomethrin

Recently, we have gained experience with two different pyrethroids: epsilon-metofluthrin (CAS-No. 240494-71-7; 2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (1R,3R)-2,2-dimethyl-3-[(1Z)-prop-1-en-1-yl]cyclopropanecarboxylate; referred to as metofluthrin in this article) and epsilon-momfluorothrin (CAS no. 1065124-65-3; 2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl(Z)-(1R,3R)-3-(2-cyanoprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate, referred to as momfluorothrin in this article), which produced hepatocellular tumours in rat carcinogenicity studies.24,25 The carcinogenicity of metofluthrin and momfluorothrin has been studied in male and female rats and mice in standard bioassays under the Good Laboratory Practice guidelines and OECD test protocols. These studies were conducted at the same laboratory (at that time, Harlan Laboratories Ltd, Itingen/Switzerland) but at different times (Table 4).

Table 4. Summary of a carcinogenicity study in rats and mice with metofluthrin and momfluorothrin.

Metofluthrin25

Male and female HanBrl:WIST rats were fed 0 (control), 20, 200, 900 or 1800 ppm metofluthrin in the diet for two years (average daily chemical intakes were 0.84, 8.24, 38.1 and 77.8 mg kg^–1 day^–1 for males; 1.03, 10.1, 47.4 and 96.1 mg kg^–1 day^–1 for females, respectively). A summary of the results of this bioassay is presented in Table 4. The incidence of hepatocellular adenomas and/or carcinomas was significantly increased in male rats given 900 or 1800 ppm metofluthrin and in female rats given 1800 ppm metofluthrin. The combined incidences of hepatocellular adenomas and carcinomas of the 0, 20, 200, 900 and 1800 ppm groups were, respectively, 2, 2, 6, 16, and 24% for males and 2, 6, 2, 10, and 24% for females. The nontumorigenic dose levels (no observed effect levels (NOEL) for tumours) in male and female rats were established at 200 ppm and 900 ppm, respectively. In contrast, metofluthrin was not carcinogenic in the liver or any other tissue in male and female CD-1 mice when administered for 78 weeks at dietary levels of 100, 1000, and 1750/2500 ppm (average chemical intakes: 11.8, 116 and 209 mg kg^–1 day^–1 for males and 15.4, 155 and 277 mg kg^–1 day^–1 for females, respectively).

Momfluorothrin24

The incidences of (the total number of animals with) hepatocellular adenomas and/or carcinomas in the 0, 200, 500, 1500 and 3000 ppm groups were 2, 0, 4, 12 and 33% for males, and 0, 0, 2, 2 and 10% for females, respectively (Table 4). Treatment with 3000 ppm momfluorothrin significantly increased the incidence of hepatocellular adenoma in both sexes and that of hepatocellular carcinoma in male rats. The combined incidence of hepatocellular adenoma and carcinoma was significantly increased in male and female rats given 3000 ppm momfluorothrin, with a non-statistically significant increase observed in male rats given 1500 ppm momfluorothrin. The incidences of hepatocellular adenoma, carcinoma, and combined incidence in males given 1500 and 3000 ppm momfluorothrin were equivalent to or higher than the maximum incidence of the historical background; the combined incidence of female rats given 3000 ppm momfluorothrin was within the historical background incidence, while the incidence of carcinoma was equivalent to the maximum incidence of the historical background. Overall, treatment with momfluorothrin in rats for 2 years produced hepatocellular tumours in males at 1500 and 3000 ppm and in females at 3000 ppm. The nontumorigenic dose levels in male and female rats were established at 500 ppm and 1500 ppm, respectively. In contrast, momfluorothrin was not carcinogenic in the liver or any other tissue in male and female CD-1 mice when administered for 78 weeks at dietary levels of 600, 2500 and 5500 ppm (average daily chemical intakes: 72, 308 and 639 mg kg^–1 day^–1 for males and 99, 427 and 853 mg kg^–1 day^–1 for females, respectively).

MOA analysis for metofluthrin- and momfluorothrin-induced rat hepatocellular tumour formation

The MOA analysis had been conducted based on the IPCS framework.9 Based on existing general toxicity data, the MOA for metofluthrin-induced rat liver tumour formation is postulated to involve activation of the CAR, which results in a pleiotropic response including the stimulation of cytochrome P450 (CYP) CYP2B subfamily enzymes, liver centrilobular hypertrophy and increased cell proliferation. Prolonged treatment results in the formation of altered hepatic foci and subsequently of liver tumours. This MOA is well known for certain other non-genotoxic agents which are CAR activators.17 Since momfluorothrin is a close structural analogue of metofluthrin, a CAR-mediated MOA was also postulated. The key and associative events in the CAR-mediated MOA for rodent liver tumour formation are shown in Table 5. Activation of CAR, altered gene expression specific to CAR activation, increased cell proliferation and clonal expansion leading to altered hepatic foci are considered to be key events in the MOA for tumour formation as they constitute necessary steps in the MOA.17 Induction of CYP2B enzymes and liver hypertrophy (both morphological changes and increases in liver weight) are considered to be associative events and as such represent reliable markers of CAR activation.17

Table 5. Key and associative events in the CAR-mediated mode of action for rodent hepatocellular tumor production.

