Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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

Constituent 1
Chemical structure
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:

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 initiation: 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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
95 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
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 organs 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, aorta, lymph nodes (mandibular and mesenteric nodes), bone (femur), bone marrow (sternum), mammary gland brain (sections of cerebrum, cerebellum, and brainstem), pituitary, pancreas, saliva gland (mandibular) esophagus, skeletal muscle with sciatic nerve, eyes and optic nerve, gonads (testes with 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-Wilks 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:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

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. LOAEL = 30 mg/kg/day.
Executive summary:

Based on a read across assessment with dibutyl maleate, a 90-day subchronic toxicity study in rats was evaluated. Dibutyl maleate (DBM) was given by oral gavage once daily 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, 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

Categories Display