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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 for a 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 (NTP, 1990).

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1982-08-01 to 1990-01-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
adopted in 1981
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of the test material used in the report: Succinic anhydride
- Appearance: white, flaky solid
- Batch no.: PE072797 (Aldrich Chemical Company)
- Purity: 99%
- Storage temperature: In refrigerator (5° C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
Appropriate amount of chemical and corn oil were homogenized with a Polytron® PT 10-ST probe at setting 7 for 2 min, followed by setting 9 for 1 min.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Frederick Cancer Research Fecility (Frederick, MD)
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Not stated
- Fasting period before study: not applicable
- Housing: Animals were housed five per cage in polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD) containing spun-bonded polyester cage filters (Snow Filtration, Cincinnati, OH) and hardwood chips for bedding (P J. Murphy Forest Products Corp., Rochelle Park, NJ). Cages of vehicle control animals were placed in the top two rows of the racks, cages of low dose animals were placed in the next two rows, and cages of high dose animals were placed in the bottom two rows. Cages were not rotated during the studies.
- 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: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°-30°C (64-86° F)
- Humidity (%): 22-84%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): fluorescent light, 12/12

IN-LIFE DATES:
From: 30 August 1982 To: 17 August 1984
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 used within 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the 2-year studies, the dose formulations were analysed 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 studies. The 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:
103 weeks (2 years)
Frequency of treatment:
5 days per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
60
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based primarily on the reduced survival for rats administered 200 mg/kg or higher doses of succinic anhydride in the 13-week studies, doses of succinic anhydride selected for the 2-year studies in rats were 50 and 100 mg/kg bw/day, administered in corn oil by gavage 5 days per week.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed two times per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed two times per day.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded once per week for the first 13 weeks of the study, and once per month thereafter. Mean body weights were calculated for each group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Animals found moribund and those surviving to the end of the studies were humanely killed.

GROSS PATHOLOGY: Yes
- A 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.


HISTOPATHOLOGY: Yes
- 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 chemically related neoplastic and nonneoplastic effects were determined by examination of the pathology data; these target organs/tissues in the low dose group were examined histopathologically.
The following tissues were examined: adrenal glands, bone, bone marrow, brain, chloral or preputial gland, epididymis/prostate/testes or ovaries/uterus, esophagus, heart, kidneys, large and small intestines, larynx, liver, lungs, lymph nodes, mammary gland, nose, pancreas, pancreatic islets, parathyroid glands, pituitary gland, salivary glands, spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Adrenal glands (male), kidneys, lungs (male), nose, pituitary gland (male), and thyroid gland (male) examined for low dose rats;
Other examinations:
When the pathology evaluation was completed by the laboratory pathologist and the pathology data entered into the Toxicology Data Management System (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 labelling 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 tumour 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 lesions for each tissue type are combined according to the guidelines of McConnell et al. (1986).
Statistics:
For details on statistics, please refer to section "Any other information on materials and methods incl. tables".
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See section "details on results" below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See section "details on results" below
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
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
See section "details on results" below
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
See section "details on results" below
Other effects:
not examined
Details on results:
Body Weights and Clinical Signs:
Mean 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.

Survival:
Estimates of the probabilities of survival for male and female rats administered succinic anhydride at the doses used in these studies and for vehicle controls were presented in the report as Kaplan and Meier curves; standard (unadjusted) survival curves were presented for comparison. No significant differences in survival were observed between any groups of either sex. The interim evaluation of 10 rats per group scheduled after 15 months of exposure was cancelled because of some early deaths due to gavage accidents. To determine whether gavage accidents were the possible cause of early death or the reason for the rats being killed in a moribund condition, a retrospective examination of sections of nose and lung, oesophagus 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 Results:
Statistically 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 tumors that 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 was not significantly greater than that in vehicle controls (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 of succinic 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 B6C3F1 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 were reduced 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 evidence of carcinogenicity of phthalic anhydride for rats or mice under the conditions of these studies. In 7-week studies in rats and mice, there were no dose-related histopathologic lesions in either species given diets containing up to 50,000 ppm phthalic anhydride. Succinic anhydride generally has been negative in assays for mutagenic or clastogenic activity. Succinic anhydride was not mutagenic in Salmonella typhimurium (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 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 in a concurrent 92 week study for murine carcinogenicity.
Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: no adverse effects were observed up to 100 mg/kg bw/day
Key result
Critical effects observed:
no
Conclusions:
In a carcinogenicity oral study (similar to OECD TG 451), test item did not show any carcinogenic effects. Based on the results, the NOAEL for carcinogenicity is considered to be higher or equal to 100 mg/kg bw/day.
Executive summary:

In a carcinogenicity study performed similar to OECD TG 451, succinic anhydride (99%) was administered to 60 Fischer 344/N rats/sex/dose by oral gavage at dose levels of 0, 50 and 100 mg/kg bw/day for 103 weeks (5 days per week).

There were no compund related effects on mortality, clinical signs or gross and histologic pathology. Mean body weights of high dose male and female rats were slightly reduced (approximately 6% and 8%, respectively) compared to vehicle controls during the second year of the study, while mean body weights of low dose and vehicle control rats were generally similar throughout the studies.

At the doses tested, there was no treatment related increase in tumor incidence when compared to controls.

The NOAEL for carcinogenicity is considered to be >100 mg/kg bw/day.

This carcinogenicity study in the rat is considered acceptable as it was performed similar to OECD TG 451.

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981-05-01 to 1990-01-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Version / remarks:
adopted in 1981
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of the test material used in the report: Succinic anhydride
- Appearance: white, flaky solid
- Batch no.: PE072797 (Aldrich Chemical Company)
- Purity: 99%
- Storage temperature: In refrigerator (5° C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
Appropriate amount of chemical and corn oil were homogenized with a Polytron® PT 10-ST probe at setting 7 for 2 min, followed by setting 9 for 1 min.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Facility (Kingston, NY)
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Not stated
- Fasting period before study: not applicable
- Housing: Animals were housed five per cage in polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD) containing spun-bonded polyester cage filters (Snow Filtration, Cincinnati, OH) and hardwood chips for bedding (P J. Murphy Forest Products Corp., Rochelle Park, NJ). Cages of vehicle control animals were placed in the top two rows of the racks, cages of low dose animals were placed in the next two rows, and cages of high dose animals were placed in the bottom two rows. Cages were not rotated during the studies.
- 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 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°-30°C (64-86° F)
- Humidity (%): 22-84%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): fluorescent light, 12/12

N-LIFE DATES:
From: 18 May 1981 To: 6 May 1983
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 used within 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the 2-year studies, the dose formulations were analysed 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 studies. The 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:
103 weeks
Frequency of treatment:
5 days per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
38 mg/kg bw/day (nominal)
Remarks:
males
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
males/females
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
females
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Because of the reduced survival of male and female mice that received 300 mg/kg and the lower weight gain for males at 150 mg/kg, doses of succinic anhydride selected for mice for the 2-year studies were 38 and 75 mg/kg for males and 75 and 150 mg/kg for females, administered in corn oil by gavage 5 days per week.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed two times per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed two times per day.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded once per week for the first 12 weeks of the study, except during week 7 for mice, and once per month thereafter. Mean body weights were calculated for each group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Animals found moribund and those surviving to the end of the studies were humanely killed.

GROSS PATHOLOGY: Yes
- A 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.