Key events	Associative events
Definition: an empirically observable causal precursor step to the adverse outcome that is itself a necessary element of the MOA.	Definition: a biological process that is not a causal necessary key event for the MOA, but is a reliable indicator or marker for a key event.
Key 1. CAR activation	• Altered epigenetic changes specific to CAR activation
Key 2. Altered gene expression specific to CAR activation	• Liver hypertrophy
Key 3. Increased hepatocellular proliferation	• Hepatic CYP2B induction
Key 4. Clonal expansion leading to altered hepatic foci	• Decreased apoptosis
Key 5. Hepatocellular adenomas/carcinomas

A number of investigative studies were performed to obtain data on the proposed key and associative events.24–27 In some MOA studies, rats were also given sodium phenobarbital (NaPB), which is known to activate CAR in the rat and to produce a variety of hepatic effects including CYP2B enzyme induction and increased cell proliferation.17 Phenobarbital (PB) (or NaPB) served as a positive control to confirm the potential responsiveness of the animals used in these MOA studies to a known CAR activator. The IPCS requirements for analyzing cancer MOA are summarized in previous publications24,28 and ESI Table† in this review. A dose–response and temporality concordance table for metofluthrin- and momfluorothrin-produced liver tumours is shown in Table 6. The key and associative events in the postulated MOA for these two pyrethroid-produced liver tumours have been established, with a strong dose–response and temporal consistency. The present data are considered adequate with a high degree of confidence to explain the development of liver tumours in rats following chronic administration of metofluthrin and momfluorothrin.

Table 6. Temporality and dose–response for MOA key events related to male and female Wistar rat liver tumors.

Temporality		Early events				Late events
		Key events #1		#2	#3	#4	#5
		CAR activation		Altered gene expression specific to CAR activation	Increased hepatocellular proliferation	Clonal expansion leading to altered hepatic foci	Hepatocellular adenomas/carcinomas
		Biomarker: CYP2B mRNA and/or activity	Associative event: Increased liver weight and/or hypertrophy
Compounds, sex	Dose (ppm)
Metofluthrin, males	20	ND	–(52 & 104 W)	ND	ND	–(52 & 104 W)	–(52 & 104 W)
	200	–(1 W)	–(1, 52 & 104 W)	ND	–(1 W)	–(52 & 104 W)	–(52 & 104 W)
	900	–(1 W) a	–(1 & 104 W), +(52 W)	ND	+(1 W)	–(52 W), +(104 W) b	–(52 W), +(104 W)
	1800	+(1 W)	+(1, 52 & 104 W)	+(1 W)	+(1 W)	–(52 W), +(104 W)	–(52 W), +(104 W)
Metofluthrin, females	20	ND	–(52 & 104 W)	ND	ND	–(52 & 104 W)	–(52 & 104 W)
	200	–(1 W)	–(1, 52 & 104 W)	ND	–(1 W)	–(52 & 104 W)	–(52 & 104 W)
	900	+(1 W)	–(1 W), +(52 & 104 W)	ND	–(1 W)	–(52 & 104 W)	–(52 & 104 W)
	1800	+(1 W)	+(1, 2, 52 & 104 W)	ND	+(1 W) c	–(52 W), +(104 W) d	–(52 W), +(104 W)
Momfluorothrin, males	200	–(1 W)	–(1, 52 & 104 W)	ND	–(1 W)	–(52 & 104 W)	–(52 & 104 W)
	500	–(1 W)	–(1, 52 & 104 W)	ND	–(1 W)	–(52 & 104 W)	–(52 & 104 W)
	1500	–(1 W) e	+(1, 52 & 104 W)	ND	+(1 W)	–(52 & 104 W)	–(52 W), +(104 W) g
	3000	+(1 W)	+(1, 2, 13, 52 & 104 W)	+(2 W)	+(1 W) f	–(52 W), +(104 W)	–(52 W), +(104 W)
Momfluorothrin, females	200	–(1 W)	–(1, 52 & 104 W)	ND	–(1 W)	–(52 & 104 W)	–(52 & 104 W)
	500	–(1 W)	+(52 W), –(1 & 104 W)	ND	–(1 W)	–(52 & 104 W)	–(52 & 104 W)
	1500	+(1 W)	+(1, 52 & 104 W)	ND	+(1 W)	–(52 & 104 W)	–(52 & 104 W)
	3000	+(1 W)	+(1, 2, 13, 52 & 104 W)	ND	+(1 W) f	–(52 W), +(104 W)	–(52 W), +(104 W)

^aThe increased CYP2B mRNA at 1 week in males at 900 ppm of metofluthrin was a marginal change without statistical significance (2.9-fold of the control).

