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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1977
Report date:
1977
Reference Type:
review article or handbook
Title:
Maleic Anhydride and Maleic Acid, SIDS Initial Assessment Report For SIAM 18
Author:
OECD SIDS
Year:
2004
Bibliographic source:
OECD SIDS Initial Assessment Report For SIAM 18, Paris, France, 20-23 April 2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
only 2 dose levels, organ weight determination after 183 days (not of all rats)
Principles of method if other than guideline:
The purpose of the present study is to determine whether lung lesions would be observed in a repeated study.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Maleic anhydride
EC Number:
203-571-6
EC Name:
Maleic anhydride
Cas Number:
108-31-6
Molecular formula:
C4H2O3
IUPAC Name:
2,5-dihydrofuran-2,5-dione
Details on test material:
- Name of test material (as cited in study report): Maleic anhydride
- Physical state: snow white briquets
- Analytical purity: >/= 99.5%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Spartan Research Animals
- Age at study initiation: 6-7 weeks
- Housing: individual
- Diet: ad libitum
- Water: ad libitum


Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly for the first 90 days and monthly thereafter according to the mean food consumption and body weight of each group of rats in order to maintain the assignated dose levels of maleic anhydride on a mg/kg/day basis.
- Mixing appropriate amounts with: A 10% premix of maleic anhydrid in ground laboratory chow was used to prepare the test diets.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A gas chromatographic method was used for analysis of the feed to confirm the concentrations.
Duration of treatment / exposure:
90 and 183 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
50/dose (treatment groups), 75 (control group)
Control animals:
yes, concurrent no treatment
Details on study design:
- Other:
Ninety-day diietary feeding studies were conducted with rats on the maleic anhydride component to define the effects of repeated ingestion and In one rat study, a singular nodular lesion observed grossly in the lung of one male rat on a diet providing 600 mg/kg/day of maleic anhydride was diagnosed microscopically as a pulmonary adenoma. A similar lesion was observed microscopically in one male at 250 mg/kg/day.
The purpose of the present study is to determine whether similar lung lesions would be observed in a repeat study conducted under similar conditions on a larger number of animals. Only male rats is used in this study since there is no evidence of similar effects in females in the previous study.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 13 weeks of the study and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood:on day 174
- How many animals: 5 rats/dose level
- Parameters checked: packed cell volme (PLV), red blood cell count (RBC), hemoglobin concentration (Hgb), white blood cell count (WBC), and differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the 90-day and 183-day necropsies
- How many animals: 10 rats/dose
- Parameters checked: blood urea nitrogen (BUN) concentration, alkaline phosphatase (AP) activity and serum glutamic pyruvic transaminase (SGPT) activity

URINALYSIS: Yes
- Time schedule for collection of urine: on days 12, 84, 174 and 176 (on days 84-85 and 176-177 a urine concentration test was conducted; the specific gravity was determined on urine samples collected over 6 hrs intervals (8 am - 2 pm) on the 2 conecutive days)



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Analyses of body weights, food consumption, hematological and clinical chemistry parameters, organ weights and organ to body weight ratio were made using a one-way analysis of variance and the treatment means were compared with control values by Dunnett's test. The level of significance chosen for all cases was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For further details, see section below "Details on results".
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For futher details, see section below "Details on results".
Urinalysis findings:
no effects observed
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):
For futher details, see section below "Details on results".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
For futher details, see section below "Details on results".
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
For futher details, see section below "Details on results".
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY:
One rat died spontaneously and lung, liver, and kidneys were exarnined microscopically.

BODY WEIGHT AND WEIGHT GAIN: the high dose group appeared to be gaining weight at a rate slightly less than that of the control group during the last 2 months of the study

CLINICAL CHEMISTRY: small but significant decrease in the alkaline phosphatase activity for both treatment groups at 90 days and for the 250 mg/kg/day group at 183 days. The significance of these decreases in alkaline phosphatase activity in relation to the other toxicological changes observed is uncertain.

URINALYSIS: On day 174, there was a statistically significant increase in the specific gravity of the urine of rats at the highest dose level. However, it is doubtful that this was physiologically or toxicologically significant since the value is not different from the values for all groups obtained two days later during the urine concentration test.

ORGAN WEIGHT: At the 90-day timepoint, the relative liver weight (+ 16%) for the rats in the high-dose level and the absolute/relative kidney weights for both the 250 and 600 mg/kg groups were significantly higher (+ 24% and + 65%, respectively) compared to the controls (p<0.05). At 183 days, there was a significant increase in the absolute and relative liver (+ 18% and +10%) and kidney weights (+ 54% and + 119%) for both treatment groups, increased relative testes, brain and heart weight (+ 5%), and decreased fasted body weights for rats at 600 mg/kg/day (p<0.05). The increase in the absolute and relative weights of the liver and kidney and the relative weight of the heart are attributed to treatment whereas the increased relative brain and testes weights are interpreted to reflect the decreased body weight rather than an effect on these organs.

GROSS PATHOLOGY: Dose dependent treatment - related changes in the kidneys were apparent in rats sacrificed at 90 and 183 days. The number of rats showing the kidney changes and the degree of involvement increased in severity with the dose and the duration on the diet containing maleic anhydride. Some rats from both dose levels at the 183-day sacrifice were smaller and lacked normal amounts of abdominal adipose tissue. No gross treatment related changes were recognized in the lungs or other organ systems at either the 90- or 183-day sacrifice.

HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related changes were present in the kidneys of rats sacrificed at 90 and 183 days. The observed changes indicated a marked accentuation of the spontaneously occurring findings seen in the control animals. The changes in the controls and treated animals included: individual tubules that were dilated and contained eosinophilic staining casts, granular degeneration of the epithelial cells lining these tubules, tubular collapse and atrophy with peritubular fibrosis, focal mononuclear inflammatory cell infiltrates, glomeruli that showed thickening of the basement membrane, thickening and epithelialization of Bowman's Capsule, and occasionally showed either focal or diffuse sclerosis of the glomerular tufts. Both tubular and glomerular changes in treated animals were more severe than in controls. In addition to the focal nature of the lesion (in controls), the tubules throughout the cortex of treated rats showed a generalized dilatation and hypertrophy. There were more degenerative tubules and tubules showing mitotic activity in treated versus controls. The degree of degenerative, hypertrophic, and regenerative changes was dose related. In many of the 600 mg/kg dose group animals, there was a marked decrease in the amount of functional tissue in the kidney. Livers of maleic anhydride-treated rats at 183 days showed changes characterized by swollen individual hepatocytes having vacuolated cytoplasm. In some of the smaller rats having decreased amounts abdominal adipose tissue, the liver contained an increased number of focal mononuclear inflammatory cell aggregats surrounded by hepatocytes showing coagulation necrosis. These rats frequently had hepatocytes with vacuolated cytoplasm.

The microscopic examination of five lobes of lungs from all control and treated rats sacrificed at 90 or 183 days did not reveal a treatment-related change. No proliferative lesion suggestive of a tumor was detected in rats sacrificed at either 90 or 183 days.

Effect levels

Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the given conditions of this subchronic oral study in male rats, the LOAEL was determined to be 250 mg/kg bw/day based on renal changes .
Executive summary:

In a subchronic toxicity study (similar to OECD 409) maleic anhydride (purity 99.5%) was administered to 50 male rats, Sprague-Dawley/dose in diet at dose levels of 250 or 600 mg/kg bw/day for 90 and 183 days. The control groups were constituted of 75 rats. The purpose of this study was to determine whether lung lesions would be observed.

 

One rat died spontaneously. The high dose group appeared to be gaining weight at a rate slightly less than that of the control group during the last 2 months of the study. Small but significant decrease in the alkaline phosphatase activity for both treatment groups at 90 days and for the 250 mg/kg/day group at 183 days was observed. The significance of these decreases in alkaline phosphatase activity in relation to the other toxicological changes observed was uncertain. On day 174, there was a statistically significant increase in the specific gravity of the urine of rats at the highest dose level. However, it is doubtful that this was physiologically or toxicologically significant since the value was not different from the values for all groups obtained two days later during the urine concentration test.

At the 90-day timepoint, the relative liver weight (+ 16%) for the rats in the high-dose level and the absolute/relative kidney weights for both the 250 and 600 mg/kg groups were significantly higher (+ 24% and + 65%, respectively) compared to the controls (p<0.05). At 183 days, there was a significant increase in the absolute and relative liver (+ 18% and +10%) and kidney weights (+ 54% and + 119%) for both treatment groups, increased relative testes, brain and heart weight (+ 5%), and decreased fasted body weights for rats at 600 mg/kg/day (p<0.05). The increase in the absolute and relative weights of the liver and kidney and the relative weight of the heart are attributed to treatment whereas the increased relative brain and testes weights are interpreted to reflect the decreased. Furthermore, dose dependent treatment-related changes in the kidneys were apparent in rats sacrificed at 90 and 183 days. The number of rats showing the kidney changes and the degree of involvement increased in severity with the dose and the duration on the diet containing maleic anhydride. Some rats from both dose levels at the 183-day sacrifice were smaller and lacked normal amounts of abdominal adipose tissue. No gross treatment related changes were recognized in the lungs or other organ systems at either the 90- or 183-day sacrifice. Treatment-related changes were also present in the kidneys of rats sacrificed at 90 and 183 days. The observed changes indicated a marked accentuation of the spontaneously occurring findings seen in the control animals. The changes in the controls and treated animals included: individual tubules that were dilated and contained eosinophilic staining casts, granular degeneration of the epithelial cells lining these tubules, tubular collapse and atrophy with peritubular fibrosis, focal mononuclear inflammatory cell infiltrates, glomeruli that showed thickening of the basement membrane, thickening and epithelialization of Bowman’s Capsule, and occasionally showed either focal or diffuse sclerosis of the glomerular tufts. Both tubular and glomerular changes in treated animals were more severe than in controls. In addition to the focal nature of the lesion (in controls), the tubules throughout the cortex of treated rats showed a generalized dilatation and hypertrophy. There were more degenerative tubules and tubules showing mitotic activity in treated versus controls. The degree of degenerative, hypertrophic, and regenerative changes was dose related. In many of the 600 mg/kg dose group animals, there was a marked decrease in the amount of functional tissue in the kidney. Livers of maleic anhydride-treated rats at 183 days showed changes characterized by swollen individual hepatocytes having vacuolated cytoplasm. In some of the smaller rats having decreased amounts abdominal adipose tissue, the liver contained an increased number of focal mononuclear inflammatory cell aggregates surrounded by hepatocytes showing coagulation necrosis. These rats frequently had hepatocytes with vacuolated cytoplasm.

The microscopic examination of five lobes of lungs from all control and treated rats sacrificed at 90 or 183 days did not reveal a treatment-related change. No proliferative lesion suggestive of a tumor was detected in rats sacrificed at either 90 or 183 day.

The LOAEL is 250 mg/kg bw/day based on renal changes.