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R.R. Maronpot, R.J.M.M. Thoolenb, B. Hansenc
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Acrylamide is an important chemical with widespread industrial and other uses in addition to generalized population exposure from certain cooked foods. Previous rat studies to assess the carcinogenic potential of acrylamide have been carried out exclusively in the Fischer 344 rat with identification of a number of tumors amongst which mesotheliomas of the tunica vaginalis is an important tumor endpoint in the classification of acrylamide as a ‘probably human carcinogen. In a rat carcinogenicity study to determine the human relevance of mesotheliomas Wistar Han rats were exposed to 0, 0.5, 1.5, or 3.0 mg acrylamide/kg body weight/day in drinking water starting at gestation day 6. At the end of two years, mammary gland fibroadenomas in females and thyroid follicular cell tumors in both sexes were the only tumors increased in acrylamide treated rats. These tumor endpoints have rat-specific modes of action suggesting less likelihood of human cancer risk than previously estimated. This study demonstrates that tunica vaginalis mesotheliomas are strain specific and not likely of genotoxic origin.

Keywords

Mammary fibroadenomas; Thyroid follicular cell tumors; In utero exposure; Neuropathy; Myopathy; Carcinogenicity

1. Introduction

Acrylamide (ACR) is a monomer used in the manufacture of polymers for mining, oil and natural gas processing, paper production, waste processing, as well as hospital, laboratory and other uses. Adverse health effects from industrial emissions and exposure has been extensively studied Lipworth et al., 2012) and no adverse effects have been reported with daily exposure up to 2.1 mg/kg/day (Erdreich and Friedman, 2004). Nevertheless, due to various risk assessments, several of the workplace permissible exposure limits (PEL) are under evaluation and may be lowered considerably.

In addition to industrial exposure to acrylamide, generalized population exposure to acrylamide in foodstuffs has been documented (Vesper et al., 2008 and Vesper et al., 2010). This exposure to ACR has become a worldwide concern because of its generation in a variety of carbohydrate rich foods when cooked at temperatures exceeding 120 °C (Mottram et al., 2002 and Friedman, 2003). At these temperatures, Maillard reaction of sugars with asparagine residues produce acrylamide (Friedman, 2005). The 64th Joint FAO/WHO Expert Committee on Food Additives concluded that an intake of 1 μg/kg body weight/day of ACR could be taken to represent the average for the general population (JECFA, 2005). The USFDA estimated a mean intake of 0.4 μg/day for ages 2 and up with a 90% confidence limit of >2 μg/kg (http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/UCM197239.pdf). This equates to an adult male exposure of 1.4 μg/day.

Acrylamide has previously been shown to cause fibroadenomas of the mammary gland and thyroid gland follicular tumors in rats (Johnson et al., 1986, Friedman et al., 1995 and Beland et al., 2013). Since these tumor sites have well documented rat-specific modes of action (Neumann, 1991, Ben-Jonathan et al., 2008 and Alison et al., 1994), a primary consideration in classification of acrylamide as a “probable human carcinogen” centers on the induction of tumors of the tunica vaginalis mesotheliomas (TVMs) in F344 rats (Johnson et al., 1986, Friedman et al., 1995 and Beland et al., 2013). These unique mesotheliomas in acrylamide-treated F344 rats might be considered a potential human health risk if they were caused by a genotoxic mechanism (Wall, 2005). However, F344 rats have an elevated incidence of TVMs secondary to their known high spontaneous incidence of Leydig cell tumors (Maronpot et al., 2009). TVMs found in the acrylamide studies have the same biological behavior, ultrastructure, and morphology as TVMs found in control rats, prompting the conclusion that acrylamide accelerates the appearance of these background tumors (Damjanov and Friedman, 1998 and Maronpot et al., 2009). It has also been shown that the time-to-tumor and incidence data for TVMs are consistent with a non-genotoxic mechanism (Maronpot et al., 2009). The mechanism of action of acrylamide induction of TVMs has been elaborated (Shipp et al., 2006).

