Dibutyl maleate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was coducted according to OECD 408 and under GLP with a few deviations. However, these deviations had no impact on the outcome or integrity of the study or the interpretation of the results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl maleate

EC Number:

203-328-4

EC Name:

Dibutyl maleate

Cas Number:

105-76-0

Molecular formula:

C12H20O4

IUPAC Name:

dibutyl but-2-enedioate

Details on test material:

Name - (as cited in study report): dibutyl maleate
Analytical purity: 100%
Lot /batch:

Specific details on test material used for the study:

Identification: Di-n-butyl Maleate (DBM; Maleic acid dibutylester;
maleic acid di-n-butyl ester; CAS Registry No. 105-76-0)
Purity (GC): 99.1
Storage Conditions: 22°C ± 5°C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River Laboratories, Inc, Portage, MI
Age at study intiation: 7 - 10 weeks
Weight at study initiation: 200-350 g
Housing: individually housed in polycarbonate cages with contact bedding
Diet: Purine certified rodent Chow # 5002 ad libitum, except prior to blood collection and scheduled sacrifice.
Water: filtered tap water ad libitum
Acclimation period: 14 days
Unique identification: yes, by tattoo or ear punch
ENVIRONMENTAL CONDITIONS
Temperature: 68F to 72F (20C to 22C)
Humidity: 30% to 70%
Light Cycle: 12 hour light/12 hour dark cycle
In-life days: from September 29, 2009 to Jan 12, 2010.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Animals assigned to the 30, 95, and 300 mg/kg/day test groups were administered either 6, 19, or 60 mg/ml solutions of the test article, respectively, by oral gavage for 90 consecutive days at the rate of 5 ml/kg/day of body weight. Dosing volumes were adjusted weekly.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90-day of treatment plus 2-week recovery period

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low-dose

Doses / concentrations 3

Dose / conc.:

95 mg/kg bw/day (nominal)

Remarks:

Mid-dose

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

High-dose

No. of animals per sex per dose:

A total of 30 animals (15 males and 15 females) per group

Control animals:

yes, concurrent vehicle

Details on study design:

Dibutyl maleate was given by oral gavage once daily for 90 days to Sprague-Dawley rats and to further evaluate the reversibility or delayed onset of any changes with a subsequent 2-week treatment free recovery period.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Toxicity was evaluated by monitoring mortality/moribundity (twice daily); cageside observations (once daily); and detailed clinical observations (once weekly), body weight, (before treatment, approx. weekly and on the day of necropsy) food consumption (once before the study start and weekly, except on days after an overnight fast), ophthalmic examination observations (on day 84 (males) and 83 (females); functional observational battery (FOB) observations (on day 72); and clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) before necropsy (on day 91 and 105). After euthanasia, animals were subjected to comprehensive necropsy and tissue collection; organ weights were also measured. Collected tissues were evaluated histopathologically, and bone marrow smears were prepared but were not evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: yes
Time schedule for gross pathology: were done on study days 91 (10 females and 10 males) and 105 (5 females and 5 males) at terminal sacrifice. All external surfaces, orifices, and organs; the cranial cavity; carcass; external and cut surfaces of the brain; the abdominal, thoracic, and pelvic cavities and their viscera; and cervical tissues and organs were examined during necropsy.

HISTOPATHOLOGY: yes
All tissues and organd listed below from all animals found dead and from all animals from vehicle control and test groups at study termination were processed and examined histologically.
Tissues and organs: adrenals, lung and mainstream bronchi, aourta, lymph nodes (mandibular and mesenteric nodes), bone (femur), bone marrow (sternum), mammary gland brain (sections of cerebrum, cerebellum, and brainstem), pituitary, pancreas, saliva grland (mandibular) esophagus, skeletal muscle with sciatic nerve, eyes and optic nerve, gonads (testes wiith epidymides or ovaries), uterus/cervix, corpus, spinal cord (mid-thoracic), spleen, heart, stomach (cardiac, fundic, and pyloric portions), intestine (sections of cecum, colon, doudenum, ileum, jejunum, and rectum), thymus (or thymic remnant), thyroids (with patathyroids), kidneys, trachea, liver, urinary bladder, any gross changes of uncertain nature or tissue masses (including adjacent tissue).

Other examinations:

ORGAN WEIGHTS: yes
Organs: adrenals, brain, gonads (testes with epididymides or ovaries), heart, kidneys, liver, and spleen or each final sacrifice animal. Paired organs were weighted separately. Organ to body and brain weight ratios were calculated. Final fasted body weights were used for organ to body weight ratio calculations.