HISTOPATHOLOGY: Yes
- Histopathologic examinations of tissues were performed on all high dose and vehicle control animals and on all mice that died before wk 92. In addition, histopathologic examinations were performed on all grossly visible lesions in all dose groups. Potential target organs for chemically related neoplastic and nonneoplastic effects were determined by examination of the pathology data; these target organs/tissues in the low dose group were examined histopathologically.
The following tissues were examined: adrenal glands, bone, bone marrow, brain, chloral or preputial gland, epididymis/prostate/testes or ovaries/uterus, esophagus, heart, kidneys, large and small intestines, larynx, liver, lungs, lymph nodes, mammary gland, nose, pancreas, pancreatic islets, parathyroid glands, pituitary gland, salivary glands, spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Kidneys, liver, and nasal cavity examined for low dose male mice, and pituitary gland and cecum examined for low dose female mice.
Other examinations:
When the pathology evaluation was completed by the laboratory pathologist and the pathology data entered into 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 lesions for each tissue type are combined according to the guidelines of McConnell et al. (1986).
Statistics:
For details on statistics, please refer to section "Any other information on materials and methods incl. tables".
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See section "details on result" below
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See section "details on result" below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See section "details on result" below
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
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
See section "details on result" below
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
See section "details on result" below
Other effects:
not examined
Details on results:
Body Weights and Clinical Signs:
Mean body weights of high dose male mice were generally 5%-12% lower than those of vehicle controls after week 11. Mean body weights of vehicle control and low dose male mice were similar throughout most of the study. Mean body weights of high dose female mice were 10%-32% lower than those of vehicle controls from week 12 to the end of the study. Mean body weights of low dose female mice were 10%-20% lower than those of vehicle controls from week 28 to the end of the study. During months 8 through 12, low and high dose male and female mice assumed arched postures immediately after dosing and became lethargic. The mice had a normal appearance about 15 minutes later. After dosing during this same period, mice were occasionally observed to rub their faces and burrow in bedding. High dose females occasionally wheezed and had rough hair coats.

Survival:
Estimates of the probabilities of survival for male and female mice administered succinic anhydride at the doses used in these studies and for vehicle controls are presented in the report as Kaplan and Meier curves. Standard (unadjusted) survival curves are also presented. The survival of the vehicle control male mice was significantly lower than that of the high dose group after week 77. No other significant differences in survival were observed between any groups of either sex. To determine whether gavage accidents were the possible cause of early death or the reason for the mice being killed in a moribund condition, a retrospective examination of sections of nose and lung, oesophagus and trachea, and heart and mediastinum was performed on all 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 mice in each dose group lived long enough to allow evaluation of the potential carcinogenicity of succinic anhydride.

Pathology and Statistical Analyses of Results:
Statistically significant or biologically noteworthy changes in the incidences of mice with nonneoplastic lesions of the nasal cavity and kidney. No significant increases in the incidences of neoplastic lesions were observed. Summaries of the incidences of neoplasms and nonneoplastic lesions, individual animal tumor diagnoses, and statistical analyses of primary tumors that occurred with an incidence of at least 5% in at least one animal group are presented for male and female mice, respectively, in the study report but not reproduced in this summary.

Nasal Cavity: Acute inflammation and foreign material were seen at increased incidences in dosed male mice (acute inflammation: vehicle control, 1/48; low dose, 9/50; high dose, 9/50; foreign material: 10/48; 24/50; 19/50). The inflammation was considered to be a consequence of the foreign material (corn oil) in the nasal cavity. Squamous metaplasia, secondary to inflammation, was observed in four high dose male mice.

Kidney: Renal mineralization was observed with a decreasing trend in male mice (vehicle control, 16/49; low dose, 6/50; high dose, 0/50).
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 of succinic 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 B6C3F1 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 were reduced 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 evidence of carcinogenicity of phthalic anhydride for rats or mice under the conditions of these studies. In 7-week studies in rats and mice, there were no dose-related histopathologic lesions in either species given diets containing up to 50,000 ppm phthalic anhydride. Succinic anhydride generally has been negative in assays for mutagenic or clastogenic activity. Succinic anhydride was not mutagenic in Salmonella typhimurium (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 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 in a concurrent 92 week study for murine carcinogenicity.
Dose descriptor:
NOAEL
Effect level:
> 75 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: no adverse effects observed in male mice up to 75 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
(systemic)
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: neoplastic
Dose descriptor:
LOAEL
Remarks:
(systemic)
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Conclusions:
In a carcinogenicity oral study (similar to OECD TG 451) in mice, test item did not show any carcinogenic effects. Based on the results, the NOAEL for carcinogenicty is considered to be higher or equal to 75 mg/kg bw/day for males and 150 mg/kg bw/day for females.
Executive summary:

In a carcinogenicity study performed similar to OECD TG 451, succinic anhydride (99%) was administered to 50 B6C3F1 mice/sex/dose by oral gavage at dose levels of 0, 38 and 75 mg/kg bw/day for male mice and 0, 75 and 150 mg/kg bw/day for female mice for 103 weeks (5 days per week).

