1. Mode of Action of Rodent
Forestomach Tumours:
Relevance to Humans.
R Cope

2. Gross anatomy of murine forestomach after NMBA
(N-nitrosomethylbenzylamine) treatment.
Zanesi N et al. PNAS 2001;98:10250-10255
©2001 by The National Academy of Sciences

3. NMBA-induced histopathology of murine forestomach .
Zanesi N et al. PNAS 2001;98:10250-10255
©2001 by The National Academy of Sciences

4. Introduction
Introduction
 Forestomach tumors/pre-neoplastic lesions in rats and mice are a
common finding in chronic toxicology studies;
 Debate over the human relevance due to:
 Dose and exposure differences between rodents and humans;
 Substantial toxicokinetic differences (exposure);
 Substantial anatomical differences;
 Substantial physiological/metabolic differences of the
forestomach epithelium;
 Different mechanisms and tumor types in humans compared with
rodents;

5. Dose and Exposure Problems
Dose and Exposure Differences
 Doses used in rodent oral carcinogenesis often far exceed normal
human environmental exposure conditions (possible rare exception is
some direct food additives);
 Doses that produce forestomach irritation in rodents really should be
considered as exceeding the MTD – i.e. poor practice in rodent
carcinogenesis studies and not according to GLP/test guidelines;
 Gavage can produce forestomach irritation and is not physiological:
 Large volumes;
 Damage to the mucosa;
 Esophageal reflux;
 Possibly replicates tablets (but not capsules);

6. Tissue specificity
Tissue Specificity
 Forestomach carcinogens divisible into at least 3 categories:
 Produce forestomach tumors and tumors at other sites when
administered by gavage;
 Produce only forestomach tumors when administered by gavage;
 Produce forestomach tumors and tumors when administered by
non-oral routes;
 In terms of human relevance, forestomach + tumors at other sites is
likely to be more important except in the case of site of first contact
carcinogens.

7. Tissue concordance/anatomical
issues
Tissue concordance/anatomical issues
 Humans do not have a forestomach or a pars esophagea:
 Roughly equivalent tissue in terms of histology is the esophagus;
 Humans do not store food in the esophagus where as rodents
store food in the forestomach;
 Transit time through the human stomach is lower than transit time
through the rodent stomach (forestomach)  difference in tissue
exposure;
 Chemicals pass quickly through the human esophagus and thus
the exposure is very limited compared with chemical exposure of
the rodent forestomach.

10. Tissue concordance/anatomical
issues
Tissue concordance/anatomical issues
 Physiological issues:
 Rodent forestomach does not have a protective mucous coating
 increased tissue exposure to chemicals and more prone to
irritant effects;
 pH in rodent forestomach is higher than the pH of the human
stomach  relevant to detoxification (e.g. hexavalent chromium to
trivalent chromium in low pH of human stomach);
 Potential metabolic differences of rodent forestomach epithelium
 conversion of 2-butoxy ethanol to 2-butoxyacetic acid in rodent
forestomach but not in human stomach;

11. Tumour types and biology issues
Tumor types and biology issues
 Rodents
 Predominant tumor types are papillomas (non-malignant) and
squamous cell (low malignancy – regional metastasis)
carcinomas;
 Typically located at the limiting ridge;
 Possibly have some relevance to human esophageal squamous
cell carcinoma BUT chemical exposure of the human esophagus
is much lower than in the rodent forestomach due to much lower
transit time (no storage in esophagus);
 Not relevant to human esophageal adenocarcinoma.

12. Tumour types and biology issues
Tumor types and biology issues
 Humans
 All human stomach cancers are gastric adenocarcinomas and
arise from the glandular epithelium;
 Rodent forestomach tumors have a different histiogenesis and are
not relevant to the human gastric tumors;

13. Genotoxicity issues
Genotoxicity issues
 Forestomach carcinogens are divisible into 3 basic groups:
 DNA reactive chemicals (classical in vivo genotoxic carcinogens)
 Site of first contact carcinogens (generally direct acting
carcinogens and are usually highly reactive chemicals;
typically direct acting alkylating agents);
 Classical pro-carcinogen DNA reactive chemicals;
 Non-DNA reactive chemicals (classical non-genotoxic
carcinogens);
 Typically irritant chemicals or chemicals that produce local
increased cell turnover.