The EPA calculation of the cancer risk for ACR relies on 2 important considerations. The first is that acrylamide acts as a mutagen. We will not discuss this assumption here. Second, that tunica vaginalis mesotheliomas (TVMs) are added to thyroid tumors in males (EPA, 2010). Depending on which study is used (Johnson, Friedman, or Beland) there are either twice as many TVMs as thyroid tumors or the same number. While the biochemistry supports an endocrine mechanism for TVMs, there are no data directly demonstrating this mechanism (EPA, 2010). In order to clarify the mode of action of acrylamide induced neoplasms, we sought out a strain of rats which did not get Leydig cell tumors and had a low level of background TVMs to examine a potential endocrine mode of action. If acrylamide acted by a genotoxic mode of action, it would necessarily produce TVMs in a second strain. Wistar Han rats were selected due to their longevity and low incidence of Leydig cell tumors. We report here the oncogenic response of Wistar Han rats to orally administered acrylamide. Furthermore, concern has been expressed that children might be at higher risk to acrylamide due to greater dietary acrylamide intake (EPA Science Advisory Board, 2008). This parameter was evaluated in the current research by initiating dosing on gestation day 6 in pregnant rats and continued to termination of the F1 offspring 2 years later.

2. Materials and methods

2.1. Study conduct

This study was conducted under GLP guidelines enacted in Germany in the ‘Chemikaliengesetz’, current edition and OECD Principles of Good Laboratory Practice’ Document Nos. 1, 8 and 13 ENV/MC/CHEM (98)17, ENV/JM/MONO(99)24, and ENV/JM/MONO (2002)9 and was externally audited. The following guidelines were considered: OECD Guideline for the Testing of Chemicals No. 453: Combined chronic toxicity/carcinogenicity studies, adopted 7 September 2009; and EC method 8.33. Combined chronic toxicity/carcinogenicity test, 88/303/EEC; Official Journal of European Communities, L 133 1988.

2.2. Test article

Acrylamide (C3H3NO, CAS no 79-06-1, 1,2-propenamide; >99.9% pure) was purchased from Sigma Aldrich as a white, odorless, crystalline solid which is stable at room temperature for at least 2 years. Acrylamide was administered daily in drinking water at the following doses: 0, 0.5, 1.5 and 3.0 mg/kg/day. Water was available ad libitum. In a separate study, solutions of acrylamide were prepared in tap water and evaluated for stability at room temperature at 6, 13, 20, 27, 41, 55 or 90 test days after preparation and recovery ranged from 96.9% to 102.6%. In the present study acrylamide solutions were prepared weekly and after concentration adjustment for body weight, water bottles were changed weekly. Aliquots for analysis were taken at the beginning and end of exposure to determine stability. Acrylamide concentration in the drinking water was determined at test week 4, 10, 16, 22, 28, 34, 40, 46, 52, 65, 78, and 91. Fig. 1 shows that acrylamide was stable in the animal cages for the weeks of exposure.

Stability of acrylamide in the test item formulation samples.

Fig. 1. Stability of acrylamide in the test item formulation samples.

2.3. Animals

Sperm positive female Wistar Han™/RccHan™:WIST rats were obtained from Harlan Laboratories GmbH, Serumweg 48, 27324 Eystrup, Germany in multiple deliveries. At gestation day 6 pregnant dams were provided acrylamide in their drinking water with exposures continuing in F1 offspring through postnatal day (pnd) 722. Rats were housed 1 per cage in MACROLON cages with granulated wood bedding (Brandenburg, 49424 Goldenstedt/Arkeburg). Study design is summarized in Table 1. Sixty male and sixty female rats were selected for the chronic study as well as 5 sentinel animals. Five rats per sex per group were sacrificed at 12 months and 18 months. Animal rooms were alternately lit (about 150 lx at approximately 1.50 m room height) and darkened in a 12-h lighting cycle. Cage side observations were conducted twice per day during the week and once per day on weekends. Ophthalmological and auditory examinations were conduct at the interim kills (12 and 18 months) and at termination.

Table1. Study design and survival and body weight gain at terminal sacrifice.

Table1. Study design and survival and body weight gain at terminal sacrifice.

On day 4 after birth, the weights of the pups were determined. The size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, five males and five females per litter. The remaining animals were allowed to remain with the dams until day 21 of lactation (weaning). On lactation day 21, the F1 animals were randomized using a computer randomization program to assign the animals to the subsets within each group.

2.4. Assessment of physical and functional development

Neurological determinations were conducted on 5 rats per sex per group. Ear opening, eye opening, cleavage of the balanopreputial gland, vaginal opening and upper incisor eruption were assessed. No significant changes were seen in any of these parameters. Functional tests that included open field behavior and passive avoidance (learning) were conducted at pnd 27 and passive avoidance (learning) at pnd 34. Functional tests included grip strength and locomotor activity. No changes were seen in these parameters.