^bThe increased incidence of eosinophilic foci (3/50, control 1/50) at 104 week in males at 900 ppm of metofluthrin was not statistically significant but the increased incidence of mixed foci (9/50, control 1/50) was statistically significant.

^cWhile hepatocyte labeling index values, determined as BrdU labeling index, in females at 1800 ppm of metofluthrin returned toward control levels with continued metofluthrin treatment for 14 days, the number of cell replications in treated animals appeared to be enhanced due to the increased total number of hepatocytes per animal. In this table, “Increased cell proliferation” means “Increased total cell proliferation”.

^dThe increased incidence of eosinophilic foci (6/49, control 3/49) at 104 week in females at 1800 ppm of metofluthrin was not statistically significant but the increased incidence of mixed foci (12/49, control 2/49) was statistically significant.

^eThe increased PROD activity at 1 week in males at 1500 ppm of momfluorothrin was a marginal change without statistical significance (1.2-fold of the control).

^fThe increased hepatocyte labeling index values, determined as BrdU labeling index, in both sexes at 3000 ppm of momfluorothrin were also observed at 2 weeks but were less than those of 1 week and thus may have returned toward control levels with continued momfluorothrin treatment for more than 14 days, the number of cell replications in treated animals appeared to be enhanced due to the increased total number of hepatocytes per animal.

^gThe combined incidence of hepatocellular adenoma and carcinoma was increased in male rats given 1500 ppm momfluorothrin, with a non-statistically significant increase. As the incidences of hepatocellular adenoma, carcinoma, and combined in males given 1500 ppm momfluorothrin were equivalent to or higher than the maximum incidence of the historical background, 1500 ppm was concluded as the tumor inducing dose level.

The postulated MOA is similar to that of certain other non-genotoxic agents which are CAR activators.17,19,29–33 Furthermore, multiple alternative MOAs have been excluded. Overall, these data strongly support that the postulated MOA for metofluthrin- and momfluorothrin-produced rat hepatocellular tumours is mediated by CAR activation.24,28

Non-genotoxic chemicals which are activators of CAR may produce liver tumours in both rats and mice.29 Although the mouse appears to be more susceptible than the rat to liver tumour formation by such compounds, not all non-genotoxic CAR activators which produce liver tumours in the rat also produce tumours in the mouse. For example, the natural pyrethrins, which are known to produce liver tumours in the rat through activation of CAR, do not produce liver tumours in the mouse.30 Rat specific liver tumour production is not specific to pyrethroids. Metazachlor (a chloroacetanilide herbicide) and fluopyram (a fungicide) also produced liver tumours in Wistar rats but not in CD-1 mice.34,35

Evaluation of the human relevance of the CAR-mediated MOA for rat hepatocellular tumour formation by metofluthrin and momfluorothrin

Since increased hepatocellular proliferation, determined as replicative DNA synthesis, is considered to be the critical key event for CAR-mediated hepatocellular tumorigenesis,17 it is important to determine whether the test compound has a mitogenic effect on human hepatocytes or not. As mentioned above, numerous publications have mentioned that the CAR-mediated MOA is qualitatively not relevant to humans, based on the lack of the key event of an increased cell proliferation in hepatocellular tumorigenesis induced by the prototypic CAR activator PB.15,17,19,20,29–33,36–39

A cultured human hepatocyte study

To evaluate the potential human carcinogenic risk of metofluthrin and momfluorothrin, the effects of these compounds on hepatocyte replicative DNA synthesis and CYP2B mRNA expression (an associative event, as a marker of CAR activation) were examined in cultured rat and human hepatocyte preparation.26,27,40 The effect of PB was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis or Ki-67 mRNA in rat and human hepatocytes. However, while PB, metofluthrin and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, none of these three compounds increased replicative DNA synthesis in human hepatocytes. PB, metofluthrin and momfluorothrin increased CYP2B1/2 mRNA levels in rat hepatocytes and also increased CYP2B6 mRNA levels in human hepatocytes, indicating that there was CAR activation. Overall, metofluthrin, momfluorothrin and PB activated CAR in cultured human hepatocytes, but none of these chemicals increased replicative DNA synthesis. As human hepatocytes are refractory to the mitogenic effects of metofluthrin and momfluorothrin, in contrast to rat hepatocytes, these data support the hypothesis that the MOA for metofluthrin- and momfluorothrin-produced rat liver tumours is not relevant to humans.26,27,40

