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Administrative data

Description of key information

Rat, oral feeding, 2 years: NOELcarcinogenicity >= 100 mg/kg bw; NOELtoxicity =10 mg/kg bw/day  (comp. OECD 451; Procter and Gamble Company, 1983)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978-1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. Reliability adopted from OECD SIDS
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
yes
Remarks:
Diets were not analyzed for maleic anhydride until 2-4 years after the study ended. Maleic anhydride may have converted to maleic acid when mixed in the feed.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
The rats were housed in groups of three per po1ycarbonate cage (19 x 10.5 x 8 in) with food and tap water ad. libitum. A table of random numbers was used to assign the animals to control and treatment groups at 7 weeks of age. At this time, the weights of the male animals ranged from 130 to 169 grams and the weights of the female animals ranged from 105 to 129 grams. To avoid contamination, the control animals were housed separately in an adjacent room. A bedding of hardwood chips (Ab-sorb-dri, Garfield, NJ) was used; cages, water bottles, and bedding were changed
once weekly for the first 22 weeks on test and then twice weekly. A light CyCle of 12 hr light: 12 hr dark was employed, however, there is a possibility that the animals were exposed to continuous light during an undefined period of the study.
Route of administration:
oral: feed
Vehicle:
not specified
Details on exposure:
The basal laboratory diet consisted of Wayne Laboratory powdered meal (No. 86-04, Supplied by Locke-Erickson, Melrose Pk, IL). The maleic anhydride bricks (approximately 2ft x lR X In) were placed in a plastic bag and broken into a powder with a mallet. The powder was then passed through a sieve (Size SO, 0.297 sq mm openings) before weighing. The various quantities of maleic anhydride, depending on the dietary levels, were first
added to 500 grams of the basal diet and premixed by hand by stirring with a large spatUla. The premix was then added to a twin-shell blender (Patterson-Kelly) and mixed fDr at least one minute per kilogram of meal to provide the appropriate dose level. The diets, based on body weight and food consumption data for each group, were prepared weekly to ensure the maintenance of a constan~ intake on a mg/kg body weight basis. The mean maleic anhydride consumed by each level and sex was calculated based on target dose levels (weekly from Week I through Week 13, bi-weekly through Week 25, and monthly thereafter).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis of the diet for maleic anhydride was conducted 2 to 4 years after completion of the in-life phase of the study. A sample of each diet was removed, refrigerated or frozen for future chemical determination of the concentration of maleic anhydride. The GC-MS measurements were (on average of random samples) 69-75% of the expected for male and female diets, respectively.
Duration of treatment / exposure:
2 years
Frequency of treatment:
7 days/week
Remarks:
Doses / Concentrations:
0, 10, 32, 100 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
126
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption : Yes
- 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


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


OPHTHALMOSCOPIC EXAMINATION: Yes


HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 animals of each sex from the control and each test group at 6 and 12 months and on approximately 20 animals from each group at 18 and 24 months.


CLINICAL CHEMISTRY: Yes
- Animals fasted: No data
- How many animals: 5 animals of each sex from the control and each test group at 6 and 12 months and on approximately 20 animals from each group at 18 and 24 months.


URINALYSIS: Yes
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data


NEUROBEHAVIOURAL EXAMINATION: No data


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At 6 and 12 months five animals from each group, and at 18 months, 20 animals from each group, were anesthe,ized, sacrificed
by exsanguination, and necropsied. Surviving animals were sacrificed and necropsied after 24 months on test. Anesthesia was induced by pentobarbital injection at the 6-month sacrifice and by exposure to carbon dioxide gas thereafter. Necropsies
were performed under the supervision of the Project Officer and a pathologist. For animals sacrificed at 6, 12, and 18 months and for 20-22 per group of those sacrificed at study termination, fresh unstained impression smears of liver were examined microscopically for porphyrin fluorescence by exposure to ultra-violet light.

The following organs of each rat sacrificed at 6, 12, and 18 months and at study termination were excised, trimmed of fascia, blotted dry, and weighed prior to fixation: brain, heart, liver, kidneys, lungs, and testes or ovaries. Organ/body weight ratios were determined for these organs.

HISTOPATHOLOGY: Yes
The following tissues from each rat were preserved in 10% neutral buffered formalin: brain (including optic nerves), peripheral nerves (sciatic and anterior tibial), pituitary, thyroid, parathyroid, SUbmaxillary salivary glands, heart, lungs, spleen, liver, pancreas, adrenals, lymph nodes (mesenteric, thoracic, and mandibular), kidneys, urinary bladder, ovaries, uterus, oviducts, stomach, small intestine (3 levels), large intestine (3 levels), skeletal muscle, skin, mammary glands, bone marrow (smear and section), aorta, ear canal, nasal turbinate, trachea, spinal cord and ganglia (thoracic and lumbar), esophagus, thymus, prostate, seminal vesicles, epididymis, and any gross lesions.
The testes were fixed in Bouin's solution. The eyes were fixed in Bouin's solution if collected during the first 16 months of the study and in 3% gluteraldehyde if collected thereafter. Testes and eyes were SUbsequently stored in 70% ethanol.