Statistics:

Statistical analysis was performed on body weights, food consumption, rearing, hindlimb and forelimb grip strength, landing foot splay, rectal body temperature, hematology, coagulation, clinical chemistry, organ weights, organ to body weight percentages, and organ to brain weight ratios. Associated numeric values collected during the functional observational battery assessment used to calculate standard deviation or mean scores, including, but not limited to, grip strength, landing foot splay, and rearing count, was reported in the summary tables out to an additional significant figure than the representative raw data collected.

To determine the appropriate statistical test, each data set was subjected to a statistical decision tree using the SAS® System. A minimum of 3 animals/sex/group per interval was required for statistical analysis.

The data was initially be tested for normality using the Shapiro-Wilk test followed by the Levene’s test for homogeneity of variance. A p = 0.05 level of significance was required for either test to reject the assumptions.

If both assumptions are fulfilled, a single-factor analysis of variance (ANOVA) was applied, with animal grouping as the factor, utilizing a p = 0.05 level of significance. If the parametric ANOVA is significant at p = 0.05, Dunnett's test was used to identify statistically significant differences between the control group and each test article-treated group at the 0.05 level of significance.

If either of the parametric assumptions was not satisfied, then the Kruskal-Wallis nonparametric ANOVA procedure was used to evaluate intergroup differences (p = 0.05). The Dunn’s multiple comparison test was applied if this ANOVA is significant, again utilizing a significance level of p = 0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

ad libitum

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:

effects observed, treatment-related

Description (incidence and severity):

kidneys and liver

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Multiple bilateral pale foci in the cortex of the kidneys at Day 91 in males and females at 300 mg/kg/day DBM and at Day-105 in one female in group 300 mg/kg/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

CPN, mineralization at the corticomedullary junction and/or medulla, tubular basophilia within the cortex, and tubular ectasia in the cortex and/or medulla.

Histopathological findings: neoplastic:

no effects observed

Details on results:

DBM-related microscopic findings were noted in the kidneys of = 30 mg/kg/day males and females at Day 91. These lesions included CPN, mineralization at the corticomedullary junction and/or medulla, tubular basophilia within the cortex, and tubular ectasia in the cortex and/or medulla. Lesions consistent with CPN of rats were seen at an increased incidence and severity in = 30 mg/kg/day males and 300 mg/kg/day females. The lesions of CPN included, but were not limited to, tubular basophilia/tubular degeneration, tubular ectasia with or without proteinaceous luminal material, interstitial fibrosis, thickening of the glomerular and/or tubular basement membranes, and mononuclear infiltrates. The lesions of CPN affected whole nephrons or segments of nephrons. Some of the lesions of CPN present in these rats (ie, tubular basophilia and tubular ectasia) were not separated out when categorized within foci of CPN. Tubular basophilia (in non-CPN affected areas) was categorized by basophilia within the cytoplasm of proximal and distal tubules within the cortex. Affected tubular epithelial cells ranged in appearance from swollen to attenuated. Many affected tubules also contained proteinaceous material within their lumens. Affected areas ranged from regionally extensive to small clusters of tubules and often appeared closest to the corticomedullary junction region. Tubular basophilia due to DBM administration was present in = 30 mg/kg/day males and = 95 mg/kg/day females. Ectasia of tubules (away from CPN-affected areas) was often noted within the cortex and medulla of 300 mg/kg/day males and females. Increased incidence of mineralization was noted at the corticomedullary junction of females at = 30 mg/kg/day. Mineralization was present in 1 vehicle-treated female.

Although many of the lesions referenced above, including CPN and tubular basophilia as well as mineralization (in females), are often considered background lesions in rat kidneys, the increased incidence and/or severity of these lesions in DBM-administered animals resulted in the lesions being considered DBM-related findings.

Moderate pyelonephritis was noted in one 300 mg/kg/day female. This animal exhibited many of the DBM-related lesions that were listed above, including lesions consistent with CPN. However, due to the severe alteration in renal architecture caused by the ascending pyelonephritis, any potential DB

Some DBM-related microscopic findings observed at Day 91 persisted at Day 105 in males and females administered 300 mg/kg/day DBM. These lesions included CPN (300 mg/kg/day in males and females), mineralization at the corticomedullary junction and/or medulla (= 30 mg/kg/day in females), and tubular ectasia in the cortex and/or medulla (300 mg/kg/day in males and females). Tubular basophilia within the cortex was still present at Day 105; however, this lesion was minimal and present in vehicle-treated animals as well as 300 mg/kg/day males and females and was, therefore, of unknown relation to DBM. All other lesions were considered incidental and not related to DBM administration.