14. Genotoxicity issuse
Genotoxicity issues
 Site of first contact carcinogens:
 Generally require no metabolism to be carcinogenic;
 Generally will produce tumors at other sites if the route of
administration is different  tumor location is the site of contact;
 Generally only produce forestomach tumors in gavage/dietary
studies because of limited/no systemic bioavailability;
 Typically alkylating agents;
 Typically genotoxicants in vitro and in vivo;
Forestomach tumours are potentially human relevant but only
at the site of first contact in humans (e.g. dermal exposures)

15. Genotoxicity issuse
Genotoxicity issues
 Classical pro-carcinogen DNA reactive chemicals;
 Generally pro-carcinogens;
 Often produce tumours at more than one anatomical site following
oral dosing (at least one systemic site + forestomach);
 Often other routes of administration also result in tumors;
 Generally systemically bioavailable;
Human relevance of forestomach tumors depends on: (a) was
there evidence of gastric irritation; (b) were the doses
excessive (> MTD); (c) were the effects only seen with gavage
dosing/diet studies and not with drinking water studies?

17. • Observation of tumours under different circumstances lends support to the
significance of the findings for animal carcinogenicity. Significance is
generally increased by the observation of more of the following factors:
•Uncommon tumour types;
•Tumours at multiple sites;
•Tumours by more than one route of administration;
•Tumours in multiple species, strains, or both sexes;
•Progression of lesions from preneoplastic to benign to malignant;
•Reduced latency of neoplastic lesions;
•Metastases (malignancy, severity of histopath);
•Unusual magnitude of tumour response;
•Proportion of malignant tumours;
•Dose-related increases;
•Tumor promulgation following the cessation of exposure.

19. Benzo(a)pyrene (IARC 1)
Parameter
Genotoxicity in vivo that is relevant to humans +
Forestomach cancers following oral dosing +
Not observed in drinking water studies, only observed with gavage/diet studies -
Only observed at doses that irritate the forestomach (> MTD) -
Uncommon tumour types; +
Tumours at multiple sites; +
Tumours by more than one route of administration; +
Tumours in multiple species, strains, or both sexes; +
Progression of lesions from preneoplastic to benign to malignant; +
Reduced latency of neoplastic lesions; +
Metastases (malignancy, severity of histopath); +
Unusual magnitude of tumour response; +
Proportion of malignant tumours; +
Dose-related increases; +
Tumour promulgation following the cessation of exposure. +

20. Ethyl Acrylate
•Oral gavage: dose related increases in the incidence of
squamous-cell papillomas and carcinomas of the forestomach
were observed in rats and mice. Exposure caused gastric
irritancy;
•Ethyl acrylate was tested by inhalation in the same strains of
mice and rats; no treatment-related neoplastic lesions were
observed;
•No treatment-related tumour was observed following skin
application of ethyl acrylate for lifespan to male mice.

21. Ethyl Acrylate

22. Ethyl acrylate (IARC 2B)
Parameter
Genotoxicity in vivo that is relevant to humans -
Forestomach cancers following oral dosing +
Not observed in drinking water studies, only observed with gavage/diet studies ?
Only observed at doses that irritate the forestomach (> MTD) +
Uncommon tumour types; -
Tumours at multiple sites; -
Tumours by more than one route of administration; -
Tumours in multiple species, strains, or both sexes; +
Progression of lesions from preneoplastic to benign to malignant; +
Reduced latency of neoplastic lesions; +
Metastases (malignancy, severity of histopath); -
Unusual magnitude of tumour response; -
Proportion of malignant tumours; -
Dose-related increases; -
Tumour promulgation following the cessation of exposure. +

23. Mercuric chloride (IARC 3)
Parameter
Genotoxicity in vivo that is relevant to humans -
Forestomach cancers following oral dosing +
Not observed in drinking water studies, only observed with gavage/diet ?
studies
Only observed at doses that irritate the forestomach (> MTD) +
Uncommon tumour types; -
Tumours at multiple sites; -
Tumours by more than one route of administration; (thyroid follicular cell adenomas)
Tumours in multiple species, strains, or both sexes; -
Progression of lesions from preneoplastic to benign to malignant; -
Reduced latency of neoplastic lesions; -
Metastases (malignancy, severity of histopath); -
Unusual magnitude of tumour response; -
Proportion of malignant tumours; -
Dose-related increases; -
Tumour promulgation following the cessation of exposure. -

24. Thanks!