Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981-1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline-compliant, GLP-compliant study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
The NTP report contains information relating to a variety of different test types and durations including sub-acute, subchronic and chronic oral exposure to rats and mice over 16 days, 13 weeks or 2 years. Information relating to a battery of in vitro genotoxicity tests is also included and a review of developmental toxicity effects is incorporated into the report. Test methods are in general compliance with EU methods B.7 and B.26 and OECD 453 where appropriate.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
White, flaky solid with purity indicated as 99%. Succinic anhydride is a colorless and odorless organic solid (melting point 119.6° C) that is soluble in ethanol, chloroform, and carbon tetrachloridebut only very slightly soluble in water. In boiling water, succinic anhydride is converted almost instantaneously to succinic acid, an endogenousmetabolic intermediate whereas at 21° C, hydrolysis of an aqueous suspension of succinic anhydride requires 30 minutes.Succinic anhydride has been produced by hydrogenation of maleic anhydride; by dehydration of succinic acid at elevated temperatures and pressure;by treating succinic acid with diketene, succinyl chloride, or acetic anhydride; or by reacting the diethyl ester with boron chloride

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Four- to 6-week old male and female rats were obtained from Charles River Breeding Laboratories, observed for 18 days, distributed to cages from weight classes and assigned to dose groups according to a table of random numbers. Five animals were assigned per polycarbonate cage containing spun-bonded polyester filters and hardwood chips for bedding. Animals were fed a diet of NIH 07 Rat Ration and water was provided via an automatic watering system. The temperature in the animal room was maintained at 70-80 F, humidity of 22-74%, fluorescent lights for 12h/d with 12-15 room air changes/hour. Rats were approximately 7-8 weeks old when placed on the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Groups of male and female rats were administered succinic anhydride by gavage for 5 days per week for 13 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic gas chromatographic analysis of the dose formulations was conducted by both the study laboratory as well as the analytical chemistry laboratory.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:Males: 0, 25, 50, 100, or 400 mg/kgBasis:actual ingested
Remarks:
Doses / Concentrations:Females: 0, 12.5, 25, 50, 100 or 200 mg/kgBasis:actual ingested
No. of animals per sex per dose:
10 males/dose and 10 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Succinic anhydride was nominated by the US National Cancer Institute for toxicology and carcinogenicity studies because of its potential to be a direct-acting acylating agent, because its extensive use may lead to human exposure, and because there was a lack of long-term toxicity and carcinogenicity information on this chemical. The gavage route of administration was selected because human exposure occurs by the oral route.Thirteen week studies of succinic anhydride were originally performed after the chemical had been ground with a mortar and pestle before being mixed with corn oil. Suspensions were constantly stirred with a magnetic stirrer during the dosing procedures. Another procedure was developed to produce a more stable suspension of succinic anhydride by using a Polytron homogenizer to reduce particle size. The thirteen week studies were repeated using the Polytron-prepared suspensions as they were found to be more toxic to rats than those prepared using the mortar and pestle. Results of the second 13-week study are presented here.
Positive control:
No information

Examinations

Observations and examinations performed and frequency:
Rats were observed once per day. Clinical observations were recorded once per week. Animals were weighed at the beginning of the study and then once per week.
Sacrifice and pathology:
Necropsy was performed on all animals and histological examinations were performed on all vehicle controls, the two highest dose groups in males, the two highest dose groups in females and animals dying before the end of the studies. The following tissues were examined microscopically: brain, cecum, esophagus, heart, kidneys, larynx, liver, lungs, mediastinum, mesenteric lymph nodes, pancreas, salivary glands, spleen, stomach, thymus, thyroid gland, and trachea. Liver weights were obtained at necropsy.
Other examinations:
No details
Statistics:
The majority of the statistical methods detailed relate to assessment of survival or carcinogeniciy parameters. Tests of significance included pairwise comparisons of each dosed group with vehicle controls and a test for an overall dose-response trend.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Deaths of 8/10 males at 400 mg/kg and 4/10 males and 5/10 females at 200 mg/kg were considered treatment related. Lethargy and abdominal distension were apparent at 200 and 400 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
Deaths of 8/10 males at 400 mg/kg and 4/10 males and 5/10 females at 200 mg/kg were considered treatment related. Lethargy and abdominal distension were apparent at 200 and 400 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slight reduction in mean bodyweight for males dosed at 200 mg/kg (-9% ) or at 400 mg/kg (-15%)
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weights were slightly increased for females dosed at 100 or 200 mg/kg bw/day
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No macroscopic lesions noted
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related microscopic changes observed
Details on results:
Deaths of 8/10 males that received 400 mg/kg and 4/10 males and 5/10 females that received 200 mg/kg were considered to be compound related. Other deaths were considered to be the result of gavage error. Lethargy and distended abdomens were seen at the two highest doses. The mean body weights at necropsy of male rats that received 200 or 400 mg/kg were 9% or 15% lower than that of vehical controls. The mean body weights at necropsy of dosed and vehicle control female rats were similar. The relative organ weights for female rats that received 100 or 200 mg/kg were slightly greater than that for vehicle controls. No compound-related lesions were seen microscopically.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Survival and mean body weights of rats in the 13 -week gavage study of succinic anhydride