Chimeric liver mouse study

To confirm whether the findings observed in the in vitro cultured human hepatocyte study were also observed in vivo, a humanized chimeric liver mouse study was conducted.27 Recently, mice with human hepatocyte chimeric livers have been produced by transplanting human hepatocytes into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic/severe combined immunodeficient (uPA/SCID) mice.41–45 The host mouse hepatocytes are replaced with human hepatocytes in the livers of the chimeric mice. Human hepatocytes in the livers of chimeric mice are susceptible to growth enhancing activities. The treatment of chimeric mice with human growth hormone (hGH) increases the repopulation speed and the replacement index of transplanted human hepatocytes, as determined by increased replicative DNA synthesis and the up-regulation of hGH-related signalling molecules.46 Furthermore, treatment with epidermal growth factor33 and partial hepatectomy (personal communication, Dr Chise Tateno) also enhanced human hepatocellular proliferation in this chimeric liver mouse model. These findings demonstrated that transplanted human hepatocytes in chimeric mice are responsive to hepatocyte mitogens.

Previously, we had reported that human hepatocytes support the hypertrophic but not the hyperplastic response to PB in an in vivo study using a chimeric mouse with human liver cells.33 The treatment of chimeric mice with 1000–1500 ppm PB resulted in plasma levels 3–5-fold higher than those observed in human subjects given therapeutic doses of PB.47 PB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in wild-type rats (Wistar Hannover), wild-type mice (CD-1) and humanized chimeric mice, indicating that the cells can undergo CAR activation. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to PB in the humanized mouse liver were clearly different from those in rodents. Although PB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originating areas of mouse chimeric liver. In addition, treatment with PB had no effect on Ki-67, PCNA, GADD45, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment with epidermal growth factor. Thus, although PB could activate CAR in both rodent and human hepatocytes, PB did not increase replicative DNA synthesis in human hepatocytes of the mouse chimeric liver, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of PB, the present data suggest that the MOA for PB-induced rodent liver tumour formation is thus not relevant for humans,33 supporting previous conclusions.15,17,19,20,29–32,36–39

Recently, in addition to cultured human hepatocytes, the effects of 7-day treatment with metofluthrin (1800 ppm) and momfluorothrin (3000 or 1100 ppm) on replicative DNA synthesis in chimeric mice transplanted with human hepatocytes from three different donors were examined.27 Significant increases of mouse Cyp2b10 mRNA expression in the liver demonstrated that treatment with metofluthrin or momfluorothrin significantly stimulated CAR in mouse hepatocytes of chimeric mice. Increased levels of human CYP2B6 mRNA were also observed in the liver of chimeric mice, but the effects were relatively weak. Under these conditions, treatment with metofluthrin or momfluorothrin did not result in any increases in replicative DNA synthesis of human hepatocytes. However, as a positive control, hEGF treatment increased replicative DNA synthesis in human hepatocytes of chimeric mice. As human hepatocytes are refractory to the mitogenic effects of metofluthrin and momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for metofluthrin- and momfluorothrin-produced rat hepatocellular tumour formation is not relevant to humans.27

Statement of confidence, analysis, and implications

In assessing the relevance of animal MOA data to humans, a concordance table has been suggested to be of considerable value.7,9 Such a table is presented in Table 7. This includes not only the data for the effects of metofluthrin and momfluorothrin in the rats and human hepatocytes, but also the available data of PB in humans.

Table 7. Comparison of key and associative events for metofluthrin and momfluorothrin hepatocellular tumor formation in rats and humans.

Key (K) and associative (A) event	Evidence in rats	Evidence in humans
CAR activation (K)	Yes	Possible at high doses (Inferred from induction of CYP2B mRNA in vitro in cultured hepatocytes and in vivo in chimeric liver mice with human hepatocytes)
Altered gene expression specific to CAR activation (K)	Yes	Possible at high doses (Experimental evidence in vivo in chimeric liver mice with human hepatocytes treated with PB)
Altered epigenetic changes specific to CAR activation (A)	Yes	Not available.
Induction of CYP2B (A)	Yes	Probable at high doses (Experimental evidence in vitro in cultured hepatocytes and in vivo in chimeric liver mice with human hepatocytes)
Hypertrophy (A)	Yes	Possible at very high doses # (Experimental evidence in vivo in chimeric liver mice with human hepatocytes treated with PB)
Increased hepatocellular proliferation (K)	Yes	Not predicted to occur (Not observed in cultured hepatocytes and in vivo in chimeric liver mice with human hepatocytes)
Clonal expansion leading to altered hepatic foci (K)	Yes	Not predicted to occur
Hepatocellular adenomas/carcinomas (K)	Yes	Not predicted to occur

