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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981-10 to 1990-01-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted in 1981
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. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Succinic anhydride
EC Number:
203-570-0
EC Name:
Succinic anhydride
Cas Number:
108-30-5
Molecular formula:
C4H4O3
IUPAC Name:
oxolane-2,5-dione
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 (4 to 8° C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
Chemical in corn oil was homogenized with a Polytron homogenizer.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
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.
- Source: Charles River Breeding Laboratories (Kingston, NY)
- Age at study initiation: 7-8 weeks
- Housing: Animals were housed five per cage in polycarbonate cages (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).
- Diet (e.g. ad libitum): NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA), ad libitum
- Water (e.g. ad libitum): water was provided via an automatic watering system (Edstrom Industries, Waterford, WI), ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 10-80° F
- Humidity (%): 22-74%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): fluorescent lights, (12h/12h)

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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
males/females
Dose / conc.:
12.5 mg/kg bw/day (nominal)
Remarks:
females
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
males/females
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
males/females
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
males/females
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
males/females
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
males
No. of animals per sex per dose:
10
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

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:
At the end of the 13-week studies, survivors were humanely killed. 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.
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):
Lethargy and abdominal distension were apparent at the two highest dose levels.
Mortality:
mortality observed, treatment-related
Description (incidence):
Deaths of 8/10 males at 400 mg/kg bw/day and 4/10 males and 5/10 females at 200 mg/kg bw/day were considered treatment related.
Other deaths were considered to be the result of gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in mean bodyweight for males dosed at 200 mg/kg bw/day (-9% ) and at 400 mg/kg bw/day (-15%) were observed.
The mean body weights at necropsy of dosed and vehicle control female rats were similar.
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:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weights were slightly increased for females dosed at 100 bw/day (36.1 mg/g) or 200 mg/kg bw/day (38.0 mg/g) compared to the vehicle control (33.3 mg/g).
No compound-related lesions were seen microscopically.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No macroscopic lesions noted
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related microscopic changes observed
Other effects:
not examined
Details on results:
Deaths of 8/10 males that received 400 mg/kg bw/day and 4/10 males and 5/10 females that received 200 mg/kg bw/day 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 bw/day 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 (liver) for female rats that received 100 or 200 mg/kg bw/day were slightly greater than that for vehicle controls. No compound-related lesions were seen microscopically.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
mortality

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 2. 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 3. 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 

 

Applicant's summary and conclusion

Conclusions:
Mortality and toxic signs as indicated by reduced body weight and an increase in relative liver weight were observed when rats were administered succinic anhydride at concentrations of 200 mg/kg and higher for 13 weeks.
Executive summary:

In a subchronic toxicity study performed for 13-weeks similar to OECD TG 408, succinic anhydride (99 %) was orally administered to 10 Fischer 344 rats/sex/dose in corn oil by gavage at dose levels of 0, 25, 50, 100, 200, 400 mg/kg bw/day (males) and 0, 12.5, 25, 50, 100, 200 mg/kg bw/day (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 considerd 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. Relative liver weights were slightly increased for females dosed at 100 or 200 mg/kg bw/day. No compound-related gross or microscopic lesions were observed. Based on these results, the NOAEL in this 13-week study was considered to be 100 mg/kg bw/day for male and female rats.

This subchronic toxicity study in rats is acceptable as it was performed similar to the requirements for a subchronic oral oral study (OECD 408).