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Carcinogenicity

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Description of key information

The carcinogenicity of succinic anhydride was evaluated by the National Toxicology Program. In 2-year gavage studies, there was no evidence of carcinogenic activity of succinic anhydride for male or female F344/N rats given 50 or 100 mg/kg succinic anhydride (NTP, 1990). In similar studies with B6C3F1 mice, there was no evidence of carcinogenic activity for males given 38 or 75 mg/kg succinic anhydride or for females given 75 or 150 mg/kg.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not stated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A review summary presented in NTP report - peer reviewed but without indications of test guideline compliance/GLP status.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Principles of method if other than guideline:
See Table 1 below
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source:Charles River Breeding Laboratories (Kingston, NY)- Age at study initiation: 8-9 weeks- Weight at study initiation: Not stated- Fasting period before study: not applicable- Housing:Polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD) - Diet (e.g. ad libitum): NIH07 Rat and Mouse Ration (Zeigler Bros., Inc ,Gardners, PA); available ad libitum- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum- Acclimation period: 19 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18°-30°C- Humidity (%):22-84%- Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: 30 August 1982 To: 17 August 1984See Table 1 below
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Dose formulations of succinic anhydride in corn oil (w/v) were prepared every 2 weeks and usedwithin 3 weeks. Because succinic anhydride forms a suspension in corn oil, the formulations were constantly stirred with a magnetic stirrer during dosing to maintain uniformity. Before the beginning of the 2-year studies, the dose formulation procedure was modified to use a Polytron® homogenizer to reduce particle size and produce more stable suspensions. However, the resulting formulations proved to be more toxic to rats than those prepared with the original method, necessitating the repetition of the short-term studies to select new doses for the 2- year studies in this species. VEHICLE- Justification for use and choice of vehicle (if other than water): corn oil selected as an appropriate suspending agent, due to insolubility of succinic anhydride in water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the 2-year studies, the dose formulations were analyzed at intervals of approximately 8 weeks. For the succinic anhydride studies, the formulations were estimated to have been prepared within +10% of the target concentrations approximately 98% of the time throughout the studiesThe stability of succinic anhydride in corn oil at concentrations of 15 or 25 mg/ml was determined at the study laboratory. The chemical was found to be stable as a suspension in corn oil for at least 18 days when stored at room temperature.
Duration of treatment / exposure:
2 years
Frequency of treatment:
5 days per week for 103 weeks
Post exposure period:
Not applicable
Remarks:
Doses / Concentrations:0,50 or 100 mg/kg bw/dayBasis:nominal conc.
No. of animals per sex per dose:
60 rats per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Refer to Table 1 below
Positive control:
Not applicable
Observations and examinations performed and frequency:
See Table 1 belowAll animals were observed two times per day.Body weights were recorded once per week for the first 12 or 13 weeks of the study and once per month thereafter. Mean body weights were calculated for each group. Animals found moribund and those surviving to the end of the studies were humanely killed.
Sacrifice and pathology:
See Table 1 belowA necropsy was performed on all animals, including those found dead.During necropsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histopathologic examinations of tissues were performed on all high dose and vehicle control animals and on all low dose rats dying before the end of the studies. In addition, histopathologic examinations were performed on all grossly visible lesions in all dose groups. Potential target organs for chemicallyrelated neoplastic and nonneoplastic effects were determined by examination of the pathology data; these target organs/tissues in the lowdose group were examined histopathologically.
Other examinations:
See Table 1 belowWhen the pathology evaluation was completed by the laboratory pathologist and the pathology data entered into the Toxicology Data ManagementSystem (rats) or the Carcinogenesis Bioassay Data System (mice), the slides, paraffin blocks, and residual formalin-fixed tissues were sent to the NTP Archives. The slides, blocks, and residual wet tissues were audited for accuracy of labeling and animal identification and for thoroughness of tissue trimming. The slides, individual animal necropsy records, and pathology tables were sent to an independent pathology quality assessment laboratory. The individual animal records and pathology tables were compared for accuracy, slides and tissue counts were verified, and histotechnique was evaluated.All tissues with a tumor diagnosis, all potential target tissues (male rats: thyroid gland, pituitary gland, adrenal gland, kidney, testes;female rats: kidney; mice: none), and all tissues from a randomly selected 10% of the animals were re-evaluated microscopically by a quality assessment pathologist. Nonneoplastic lesions were evaluated for accuracy and consistency of diagnosis only in the potential target organs, the randomly selected 10% of animals, and in tissues with unusual incidence patterns or trends The quality assessment report and slides were submitted to a Pathology Working Group (PWG) Chairperson, who reviewed microscopically all potential target tissues and any other tissues for which there was a disagreement in diagnosis between the laboratory and quality assessment pathologists. Representative examples of potential chemical-related nonneoplastic lesions and neoplasms and examples of disagreements in diagnosis between the laboratory and quality assessment pathologists were shown to the PWG. For the rat studies, the PWG examined all lesions diagnosed as mesotheliomas, preputial gland adenomas, squamous cell papillomas of the skin, neoplastic nodules of the liver, and two renal nephroblastomas, as well as several sections of adrenal gland and kidney. For the mouse studies, the PWG examined sections of kidney, nasal cavity, and lung. The PWG included the quality assessment pathologist and other pathologists experienced in rodent toxicology, who examined the tissues without knowledge of dose group or previously rendered diagnoses. When the consensus diagnosis of the PWG differed from that of the laboratory pathologist, the diagnosis was changed to reflect the opinion of the PWG. This procedure has been described, in part, by Maronpot and Boorman (1982) and Boorman et al. (1985). The final pathology data represent a consensus of contractor pathologists and the NTP Pathology Working Group. For subsequent analysis of pathology data, the diagnosed lesionsfor each tissue type are combined according to the guidelines of McConnell et al. (1986).