Based on the data described above, there is strong evidence to support a plausible MOA for metofluthrin- and momfluorothrin-produced hepatocellular tumour formation in male and female rats. This MOA involves activation of CAR, resulting in increased cell proliferation and the development of altered hepatic foci. The induction of CYP2B enzymes and liver hypertrophy comprise associative events and represent reliable markers of CAR activation. Another CAR activator, PB, is not considered to produce an increase in human hepatocellular proliferation, and therefore, would not result in an increase in hepatocellular tumours in humans, which is also supported by epidemiological data.17,20,48,49 Hence, by analogy with the CAR activator PB, it is considered that the proposed MOA for metofluthrin- and momfluorothrin-produced rat hepatocellular tumour formation is not qualitatively plausible for humans. Consequently, metofluthrin and momfluorothrin are of low carcinogenic risk for humans. Our proposal from this assessment with these compounds is in agreement with the classification done by the regulatory authorities such as ECHA, US EPA, etc.50–52

Evaluation of the human relevance of the CAR-mediated MOA for rat hepatocellular tumour formation by pyrethrins30,53,54

Pyrethrum has been used for many years as an insecticide for household, agricultural, and other applications. The six active insecticide components of Pyrethrum comprise two groups of Pyrethrins, namely Pyrethrin I (pyrethrin I, cinerin I and jasmolin I) and Pyrethrin II (pyrethrin II, cinerin II, and jasmolin II). A pyrethrin mixture (pyrethrin I 46.9%, pyrethrin II 23.0%, cinerin I 14.0%, cinerin II 7.8%, jasmolin I 4.2% and jasmolin II 4.1%) was used in the bioassay and the MOA study. In a rat carcinogenicity study, male and female Sprague-Dawley rats were fed 0 (control), 100, 1000 or 3000 ppm pyrethrins in the diet for two years (average daily chemical intakes were 0, 4.37, 42.9, and 130 mg kg^–1 day^–1 for males and 0, 5.39, 55.5, and 173 mg kg^–1 day^–1 for females, respectively). A small increase in the incidence of hepatocellular adenoma was observed in female rats given 3000 ppm pyrethrins. Treatment with pyrethrins did not affect the incidence of hepatocellular carcinoma in female rats and had no significant effect on the incidence of hepatocellular adenoma or carcinoma in male rats. Furthermore, a significantly increased combined incidence of thyroid follicular cell adenomas and/or carcinomas was observed in male rats given 1000 and 3000 ppm pyrethrins and in female rats given 3000 ppm pyrethrins. The nontumorigenic dose levels in male and female rats were established at 100 ppm and 1000 ppm, respectively. In a mouse 18-month study, pyrethrins were not carcinogenic to male or female CD-1 mice when administered at dietary levels of 0 (control), 100, 2500, and 5000 ppm (average daily chemical intakes: 0, 13.8, 346, and 686 mg kg^–1 day^–1 for males and 0, 16.6, 413, and 834 mg kg^–1 day^–1 for females, respectively).

To understand the MOA for liver tumor formation, the hepatic effects of pyrethrins have been investigated.53,54 Male Sprague-Dawley rats were fed diets containing 0 (control) and 8000 ppm pyrethrins and female rats diets containing 0, 100, 3000, and 8000 ppm pyrethrins for periods of 7, 14, and 42 days, and 42 days followed by 42 days of reversal. Rats were also fed diets containing 1200–1558 ppm sodium phenobarbital for 7 and 14 days as a positive control. The treatment of male rats with 8000 ppm pyrethrins, female rats with 3000 and 8000 ppm pyrethrins, and both sexes with PB resulted in increased liver weights, which were associated with hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis was also increased on treatment with pyrethrins and PB. Treatment of male and female rats with pyrethrins and PB produced significant increases in hepatic CYP content and a marked induction of CYP2B-dependent 7-pentoxyresorufin O-depentylase and testosterone 16β-hydroxylase activities. Significant increases were also observed in CYP3A-dependent testosterone 6β-hydroxylase activity. The hepatic effects of pyrethrins were dose-dependent in female rats, with 100 ppm being a no-effect level, and on cessation of treatment the effects were reversible in both sexes. This study demonstrates that the postulated MOA for pyrethrins-induced rat liver tumor formation is similar to that of certain other non-genotoxic agents which are CAR activators.30 Other possible MOAs including mutagenicity, cytotoxicity, hepatic peroxisome proliferation, porphyria, and hormonal perturbation were excluded.30 Furthermore, while pyrethrins increased CYP2B enzymes in both cultured rat and human hepatocytes, increased replicative DNA synthesis was only observed in cultured rat hepatocytes and not in cultured human hepatocytes.54,55 Therefore, by analogy with the CAR activator PB, it is considered that the proposed MOA for pyrethrins-produced rat hepatocellular tumour formation is not qualitatively plausible for humans.30,55