   

       Mean Body Weights (grams)

 
 Dose (mg/kg)  Survivala  Initialb  Final  Changec  Final weight relative to vehicle controls (%)
MALE               
10/10  143 + 326 + + 213 + -- 
 25 8/10d   143 + 2 352 +  + 210 + 4 99
 50  8/10d 144 + 357 +  + 212 + 10 100
 100 9/10d   143 + 2 340 + 12  + 197 + 13  96 
 200  6/10e 141 + 324 + 5 + 183 + 91 
400   2/10f 140 + 302 + 15  + 163 + 14  85 
 FEMALE              
 0 10/10   114 + 1 194 + + 80 + -- 
 12.5 10/10  115 + 199 + + 84 + 103 
 25 10/10  115 + 201 + + 86 + 104 
 50  9/10d  115 + 1 198 + + 83 + 102 
 100  9/10d  115 + 1 189 + + 74 + 97 
 200  3/10g 114 + 192 + + 78 + 99 
a Number surviving/number initially in group; b Initial group mean body weight + standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study; c Mean body weight change of the survivors + standard error of the mean; d Deaths may have been gavage related; e Week of death : 1, 10, 12, 12; f Week of death: 1, 1, 1, 2, 2, 3, 3, 9; g Week of death: 1,1,2,2,3 --two additional deaths may have been gavage related.

Table 2. Liver weights of rats in the 13 -week gavage study of succinic anhydridea

 Dose (mg/kg)  Number weighed  Necropsy Body Weight (grams)  Liver Weight (mg)  Liver Weight/Necropsy Body Weight (mg/g)
 MALE           
 0  10 356.4 + 5.7  14125 + 172.6  39.7 + 0.70 
 25  8 352.1 + 5.6   13624 + 554.0 38.7 + 1.48 
 50  8 356.6 + 8.0  14363 + 552.8  40.2 + 1.02 
 100 8b  336.5 + 13.3  13835 + 788.1  41.0 + 1.10 
 200  6 323.8 + 4.7 *  13343 + 386.5 41.2 + 0.78 
 400 302.0 + 15.0 *  12455 + 135.0  41.4 + 2.50 
 FEMALE           
 0 10  193.5 + 2.0  6437 + 138.4  33.3 + 0.68 
 12.5 10  198.8 + 1.7  6765 + 161.8  34.0 + 0.66
 25 10  201.2 + 3.8  6982 + 125.1  34.7 + 0.37 
 50  9 198.1 + 2.4  6829 + 170.6  34.5 + 0.85 
 100  9 188.8 + 3.0  6829 + 182.9  36.1 + 0.62* 
 200 192.3 + 5.2  7303 + 89.5*  38.0 + 0.96** 
a Mean + standard error; p values versus the vehicle controls by Dunnett's test; b The liver of a ninth animal was not weighed--the necropsy body weight of this animal has been excluded from the analysis; * p<0.05; ** p< 0.01 

TABLE 3. EXPERIMENTAL DESIGN AND MATERIALS AND METHODS IN THE GAVAGE STUDIES OF

SUCCINIC ANHYDRIDE

Sixteen-Day

Studies in Mice

Twenty-Day

Studies in Rats

Thirteen-Week

Studies in Mice

Thirteen-Week

Studies in Rats

Two-Year

Studies - mouse

Two-Year

Studies - rat

EXPERIMENTAL DESIGN

Size of Study Groups

5 male and 5 female mice

 