Statistics:
Survival Analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier. Animals were censored from the survival analyses at the time they were found to be missing or dead; animals dying from natural causes were not censored. Statistical analyses for a possible dose-related effect on survival used method for testing two groups for equality Cox (1972) and Tarone's (1975) life table test for a doserelated trend. When significant survival differences were detected, additional analyses using these procedures were carried out to determine the time point at which significant differences in the survival curves were first detected. P values for the survival analysis are two-sided.Calculation of Incidence: The incidence of neoplastic or nonneoplastic lesions is the ratio of number of animals bearing a lesion at a specific anatomic site to the number of animals in which that site was examined. Normally the denominators include only animals where the site was examined histologically. However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumors), or when lesions could have appeared at multiple sites (e.g., lymphomas), the denominators consist of the number of animals on which a necropsy was performed.Analysis of Tumor Incidence: The majority of tumors in this study were considered to be incidental to the cause of death or not rapidly lethal.Thus, the primary statistical method used was an incidental tumor analysis, which assumed that the diagnosed tumors were discovered as the result of death from an unrelated cause and thus did not affect the risk of death. The proportions of tumor-bearing animals in dosed and vehicle control groups were compared in each of five time intervals:weeks 0-52, weeks 53-78, weeks 79-92, week 93 to week prior to scheduled termination and the terminal kill period.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
the incidence in the high dose group was well within the range of the historical incidences
Mortality:
mortality observed, treatment-related
Description (incidence):
the incidence in the high dose group was well within the range of the historical incidences
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of high dose male and female rats were approximately 6% resp. 8% lower than those of vehicle controls during the second year of the study
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Behaviour (functional findings):
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
the incidence in the high dose group was well within the range of the historical incidences
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic or nonneoplastic lesions appeared to be related to chemical administration.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic or nonneoplastic lesions appeared to be related to chemical administration.
Details on results:
Body Weights and Clinical SignsMean body weights of high dose male rats were approximately 6% lower than those of vehicle controls during the second year of the study;mean body weights of high dose female rats were approximately 8% lower than those of vehicle controls during the second year of the study. Mean body weights of low dose and vehicle control rats were generally similar throughout the studies.SurvivalEstimates of the probabilities of survival for male and female rats administered succinic anhydride at the doses used in these studies and forvehicle controls were presented in the report as Kaplan and Meier curves; standard (unadjusted) survival curves were presented forcomparison. No significant differences in survival were observed between any groups of either sex. The interim evaluation of10 rats per group scheduled after 15 months of exposure was cancelled because of some early deaths due to gavage accidents. To determinewhether gavage accidents were the possible cause of early death or the reason for the rats being killed in a moribund condition, a retrospectiveexamination of sections of nose and lung, esophagus and trachea, and heart and mediastinum was performed on animals that died early. The detection of small oil droplets in the lung of certain animals coded as natural death or moribund kill indicates that some of these deaths may have been related to the dosing (gavage) procedure. A sufficient number of rats in each dose group lived long enough to allow evaluation of the potential carcinogenicity of succinic anhydride.Pathology and Statistical Analyses of ResultsStatistically significant changes were noted in the incidences of rats with neoplastic lesions. Marginal increases were seen for the skin and mammary gland. No neoplastic or nonneoplastic lesions appeared to be related to chemical administration.Summaries of the incidences of neoplasms and nonneoplastic lesions, individual animal tumor diagnoses, statistical analyses of primary tumorsthat occurred with an incidence of at least 5% in at least one animal group, and historical control incidences for the neoplasms mentioned in this section are presented in the report.Skin: Keratoacanthomas in male rats occurred with a significant positive trend; however, the incidence in the high dose group wa(vehicle control, 2/60; low dose, 0/60; high dose, 6/60) and is lower than the highest historical incidence observed in corn oil gavage vehicle control male F344/N rats (6/50).Mammary Gland: Fibroadenomas in female rats occurred with a marginally significant negative trend, and the incidence in the high dose group was marginally lower than that in the vehicle controls. This decrease was not considered to be chemical related because the difference between the high dose and vehicle control groups was not statistically significant when the incidence of mammary gland fibroadenomas was combined with the incidences of adenomas and adenocarcinomas. Furthermore, the incidence in the high dose group was well within the range of the historical incidences of mammary gland neoplasms in corn oil vehicle control female F344/N rats in recent National Toxicology Program (NTP) 2-year studies (14%-56%;)