Using mode of action information for regulatory decision-making

The classification and labelling of certain hazardous chemicals including carcinogenicity must be harmonized to ensure adequate risk management.56 The Committee for Risk Assessment (RAC) prepares the opinions of the European Chemicals Agency (ECHA) related to the risks of substances to human health and the environment following REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) and the Classification, Labelling and Packaging (CLP) processes (; https://echa.europa.eu/about-us/who; -we-are; /committee-for-risk-assessment). The RAC examines the available evidence for all hazard classes proposed and may consider another category to be more appropriate for the classification of the substance after having examined the available information. Metofluthrin and momfluorothrin have been evaluated by the RAC.51,52

Regarding the carcinogenicity of metofluthrin, the RAC discussed whether the MOA related to liver tumours observed in rats are relevant to humans. Based on a detailed analysis of the Rapporteur, the Committee confirmed that CAR activation was the most plausible mechanism behind liver tumour formation in the rat. This MOA was considered by the RAC to be relevant for humans, but it was also recognized that the last key event in this MOA (the induction of hepatocellular proliferation, a prerequisite for tumour formation) was not observed in human cells. Based on these findings, the RAC concluded that a classification for carcinogenicity was not justified for metofluthrin.51 It was finally noted that the findings and the conclusions drawn were consistent with the close structural analogue momfluorothrin also evaluated by the RAC based on detailed data for the MOA analysis of momfluorothrin rat liver tumorigenicity.52

Apart from metofluthrin and momfluorothrin, the RAC has recently evaluated several compounds for which the applicants have proposed the CAR-mediated MOA in rodent liver carcinogenicity and evaluated their human relevance (Table 8). According to the reports in the Web, the RAC appears to base conclusions on a weight of evidence policy. For the evaluation of the postulated MOA, the IPCS framework9 is applied. Regarding human relevance of the CAR-mediated MOA, most member states appear to agree with the conclusions of the Nuclear Receptor Workshop and the Toxicology Forum Workshop on nongenotoxic modes of action that the CAR-mediated MOA is qualitatively not relevant to humans, based on the lack of the key event of an increased cell proliferation in PB-induced hepatocellular tumorigenesis.17,20 However, some member states appear not to be in agreement with this conclusion.57

Table 8. Case examples of an evaluation by the Committee for Risk Assessment (RAC) of the opinions of the European Chemicals Agency (ECHA) on the human relevance of the chemical-induced liver tumours in rodents based on the mode of action proposed as “CAR-mediated”.

Compounds	Animals observed for liver tumorigenicity	RAC conclusion on classification for carcinogenicity	Key issues in decision making	Published Ref.
Carbetamide	Mouse	Carc. 2-H351(CLP) (RAC, 2015)84	For liver and thyroid tumours, carbetamide leads to sustained liver activation, likely via interaction with the CAR or PXR-receptor. However, cell proliferation was not investigated in mouse and human hepatocytes. In addition to these, several rare tumors including carcinomas occurred in different tissues (brain astrocytoma, liver cholangiosarcoma and adrenal phaeochromocytoma) in mice, and rats were observed at the high dose of carbetamide (exceeding MTD).
Cyproconazole	Mouse	No classification (RAC, 2015)85	Species difference in hepatocellular proliferation was observed: an increase in mouse hepatocytes but not in cultured human hepatocytes and in hepatocytes of hPXR/hCAR mice (a very slight and non-statistically significant increase).	66, 86–88
Etridiazole	Mouse, rat	Carc. 2-H351(CLP) (RAC, 2013)89	Not enough evidence for MOA identification: different enzyme induction patterns to PB, and cell proliferation data are not available. Thyroid and testis tumours were also observed in rat.
Fluopyram	Rat	No classification (RAC, 2014)90	Species difference of hepatocellular proliferation: increased in mouse hepatocytes but not increased in cultured human hepatocytes.	35
Imazalil	Mouse, rat	Carc. 2-H351(CLP) (RAC, 2013)91	Not increased cell proliferation in cultured human hepatocytes but increased in hepatocytes of hPXR/hCAR mice.	92–94
Metazachlor	Rat	Carc. 2-H351(CLP) (RAC, 2011)95	Data are not yet sufficient to conclude that CYP mediated CAR activation is the only critical key event. Kidney tumours were also observed in mice.
Metofluthrin	Rat	No classification (RAC, 2016)96	Species difference of hepatocellular proliferation: increased in rat hepatocytes but not increased in cultured human hepatocytes and human hepatocytes of humanized liver chimeric mice.	25–28 and 40
Momfluorothrin	Rat	No classification (RAC, 2015)97	Species difference of hepatocellular proliferation: increased in rat hepatocytes but not increased in cultured human hepatocytes and human hepatocytes of humanized liver chimeric mice.	24 and 27
Propiconazole	Mouse	No classification (RAC, 2016)98	Species difference of CAR activation: activated in mouse and human but responsiveness of human CAR to propiconazole was much weaker than in mice.	66, 86, 87, 99–101
			Species difference of hepatocellular proliferation: increased in mouse hepatocytes but not increased in cultured human hepatocytes
Sulfoxaflor	Mouse, rat	No classification (RAC, 2013)102	Species difference of hepatocellular proliferation: increased in rodent hepatocytes but not increased in cultured human hepatocytes and hepatocytes of hPXR/hCAR mice.	31 and 71