2.5. Gross necropsy

After 52, 78 or 104 test weeks the respective animals were sacrificed under ether anesthesia by cutting the abdominal aorta, exsanguinated, weighed, and necropsied under the direction of a pathologist. For rats that died or were sacrificed prematurely, necropsy examinations were performed immediately after the animals were found dead or after sacrifice. All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After a ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic and abdominal viscera was noted with due attention to the thymus, lymph nodes and heart.

The weights of the following organs of all animals assigned for interim dissection and the first 10 surviving main study animals/sex/group of the main dissection were determined before fixation: adrenal gland (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), spleen, testis (2), thyroid including parathyroid (1), and uterus. Adrenals, gonads and kidneys were weighed individually and identified as left or right. Organ weights of rats that died or were sacrificed prematurely were recorded but not included into the mean value comparison.

2.6. Histopathological examination

The following organs of all animals (including deceased or sacrificed animals) were preserved in 7% buffered formalin: adrenal (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris, sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, clitoral gland (2), coagulating gland with seminal vesicle, epididymis (2), extraorbital lacrimal gland (2), eye with optic nerve and Harderian gland (2), heart (left and right ventricles, septum), intestine, small (duodenum, jejunum, ileum—Swiss roll method), intestine, large (colon, rectum), kidney (2) and ureter, all macroscopically visible lesions, liver, lungs (with mainstem bronchi and bronchioles), lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal, leg), nasal cavity with nasopharynx, nerve (sciatic), esophagus, ovary (2), pancreas, pituitary, preputial gland (2), prostate, salivary glands (mandibular, sublingual and parotid gland), seminal vesicle, skin (left flank), spinal cord (3 sections), spleen, sternum, stomach (forestomach and glandular stomach), testis (2), thymus, thyroid (2, incl. parathyroids), tissue masses or tumors (incl. regional lymph nodes), tongue (incl. base), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts), vagina, and Zymbal’s gland. Parathyroids cannot always be identified macroscopically. The eyes were preserved in Davidson’s and the testes in Bouin’s solution for optimum fixation. Other tissues were fixed in 7% buffered formalin and stained with hematoxylin and eosin (H&E) or other appropriate stains for preparation of microscopic slides. All slides from control and high dose rats were evaluated as well as rats that died prematurely.

2.7. Statistics

The following statistical methods were used: χ2 test, Dunnett’s test, t-test Fisher exact test, and Peto analysis. These statistical procedures were used for all data.

3. Results

3.1. Clinical signs (F0 dams) and morphological landmarks (F1 pups)

For F0 generation dams no test article-related clinical signs or changes in body weight, food consumption, drinking water consumption or reproductive parameters from implantation until weaning were present. No litter values, body weight changes between groups, or test article-related physical development alterations in the F1 pups were present during the three-week lactation period. Neurological screening and post-natal functional development 21 days after birth were similar among all groups.

3.2. Main study mortality

No test article-related deaths were present in rats prior to test week 78. By the end of the study at 104 weeks, there was excellent survival ranging between 70 and 84% with acceptable body weight gain and no test article-related effect on mortality/survival among any of the treated groups and the controls (Table 1). Factors contributory to death or premature sacrifice were either of age-related origin (e.g. chronic renal disease, thrombo-endocarditis) and/or a result of poor clinical condition in tumor-bearing (e.g. pituitary adenoma of pars distalis, various soft tissue tumors) animals. There were no factors contributory to death that could be related to treatment with the test article. Interestingly, while there was no effect on longevity, there was a statistically significant decreased in mean survival in high dose (3.0 mg/kg) females (80.4 weeks) versus controls (94.5 weeks) specifically among decedents.

3.3. Main study parameters

Hind limb paralysis was present in 12 of 50 high dose (3.0 mg/kg) male rats starting at test week 79. Hind limb paralysis was not present in females. Body weight was reduced by up to 11% in high-dose males and up to 13% in high-dose females starting between 83 and 86 weeks on study with slightly reduced body weight and body weight gain at study termination. No test article-related changes in food or water consumption, ophthalmological findings, or auditory changes were present in main study rats. Calculation of acrylamide intake via drinking water confirmed that achieved dose levels were obtained and test article analyses were within 91 and 106% of nominal concentrations during the course of the study.