Relevance of carcinogenic effects / potential:
Because succinic anhydride is readily hydrolyzed to succinic acid, the most likely site of reactivity of succinic anhydride in a biologic system is its primary site of contact. When applied to the cornea of rabbits, succinic anhydride caused severe eye irritation (Carpenter and Smyth, 1946). In a gavage study, the most likely site of tissue acylation by succinic anhydride is in the gastrointestinal tract. In the 13-week study of succinic anhydride in male mice, the incidences of inflammation of the stomach were increased; however, there were no apparent effects in the stomach of rats or mice after 2 years of exposure to succinic anhydride. Maleic anhydride, an analog of succinic anhydride, caused nasal and ocular irritation but no evidence of systemic toxicity in an inhalation study in which CD® rats, Engle hamsters, or rhesus monkeys were exposed 6 hours per day, 5 days per week for 6 months, at target concentrations of 1, 3, or 10 mg/m3 (Short et al., 1988). In the only previous extended study of succinic anhydride, Dickens and Jones (1965) observed local transplantable sarcomas in male rats given subcutaneous injections of succinic anhydride for 65 weeks (2 mg per injection, two times per week). Although that study contained too few animals and was too short to evaluate adequately the carcinogenicity ofsuccinic anhydride, it raises concern that direct tissue interaction with succinic anhydride may result in a carcinogenic response. In the current studies, succinic anhydride was not carcinogenic in rats or mice after oral administration.Phthalic anhydride was the only other anhydride evaluated for carcinogenicity in F344 rats and B6C3Fi mice in 2-year studies (NCI, 1979). In these studies, rats were fed diets containing 0, 7500, or 15000 ppm phthalic anhydride for 2 years, and mice were fed diets containing 0, 25,000, or 50,000 ppm. Because of excessive decreases in body weight gain in dosed mice compared with that in controls, doses for males werereduced to 12,500 and 25,000 ppm after week 32 of the study, and doses for females were reduced to 6,250 and 12,500 ppm. There was no evidenceof carcinogenicity of phthalic anhydride for rats or mice under the conditions of these studies. In 7-week studies in rats and mice, there were nodose-related histopathologic lesions in either species given diets containing up to 50,000 ppmphthalic anhydride.Succinic anhydride generally has been negative in assays for mutagenic or clastogenic activity. Succinic anhydride was not mutagenic in Salmonellatyphimurium (McCann et al., 1975; Kawachi et al., 1978,1980; Rosenkranz and Poirier, 1979; Simmon 1979a; Ishidate et al., 1981;Zeiger et al., 1987), Escherichia coll (Rosenkranz and Poirier, 1979; Rosenkranz and Leifer, 1980; Leifer et al., 1981), Saccharomyces cerevisiae(Simmon, 1979b; Simmon et al., 1979), or mouse lymphoma cells (Clive et al., 1979); it was not clastogenic in Chinese hamster lung cells (Ishidateet al., 1981), hamster lung fibroblast or rat bone marrow cells (Kawachi et al., 1978, 1980), or rat hepatocytes (Sina et al., 1983). Succinic anhydride induced morphologically transformed colonies of Syrian golden hamster embryo cells (Pienta et al., 1977; Pienta, 1980) and was positive in the replicative killing test with E. coli strain CHY832 (Hayes et al., 1984). Other anhydrides, including acetic anhydride, phthalic anhydride, and maleic anhydride, were also not mutagenic in S. typhimurium.Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of succinic anhydride for male or femaleF344/N rats given 50 or 100 mg/kg succinic anhydride.There was no evidence of carcinogenic activity for male B6C3F1 mice given 38 or 75 mg/kg succinic anhydride or for female B6C3F1 mice given 75 or 150 mg/kg in a concurrent 92 week study for murine carcinogenicity.
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Conclusions:
In the 2-year or 18 month studies in rats and mice, there were no significant decreases in survival between any groups of rats or mice. Male and female rats had an apparent dose-related increase in the number of early deaths attributed to chemical-gavage accidents.Most of the accidental deaths of female rats occurred during the first 2 weeks of the study. Because animals that died from other than naturalcauses were censored from the survival analyses and because the life table and incidental tumor analyses adjust for intercurrent mortality, theseearly deaths do not weaken the analyses of the potential carcinogenesis of succinic anhydride. However, to ensure that there would be an adequatenumber of animals at risk to detect any compound-related neoplastic lesions, the interim evaluation of 10 rats per group scheduled after 15 months of exposure was canceled and all animals surviving to that time were continued on study for the remainder of the exposure period.The design of the studies in mice did not include an interim evaluation. For rats and mice, a sufficient number of animals in each dose group lived long enough to allow evaluation of the potential carcinogenicity of succinic anhydride.There was a chemical-related effect on body weights for both rats and mice in the 2-year studies. For rats, mean body weights of high dose males and females were lower than those of vehicle controls during the second year of the studies. For mice, mean body weights of high dose males and dosed females were lower than those of vehicle controls throughout most of the studies.At no site in rats or mice was there a chemical related increase in the incidences of nonneoplastic or neoplastic lesions. Squamous metaplasia in the tissues of the nasal cavity was observed in four high dose male mice; however, this effect was considered to be secondary to inflammation resulting from a foreign material (probably the succinic anhydride corn oil mixture) in the nasal cavity. Results of serologic analyses made at three separate intervals during the study were negative for antibodies to murine viruses. The incidence of renal mineralization was decreased in dosed male mice compared with that in vehicle controls; the cause and significance of this change are not known.Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of succinic anhydride for male or female F344/N rats given 50 or 100 mg/kg succinic anhydride.There was also no evidence of carcinogenic activity for male B6C3F1 mice given 38 or 75 mg/kg succinic anhydride or for female B6C3F1 mice given 75 or 150 mg/kg.
Executive summary:

Under the conditions of these 2-year gavage studies, there was no evidence of carcinogenic activity of succinic anhydride for male or female F344/N rats given 50 or 100 mg/kg succinic anhydride. There was no evidence of carcinogenic activity for male B6C3F1 mice given 38 or 75 mg/kg succinic anhydride or for female B6C3F1 mice given 75 or 150 mg/kg.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
chronic
Species:
rat

Additional information

The carcinogenicity of succinic anhydride was evaluated by the National Toxicology Program. In 2-year gavage studies, there was no evidence of carcinogenic activity of succinic anhydride for male or female F344/N rats given 50 or 100 mg/kg succinic anhydride (NTP, 1990). In similar studies with B6C3F1 mice, there was no evidence of carcinogenic activity for males given 38 or 75 mg/kg succinic anhydride or for females given 75 or 150 mg/kg.

Justification for classification or non-classification

No evidence for carcinogenesis was found in a two year or 90 week study in rats or mice respectively. No evidence of mutagenesis was determined in battery of genotoxicity tests. The rapid hydrolysis of succinic anhydride to the human endogenous metabolite succinic acid, which acts as a ubiquitous substrate for ATP generation via the Kreb's cycle, would also indicate no human carcinogenic risks.

No classification is required based on the absence of any postive indicators.