As shown in Table 8, if no significant increase of hepatocyte proliferation was observed in human hepatocytes, the compounds were concluded to be “Not classified” (cyproconazole, fluopyram, metofluthrin, momfluorothrin, propiconazole and sulfoxaflor). Based on these cases, results from the cultured human hepatocytes or the humanized mouse model appear to be effective in leading to a decision not to classify a compound as a human carcinogen. Thus, selection of the experimental model for an in vivo human hepatocyte study, namely genetically humanized or humanized chimeric mouse liver, is critical. This issue is discussed in a later part of this review.

Further evaluation of molecular key events of CAR-mediated MOA

Is phenobarbital a potential liver cancer risk factor for humans?

Regarding the human relevance of the CAR-mediated MOA in hepatocellular tumour production, numerous publications have concluded that the CAR-mediated MOA is qualitatively not relevant to humans, based on a lack of the key event of an increased cell proliferation in PB-induced hepatocellular tumorigenesis.15,17,19,20,29–33,36–39,58

Recently, an in vitro high content imaging-based assay was developed for the quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species.59 Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on the increased gene expression of Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of labeling of the nucleoside analog 5-ethynyl-2′-deoxyuridine in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The authors concluded that the observed species differences are consistent with the CAR activators inducing a proliferative response in rodents, a key event in the liver tumour MOA that is not observed in humans.59

In contrast, some studies of PB treatment indicated that PB induces cell cycle transcriptional responses in humanized CAR (referred to as hCAR) mouse liver60 and humanized CAR/PXR (referred to as hCAR/hPXR) mouse liver,61 and furthermore, that PB-treatment produced liver tumours in hCAR/hPXR mice similar to wild type mice but to a significantly lower extent than in the wild type.62 Schwartz and Braeuning's group indicated that the human relevance of the tumorigenicity of PB through CAR activation remains the subject of an ongoing debate.63–67

The genetically humanized model may be a useful model for human prediction in some cases,68 but we should also recognize its disadvantages. A comparison between genetically humanized and chimeric humanized mouse liver models was discussed from the perspective of studies on drug metabolism and toxicity.68 In that review, the advantages and disadvantages of genetically and chimeric humanized mouse liver models were also comprehensively presented. As shown in Fig. 1, it is important to recognize that the human receptors in the transgenic hCAR/hPXR mouse model are operational in a mouse hepatocyte environment.69 Human liver CAR (hCAR) is highly homologous to mouse CAR (mCAR) on a protein level.70 Although the hCAR/hPXR genes have been inserted genetically, the downstream genes are still mouse and may be the basis for those studies still producing cell proliferation (Fig. 1). Therefore, any extrapolation of data in the hCAR/hPXR mouse model to the biology of human liver tumours has to be done with caution.69

Fig. 1. Effects of phenobarbital on different animal models: (A) wild-type mouse, (B) hCAR/hPXR mouse and (C) humanized chimeric mouse liver. Phenobarbital increases CYP2B levels in all models. In contrast to CYP2B, phenobarbital increases hepatocyte proliferation in wild-type mice and in some studies using hCAR/hPXR mice but not in the humanized chimeric mouse liver.