All fixed tissues from the scheduled sacrifice animals in the control and high dose groups were stained with hematoxylin and eosin and examined microscopically. In addition, tissues from all animals from the control and high dose group that died spontaneously or were sacrificed due to a moribund state or large masses were processed through histopathologic examination. For animals in the low and middle dose groups that died or were sacrificed during the first 18 months of the study, histopathologic evaluation was performed if selected gross lesions were present. The basis for selection was a mass or organ enlargements that might have been indicative of a mass.
Tissues from the remaining animals were held for possible future consideration.
Other examinations:
Hematology, clinical chemistry, and urinalyses were performed on 5 animals of each sex from the control and each test group at 6 and 12 months and on approximately 20 animals from each group at 18 and 24 months. Blood and urine samples were collected from animals fasted for 16 hours. Water consumption and urine output were also recorded during these time periods. Animals were anesthetized by pentobarbital injection at the
6-month sacrifice and by exposure to carbon dioxide gas thereafter; blood samples were obtained from the abdominal aorta at the time of sacrifice.

The hematology parameters evaluated were hemoglobin, hematocrit, and erythrocyte, leukocyte (total and differential) and reticulocyte counts. Serum samples for clinical chemistry were analyzea for glucose, urea nitrogen (BUN), glutamic pyruvic transaminase (SGPT), and alkaline phosphat ase (AP).

Urine was collected from the animals individually housed in stainless steel metabolism cages and receiving tap water 4d libitum. The parameters evaluated were appearance, occult blood, specific gravity, total protein, pH, ketones, glucose, and microscopic examination of spun sediment for erythrocytes, leukocytes, casts, crystals and bacteria. References for the clinical laboratory methods are presented in Appendix J (Vol. VJ.

Both eyes of each animal to be sacrificed wer~ examined with an indirect ophthalmoscope prior to the 6, 12, and 18-month and terminal sacrifices. Mydriasis was induced with Mydriacyl (Alcon Laboratories, Inc., Ft. Worth, TX).

An evaluation of neurological function was made on the animals which were sacrificed at the 6, 12, and 18 month intervals and on animals that survived to the terminal sacrifice. This included an assessment of posture and gait, tone of facial muscles and an examination of pupillary, palpebral, extensor thrust, and crossed extensor reflexes. Only abnormal responses were recorded.

Statistics:
All data obtained by quantitative methods were statistically evaluated by the analysis of variance. All comparisons were limited to within-sex analysis. A "two-tail" distribution was used for evaluation of mean differences. If significant differences (p < 0.05) occurred -in the preliminary analysis,
Tukey's procedure was employed to determine the differences between control and treated groups.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
the differences between control males and those in the high dose group were less than 6%.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly lower food consumption by treated groups occurred sporadically during the first year of the study for both males and females
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Lowered RBC counts and hematocrit in males at 6 months may have indicated a mild anemia but was not accompanied by alterations in hemoglobin.


BODY WEIGHT AND WEIGHT GAIN: small (<6%), but dose-related, decrease in body weights of male rats fed 32 and 100 mg/kg/day compared to the controls. The female rats fed 32 and 100 mg/kg/day also had reduced body weights, but the reductions were smaller and of shorter duration than those observed in males.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): slightly reduced during limited periods during the study for animals in the mid- and high-dose groups.


FOOD EFFICIENCY: no data


OPHTHALMOSCOPIC EXAMINATION: no data


HAEMATOLOGY: no data


CLINICAL CHEMISTRY: no data


URINALYSIS: no data


ORGAN WEIGHTS: no data


GROSS PATHOLOGY: no data


HISTOPATHOLOGY: NON-NEOPLASTIC: no data


HISTOPATHOLOGY: NEOPLASTIC: No increases in tumor incidence that were considered related to maleic anhydride exposure. There was an unusally high incidence of uterine adenocarcinomas in this study. The tumors were present in similar numbers in both the control and treated animals (23/86 and 20/82, respectively). The report stated that Uterine adenocarcinomas is not a common spontaneous lesion in this strain of rat, although no historical control data was provided. The authors speculated that this may have been related to altered hormonal status caused by the continuous light exposure.