Effect levels

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Summary Of DBM-related Microscopic Findings - Day 91
Group/Dose Sex Organ/Finding       No. of Animals	1 (Vehicle)		2 (30 mg/kg)		3 (95 mg/kg)		4 (300 mg/kg)
	M	F	M	F	M	F	M	F
	10	10	10	10	10	10	7	10
Kidney/Chronic Progressive Nephropathy
Minimal	-	-	3	-	2	-	1	2
Mild	-	-	-	-	-	-	1	3
Moderate	-	-	-	-	-	-	1	-
Kidney/Mineralization,corticomedullaryjunction/medulla, bilateral
Minimal	-	-	-	3	-	-	-	1
Mild	-	1	-	-	-	-	-	3
Kidney/Basophilia, tubular, cortex, multifocal/focal
Minimal	2	-	1	-	4	3	-	1
Mild	-	-	1	-	-	-	3	3
Moderate	-	-	1	-	-	-	4	4
Kidney/Ectasia, tubular, cortex/medulla
Minimal	-	-	-	-	-	1	1	1
Mild	1	-	-	-	-	-	3	5
Moderate	-	-	-	-	-	-	-	2
Kidney/Pyelonephritis*
Moderate	-	-	-	-	-	-	-	1
No. = Number; M = Male; F = Female; - = No Finding; NA = Not applicable; * = Possible DBM-related finding.

 

Applicant's summary and conclusion

Conclusions:

In conclusion, oral administration of DBM to rats for 90 days was associated with persistent kidney findings at all doses during the recovery phase. There were no DBM-related effects or effects which were considered adverse identified in clinical observations, body weights, food consumption, ophthalmic examinations, FOB assessments, hematology parameters, coagulation, and urinalysis during the dosing or recovery period of this study. Morbidity and mortality observed during this study were of uncertain relation to DBM administration. At Day 91, DBM-related microscopic findings were observed within the kidneys of male and female animals. The renal lesions included CPN, mineralization at the corticomedullary junction and/or medulla, tubular basophilia within the cortex, and tubular ectasia in the cortex and/or medulla. After a 2-week recovery period, DBM-related renal microscopic findings, previously identified at Day 91, also persisted to Day 105 in male and female animals. Due to the renal lesion, the LOAEL was established at 30 mg/kg/day.

Executive summary:

Dibutyl maleate was given by oral gavage once daily for 90 days to Sprague-Dawley rats and to further evaluate the reversibility or delayed onset of any changes with a subsequent 2-week, treatment-free recovery period. Four groups of 15 animals/sex/group animals were designated as main study (10 animals/sex/group) or recovery (5 animals/sex/group). Animals were dosed once daily for 90 days via oral gavage. Group 1 received the control article, corn oil. Groups 2, 3, and 4 received the test article, dibutyl maleate (DBM), at doses of 30, 95, and 300 mg/kg/day, respectively. Toxicity was evaluated according to OECD 406 guidelines. Main study animals were euthanized on Day 91, and recovery animals were euthanized on Day 105 (2-week recovery period). After euthanasia, animals were subjected to comprehensive necropsy and tissue collection; organ weights were also measured. Collected tissues were evaluated histopathologically, and bone marrow smears were prepared for possible future evaluation. There were 4 unscheduled deaths during the study. One animal was found dead. The cause of death in this animal was of uncertain relation to DBM administration. There were no DBM-related effects or effects which were considered adverse identified in clinical observations, body weights, food consumption, ophthalmic examinations, FOB assessments, hematology parameters, coagulation, and urinalysis during the dosing or recovery period of this study. Morbidity and mortality observed during this study were of uncertain relation to DBM administration. Increased kidney weights in males and females at 300 mg/kg/day relative to vehicle-treated animals were observed on Day 91 and persisted in the males at 300 mg/kg/day on Day 105. These increased kidney weights correlated histologically to chronic progressive nephropathy (CPN) as well as tubular basophilia within the renal cortex. At Day 91, DBM-related microscopic findings were observed within the kidneys of male and female animals. The renal lesions included CPN, mineralization at the corticomedullary junction and/or medulla, tubular basophilia within the cortex, and tubular ectasia in the cortex and/or medulla. After a 2-week recovery period, DBM-related renal microscopic findings, previously identified at Day 91, also persisted to Day 105 in male and female animals. Overall, oral administration of DBM to rats for 90 days was associated with persistent kidney findings at all doses during the recovery phase. Based on the kidney effects, the lowest-observed-adverse-effect level (LOAEL) for DBM was considered to be 30 mg/kg/day in this study.