Observations indicate that mouse hepatocytes expressing human CAR appear to be less sensitive to some CAR agonists39,62,66,71 compared to their murine CAR counterparts. Marx-Stoelting et al. suggested that being less sensitive to some CAR agonists does not necessarily absolve all CAR agonists from being potentially harmful for humans.66 However, PB, chlordane, cyproconazole and sulfoxafor did not stimulate replicative DNA synthesis in hCAR/hPXR mice but did in wild type mice.31,39,66 Some have suggested that the sensitivity of the hCAR/hPXR mouse to the CAR agonists is in between that of the wild-type mouse and human, and that this model may be useful as an initial screening tool for CAR-mediated proliferation enhancers in human hepatocytes. However, given the mixed results of this model regarding cell proliferation and the fact that these human genes are acting within the context of a mouse genome rather than a human genome, its usefulness as a screening model for human relevance is limited. A speaker in the workshop recommended that the hCAR/hPXR mouse [should] not be used for chemical risk evaluation, although it is [might be] a useful model for understanding why some responses are turned on when the human receptor is placed in the mouse20 (the text was modified as in brackets based on personal communication with the speaker Prof. Brian Lake). If an increased replicative DNA synthesis is observed in the hCAR/hPXR mice, the compound should be examined in a more suitable model (a more physiological model), humanized chimeric mice. To resolve differences in understanding the human relevance of CAR-mediated rodent liver tumorigenesis, the mechanism of action for CAR-mediated liver tumorigenesis would be helpful. Thus, it is important to determine key genes, gene products or proteins involved in CAR-mediated hepatocellular tumorigenesis in rodents and to investigate their human relevance.

Integrated analysis of DNA modification and gene expression in hepatoocellular adenomas produced by phenobarbital

While epigenetics are suggested to be related to PB-mediated liver tumorigenesis,72–79 our recent study focused on genes with alterations of DNA methylation and hydroxymethylation with concomitant alteration of expression levels.80 Comprehensive analyses of DNA methylation, hydroxymethylation and gene expression using microarrays were performed on mouse hepatocellular adenomas induced by a single 90 mg kg^–1 intraperitoneal injection of diethylnitrosamine (DEN) followed by 500 ppm PB in the diet for 27 weeks. DNA modification and expression of hundreds of genes are coordinately altered in PB-induced mouse hepatocellular adenomas. Of these, gene network analysis showed alterations of CAR signaling and tumour development-related genes.

Furthermore, overlap between genes with altered expression compared with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma and in liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days was evaluated.80 With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis while a functional analysis of the selected genes remains to be evaluated.80 Interestingly, these genes did not overlap with genes altered by PB treatment of the humanized chimeric mouse liver, suggesting a species difference between the effects of PB in mouse and human hepatocytes.80 Therefore, these findings are consistent with the previous conclusion that the CAR-mediated MOA for rodent liver tumorigenesis is not relevant to humans.15,17,19,20,26–33,36–38,40,58,69,81

Conclusion

We have obtained informative data from case examples on the evaluation of the human relevance of metofluthrin-, momfluorothrin- and pyrethrins-induced liver tumours in rats based on MOA. The MOA analysis, conducted based on the IPCS framework,9 concluded that experimental data strongly support that the postulated MOA for metofluthrin- and momfluorothrin-produced rat hepatocellular tumours is mediated by CAR activation. Since metofluthrin and momfluorothrin are close structural analogues, reproducible outcomes for both chemicals provide confidence in the MOA findings. Furthermore, cultured human hepatocyte studies and/or humanized chimeric mouse studies demonstrated species differences indicating that human hepatocytes are refractory to the mitogenic effects of metofluthrin, momfluorothrin and pyrethrins, in contrast to rat hepatocytes. The mitogenic effect is a necessary key event in the production of liver tumors by this MOA. These data support the hypothesis that the MOA for these chemical-produced rat liver tumours is not relevant for humans. Furthermore, in addition to the cultured human hepatocyte study, lack of an increased hepatocyte proliferation by metofluthrin and momfluorothrin was observed in the recently developed humanized chimeric mouse liver, and these findings substantially influenced decisions in regulatory toxicology. This conclusion is also supported by recent integrated analyses of DNA modifications and gene expression in hepatocellular adenomas produced by PB and the lack of overlap between the altered genes in the liver of wild-type mice and the humanized chimeric mouse liver treated with PB for 7 days. Therefore, these data contribute substantially to addressing the long-lasting discussions on the human relevance of liver tumours induced by PB and other CAR activators in rodents. Our experimental data strongly support the conclusion that PB-produced liver tumours are not relevant to humans, which is also supported by epidemiological data.17,48,49

Conflicts of interest

There are no conflicts of interest to declare.

Biography

Tomoya Yamada

Dr Tomoya Yamada is a Senior Research Specialist of the Environmental Health Sciences Laboratory at Sumitomo Chemical Company, Ltd in Japan. He received his B.Sc. (1985), M.Sc. (1987), D.V.M. (1987) and Ph.D. (1994) in veterinary science from Osaka Prefecture University (Japan). After joining Sumitomo Chemical Company in 1987, he has worked as a researcher in experimental toxicology focusing on mechanisms of chemical-induced carcinogenicity. His research has provided data on analyzing the mode of action for chemical-induced rodent tumours in the testis, lung and liver and their human relevance, contributing to regulatory decision making.