Relevance of carcinogenic effects / potential:
Maleic anhydride did not show any carcinogenic effects.
Dose descriptor:
NOAEL
Effect level:
>= 100 other: mg/kg/day
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: reduced body weight (& food consumption)
Remarks on result:
other: Effect type: toxicity (migrated information)

There was a high incidence of cataracts in the animals of this study with 100% of the animals examined at 18 months and at study termination bearing cataracts, however, the severity of these was independent of maleic anydride consumption.

Conclusions:
1. Maleic anhydride produced only marginal toxicity when fed to Fischer 344 rats for 2 years at dietary concentrations calculated to yield daily doses of 100 and 32 mg/kg. The toxicity was evidenced primarily by lower than normal body weights and posssibly by slight decreases in food consumption.
2. Diets calculated to yield a daily dose of 10 mg/kg had no substantial effects on body weights.
3. Body weights of males were more affected than those of females fed comparable doses of maleic anhydride.
4. Lowered RBC counts and hematocrit in males at 6 months may have indicated a mild anemia but was not accompanied by alterations in hemoglobin.
5. There were other occasional significant differences between control and treated rats but the lack of relation to dose, lack of repetition of effects over time, and/or limitation of the effect to one or the other sex suggests that these were probably spurious findings of significance and need not be considered evidence of toxicity.
Executive summary:

Maleic anhydride was evaluated for toxicologic and/or carcinogenic effects in male and female Fischer 344 r.ats

following dietary administration at levels of 100, 32, and 10 mg/kg/day. A control group received only the basal diet (Wayne

Laboratory Meal).

The criteria evaluated for compound related effects included: mortality, gross and behavioral signs of toxic or pharmacologic effects, body weights, food and compound consumption, clinical laboratory analysis (hematology, biochemistry, urinalyses), ophthalmology, neurological signs, organ weights and organ/100 g body weight, and gross and microscopic pathology.

Under the conditions tested, maleic anhydride produced only marginal toxicity which was evidenced by small (< 6%) but dose

related decreases, compared to controls, in the body weights of male rats fed 100 and 32 mg/kg/day. The female rats in these two groups also had reduced body weights but the reductions were even smaller and of shorter duration than those observed in males.

Food consumption was also slightly reduced during limited periods during the study for animals in the two highest dose groups. The

10 mg/kg/day dose had no sustained significant effects on body weights.

Neither neurologic nor ophthalmologic evaluations revealed differences between control and maleic anhydride treated rats.

There was a high incidence of cataracts in the animals of this study with 100% of the animals examined at 18 months and at study

termination bearing cataracts, however, the severity of these was independent of maleic anydride consumption.

For male animals sacrificed at 6 months, a decrease in hematocrit and red blood cell counts for males treated with maleic anhydride may have indicated a mild anemia but this was not detected at any other period in the study. Although statistical differences between control and treated rats were occasionally observed in the clinical laboratory data, dose-response relationships were not present. The organ weight data similarly revealed no distinct treatment-related differences.

From the gross and histopathologic evaluations, there were no lesions that were attributed to treatment with maleic anhydride. There was an atypically high incidence of lesions of the eye and of uterine adenocarcinomas in the rats of this study.

However, in neither case was there any question of the compound having caused these lesions, nor were there other lesions in the

gross and histopathologic findings that could be attributed to the test material.

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:
2

Additional information

Male and female Fischer 344 rats were exposed to 0, 10, 32, or 100 mg/kg/day maleic anhydride in feed, seven days a week (Procter and Gamble Company, 1983). The study was performed with 126 animals/ sex/ group and comparable to OECD guideline 451; however, deviations cannot be defined since details were missing in the OECD SIDS.

There was only marginal toxicity, which was evidenced by small (<6%), but dose-related, decrease in body weights of male rats fed 32 and 100 mg/kg/day compared to the controls. The female rats fed 32 and 100 mg/kg/day also had reduced body weights, but the reductions were smaller and of shorter duration than those observed in males. Food consumption was also slightly reduced during limited periods during the study for animals in the mid- and high-dose groups. There was an unusually high incidence of uterine adenocarcinomas in this study. These lesions were present in similar numbers in control and treated animals (23/86 and 20/82, respectively). The report stated that uterine adenocarcinomas is not a common spontaneous lesion in this strain of rats, although no historical control data was provided. It is unclear why the incidence of uterine adenocarcinomas was high in this particular study both in the controls and the treated animals. But, it does not appear to be related to exposure to maleic anhydride. The authors speculated that this may have been related to altered hormonal status caused by the continuous light exposure since there were problems with the animal room lighting control system which resulted in exposure to continuous light for an unknown period.


Justification for selection of carcinogenicity via oral route endpoint:
2 year rat study, detailed description of experimental conditions.

Justification for classification or non-classification

From the available data, there is no indication given for a carcinogenic potential of maleic anhydride. Therefore criteria for classification according to REGULATION (EC) No 1272/2008 or REGULATION (EC) No 67/548 are not met.