3.4. Macroscopic post mortem findings and organ weights

The only treatment-related macroscopic change was an increase in mammary gland enlargements in 21 of 50 high dose females versus 12 of 50 in control females. No test article-related changes in absolute or relative organ weights were present at the interim or final dissections.

3.5. Histopathology

Non-neoplastic lesions related to treatment were present only in rats at the terminal 104-week dissection. At this interval adrenal cortical vacuolation was present in high-dose (3.0 mg/kg) females (Table 2). Adrenal cortical vacuolation was represented by a discrete focal change comprised of enlarged, vacuolated cells with a mixture of clear and pale pink wispy cytoplasm and a centrally located condensed nucleus. Adrenal cortical vacuolation is an age-associated change common in rats (Laast et al., 2014). Some vacuolated focal lesions were associated with a few hypertrophied cells with homogeneous pink cytoplasm within the same focal lesion. Hemorrhage was present in occasional focal vacuolated lesions.

Table 2. Non-neoplastic lesions in target tissues in Wistar Han rat 2-year dose–water carcinogenicity study of acrylamide.

Table 2. Non-neoplastic lesions in target tissues in Wistar Han rat 2-year dose–water carcinogenicity study of acrylamide.

Hind limb skeletal muscle myopathy, sciatic nerve neuropathy, and spinal cord degeneration were present in both sexes (Table 2). Histopathological findings in hind limb skeletal muscle are consistent with a basic process of segmental muscle fiber degeneration with progression to necrosis and accompanied by repair. A constellation of changes including degeneration, necrosis, mixed inflammatory cell response to degeneration and necrosis, and repair identified by increased sarcolemma nuclei was identified in most cases. Muscle fiber atrophy and interstitial fibrosis was present in only a few cases.

The increased incidence of neuropathy was present in sciatic nerves from groups 3 and 4 males and group 4 females. Changes in affected rats consisted of axonal degeneration with localized fragmentation of neurofibers, loss of myelin, and inflammatory cell infiltrates, occasionally accompanied by cholesterol clefts and giant cells. Spinal cord vacuolar degeneration and/or increased cell density representative of gliosis was present in 18% of group 4 males and at 4 to 8% in groups 3 and 4 females, respectively. These changes were minimal and present in the dorsal column of one of three spinal cord cross sections.

A variety of other non-neoplastic spontaneous lesions typical for Wistar Han rats were equally present among treated and control groups.

Treatment-related neoplastic lesions were present in female mammary glands and in male and female thyroid glands in the present study (Table 3). Mammary fibroadenomas were increased in group 3 (12/50) and group 4 (15/50) females versus control (6/50) females attaining statistical significance (p = 0.0479) for group 4. The majority of the fibroadenomas were present in rats surviving to the 24-month terminal dissection. For the terminal sacrificed animals specifically this was 11/37 in group 4 versus 5/37 in group 1, not attaining statistical significance for this observation.

Table 2. Non-neoplastic lesions in target tissues in Wistar Han rat 2-year dose–water carcinogenicity study of acrylamide.

Table 2. Non-neoplastic lesions in target tissues in Wistar Han rat 2-year dose–water carcinogenicity study of acrylamide.

A dose-related increase in thyroid follicular cell neoplasms was present in all dosed male groups and in the mid and high dose female groups (Table 3). The majority of the neoplasms were follicular cell adenomas with a few follicular cell adenocarcinomas in the high dose males and females. With only a very few exceptions, these neoplasms were present at the terminal 24-month dissection interval. Follicular cell hyperplasia was equally present at a low frequency in all groups. The morphological features of both follicular cell adenomas and adenocarcinomas were typical of previously published reports (Rosol et al., 2013, Johnson et al., 1986, Friedman et al., 1995 and Wall, 2005). A spectrum of other tumors reflective of spontaneous background responses in chronic rat studies was observed in the present study.

While in each of the previous studies in Fischer 344 rats, mesotheliomas of the tunica vaginalis testes were observed, none were seen in this study (Table 4). Similarly, there was only one Leydig cell tumor in the testes. This is clearly a strain difference between the Fischer 344 rat and the Wistar Han rat. Similarly, other possible treatment-related neoplasia identified in previous Fischer 344 rat carcinogenicity studies, including clitoral gland carcinomas, cardiac schwannomas, islet cell adenomas, and oral cavity papillomas and carcinomas did not occur in the present Wistar Han study.