10 male and 10

 female rats

10 male and 10 female mice

10 male and 10 female rats

50 male and 50 female mice

60 male and 60 female rats

Doses

0,219,438,875, 1,750, or 3,500 mg/kg succinic anhydride in corn oil by gavage; dose vol dose -10 ml/kg

0, 47, 94, 187, 375 or 750 mg/kg succinic anhydride in corn oil by gavage; dose vol dose -
5 ml/kg

0,37,75,150,300, or

600 mg/kg succinic

anhydride in corn

oil by gavage; dose

vol-10 ml/kg

Male-0,25,50,100,

200, or 400 mg/kg succinic anhydride in corn oil by gavage;

female-0,12.5,25,50,

100, or 200 mg/kg;

dose vol-5 ml/kg

mice-male: 0,

38, or 75 mg/kg; female: 0,

75, or 150 mg/kg; dose vol - 10 ml/kg

Rats~0,50, or 100 mg/kg succinic anhydride in corn oil by gavage;
dose vol -5 ml/kg

Dates of first dose

26/11/79

9/7/81

31/3/80

5/10/81

18/5/81

30/8/82

Date of last dose

11/12/79

28/7/81

27/6/80

3/1/82

6/5/83

17/8/84

Duration of dosing

12 doses over 16 d

14 doses over 20 d

5d/wk for l3wk

5d/wk for l3wk

5d/wk for l03 wk

5d/wk for l03 wk

Type and frequency of observations

Observed 1 x d; Same as 16-d studiesweighed 1 x wk in mice

Observed 1 x d; Same as 16-d studiesweighed 1 x wk in mice

Observed 2 x d; weighed initiallyand 1 x wkthereafter

Observed 1 x d; weighed initiallyand 1 x wkthereafter

Observed 2 x d, weighed 1 x wk for 13 wk (rats) or 12 wk (mice) and 1 x month thereafter

Observed 2 x d, weighed 1 x wk for 13 wk (rats) or 12 wk (mice) and 1 x month thereafter

Necropsy and histology examinations

Necropsy performed on all animals; histologic exams performed on 4 males and 2 females from the 438 mg/kg

groups

Necropsy performed on all animals in the 0, 187,375, and 750 mg/kg groups, histologic exams performed on all vehicle controls, animals from the 375 and 750 mg/kg groups, and animals in the 187 mg/kg groups dying before the end of the studies. Tissues examined include oesophagus,

kidneys, larynx,

lungs, parathyroid

glands, stomach, and

trachea; nasal cavity

or intestinal tract for

rats with gross lesions

Necropsy performed on all animals; the

following tissues examined histologically for all vehicle control and high dose animals and for animals dying before the end of the studies: adrenal glands, bone, bone marrow, brain, colon, oesophagus, gallbladder, heart, kidneys, liver, lungs, mammary gland, mandibular and mesenteric lymph nodes, pancreas, parathyroid glands, pituitary gland, salivary glands, skin, small intestine, spleen, stomach, testes/epididymis/ prostate or ovaries/uterus, thymus, thyroid gland, trachea, and urinary bladder

Necropsy performed

on all animals; histologic exams performed on all vehicle controls, 200 and 400 mg/kg males, 100 and 200 mg/kg females, and animals dying before the end of the studies. The following tissues were examined microscopically: brain, caecum, oesophagus, heart, kidneys, larynx, liver, lungs, mediastinum, mesenteric lymph nodes, pancreas, salivary glands, spleen, stomach, thymus, thyroid gland, and trachea. Liver weights obtained at necropsy

Necropsy performed on all animals; histologic exams performed on all vehicle control and high dose animals, all mice that died before wk 92, and all animals with gross lesions, the following tissues were examined adrenal glands, bone, bone marrow, brain, chloral or Preputial gland, epididymis/prostate/ testes or ovaries/uterus, oesophagus, 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 caecum examined for low dose female mice

Necropsy performed on all animals; histologic exams performed on all vehicle control and high dose animals, all rats that died before scheduled termination, and all animals with gross lesions, the following tissues were examined adrenal glands, bone, bone marrow, brain, chloral or Preputial gland, epididymis/prostate/ testes or ovaries/uterus, oesophagus, 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;

Animals and maintenance

Strain and species

B6C3F1 mice

F344/N rats

B6C3F1 mice

F344/N rats

B6C3F1 mice

F344/N rats

Animal source

Charles River Breeding Laboratories (Portage, MI)

Charles River Breeding Laboratories (Kingston, NY)