There were no compund related effects on mortality. Clinical signs included arched postures immediately after dosing and lethargy in low and high dose male and female mice during months 8 and 12. The mice had a normal appearance about 15 minutes later. After dosing during this same period, mice were occasionally observed to rub their faces and burrow in bedding. High dose females occasionally wheezed and had rough hair coats. Mean body weights of high dose male mice were generally 5%-12% lower than those of vehicle controls after week 11, while mean body weights of vehicle control and low dose male mice were similar throughout most of the study. Mean body weights of high dose female mice were 10%-32% lower than those of vehicle controls from week 12 to the end of the study. Mean body weights of low dose female mice were 10%-20% lower than those of vehicle controls from week 28 to the end of the study.

Acute inflammation and foreign material were seen at increased incidences in dosed male mice. The inflammation was considered to be a consequence of the foreign material (corn oil) in the nasal cavity. Squamous metaplasia, secondary to inflammation, was observed in four high dose male mice. Based on these results, the NOAEL for male mice was considered to be >75 mg/kg bw/day, while for female mice no NOAEL could be determined due to increased body weight loss at the lowest dose administered.

At the doses tested, there was no treatment related increase in tumor incidence when compared to controls. Therefore, the NOAEL for carcinogenicty is considered to be higher or equal to 75 mg/kg bw/day for males and 150 mg/kg bw/day for females.

This carcinogenicity study in the mouse is considered acceptable as it was performed similar to OECD TG 451.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Study performed similar to OECD TG 451.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results from carcinogenicity studies with rats and mice, no classification of succinic acid is required for carcinogenicity based on the CLP criteria as set out in Regulation (EC) No. 1272/2008.

Additional information

In a carcinogenicity study performed similar to OECD TG 451, succinic anhydride (99%) was administered to 60 Fischer 344/N rats/sex/dose by oral gavage at dose levels of 0, 50 and 100 mg/kg bw/day for 103 weeks (5 days per week).

There were no compund related effects on mortality, clinical signs or gross and histologic pathology. Mean body weights of high dose male and female rats were slightly reduced (approximately 6% and 8%, respectively) compared to vehicle controls during the second year of the study, whilel mean body weights of low dose and vehicle control rats were generally similar throughout the studies.

At the doses tested, there was no treatment related increase in tumor incidence when compared to controls.

The NOAEL for carcinogenicity is considered to be >100 mg/kg bw/day.

In a carcinogenicity study performed similar to OECD TG 451, succinic anhydride (99%) was administered to 50 B6C3F1 mice/sex/dose by oral gavage at dose levels of 0, 38 and 75 mg/kg bw/day for male mice and 0, 75 and 150 mg/kg bw/day for female mice for 103 weeks (5 days per week).

There were no compund related effects on mortality. Clinical signs included arched postures immediately after dosing and lethargy in low and high dose male and female mice during months 8 and 12. The mice had a normal appearance about 15 minutes later. After dosing during this same period, mice were occasionally observed to rub their faces and burrow in bedding. High dose females occasionally wheezed and had rough hair coats. Mean body weights of high dose male mice were generally 5%-12% lower than those of vehicle controls after week 11, while mean body weights of vehicle control and low dose male mice were similar throughout most of the study. Mean body weights of high dose female mice were 10%-32% lower than those of vehicle controls from week 12 to the end of the study. Mean body weights of low dose female mice were 10%-20% lower than those of vehicle controls from week 28 to the end of the study.

Acute inflammation and foreign material were seen at increased incidences in dosed male mice. The inflammation was considered to be a consequence of the foreign material (corn oil) in the nasal cavity. Squamous metaplasia, secondary to inflammation, was observed in four high dose male mice. Based on these results, the NOAEL for male mice was considered to be >75 mg/kg bw/day, while for female mice no NOAEL could be determined due to increased body weight loss at the lowest dose administered.

At the doses tested, there was no treatment related increase in tumor incidence when compared to controls. Therefore, the NOAEL for carcinogenicty is considered to be higher or equal to 75 mg/kg bw/day for males and 150 mg/kg bw/day for females.

No evidence for carcinogenesis was found in studies with 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.