Charles River Breeding Laboratories (Kingston, NY)

Charles River Breeding Laboratories (Kingston, NY)

Frederick Cancer Research Facility (Frederick, MD)

Charles River Breeding Laboratories (Kingston, NY)

Laboratory

Microbiological Associates Inc

Microbiological Associates Inc

Microbiological Associates Inc

Microbiological Associates Inc

Microbiological Associates Inc

Microbiological Associates Inc

Acclimatisation period

18d

21d

19d

18d

0d

19d

Age at study commencement

7-9 wk

7-8 wk

7-9 wk

7-8 wk

8-9 wk

8-9 wk

Age at termination

9-11 wk

10-11 wk

20-22 wk

20-21 wk

112-114 wk

112-114 wk

Diet

NIH07 Rat and Mouse Ration (Zeigler Bros., Inc ,Gardners, PA); availablead libitum

NIH07 Rat and Mouse Ration (Zeigler Bros., Inc ,Gardners, PA); availablead libitum

NIH07 Rat and Mouse Ration (Zeigler Bros., Inc ,Gardners, PA); availablead libitum

NIH07 Rat and Mouse Ration (Zeigler Bros., Inc ,Gardners, PA); availablead libitum

NIH07 Rat and Mouse Ration (Zeigler Bros., Inc ,Gardners, PA); availablead libitum

NIH07 Rat and Mouse Ration (Zeigler Bros., Inc ,Gardners, PA); availablead libitum

Bedding

Hardwood chips

Hardwood chips

Hardwood chips

Hardwood chips

Hardwood chips

Hardwood chips

Water

Automatic watering system (Edstrom Industries, Waterford, WI); availablead libitum

Automatic watering system (Edstrom Industries, Waterford, WI); availablead libitum

Automatic watering system (Edstrom Industries, Waterford, WI); availablead libitum

Automatic watering system (Edstrom Industries, Waterford, WI); availablead libitum

Automatic watering system (Edstrom Industries, Waterford, WI); availablead libitum

Automatic watering system (Edstrom Industries, Waterford, WI); availablead libitum

Cages

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD)

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD)

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD)

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD)

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD)

Polycarbonate (Lab Products, Inc., Rochelle Park, NJ, or Hazleton Systems, Inc., Aberdeen, MD)

Cage filters

Spun-bonded polyester (Snow Filtration, Cincinnati, OH)

Spun-bonded polyester (Snow Filtration, Cincinnati, OH)

Spun-bonded polyester (Snow Filtration, Cincinnati, OH)

Spun-bonded polyester (Snow Filtration, Cincinnati, OH)

Spun-bonded polyester (Snow Filtration, Cincinnati, OH)

Spun-bonded polyester (Snow Filtration, Cincinnati, OH)

Number of animals per cage

5

5

5

5

5

5

Animal room environment

Temp: 62°-74°F;

Humid: 45%-70%;

fluorescent light 12h/d;
12-15 room air changes/h

Temp: 70°-80°F;

Humid: 55%-73%;

fluorescent light 12h/d;
12-15 room air changes/h

Temp: 68°-87°F;

Humid: 35%-90%;

fluorescent light 12h/d;
12-15 room air changes/h

Temp: 70°-80°F;

Humid: 22%-74%;

fluorescent light 12h/d;
12-15 room air changes/h

Temp: 64°-86°F;

Humid: 22%-84%;

fluorescent light 12h/d;
12-15 room air changes/h

Temp: 64°-86°F;

Humid: 22%-84%;

fluorescent light 12h/d;
12-15 room air changes/h

 

Applicant's summary and conclusion

Conclusions:
Mortality and toxic signs were observed when rats were administered succinic anhydride at concentrations of 200 mg/kg and higher for 13 weeks.
Executive summary:

In a 13 -week study in rats conducted by the US National Toxicology Program, doses of succinic anhydride ranged from 25 to 400 mg/kg for males and from 12.5 to 200 mg/kg for females. Deaths of 8/10 male rats that received 400 mg/kg and 4/10 males and 5/10 females that received 200 mg/kg were compound related. At necropsy, the mean body weights of male rats that received 200 or 400 mg/kg were 9% or 15% lower than that of vehicle controls, whereas the mean body weights of dosed and vehicle control female rats were similar. No compound-related gross or microscopic lesions were observed. The NOAEL in the 13 -week investigation was 100 mg/kg bw/day for male and female rats.