Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear colourless liquid
Details on test material:
- Name of test material (as cited in study report): Dioctyl fumarate
- Substance type: Clear colourless liquid
- Physical state: liquid
- Purity: 99.3% (GC area)
- Lot/batch No.: LEDF1C6046 (taken from label)
- Expiration date of the lot/batch: 31 October 2013 (Retest date)
- Purity/composition correction factor required: No
- Hygroscopic: No
- Volatile: No
- Specific Gravity / Density: 0.942

Test animals

Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx.6 weeks old)
- Weight at study initiation: Body weight variation was within ± 20% of the sex mean (males: 144 grams; females: 126 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days
Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 11 February - 14 May 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dosed as delivered by the sponsor. No correction was made for the purity/composition of the test substance.

DOSE VOLUME:
Dose volume (mL/kg body weight) was calculated as dose level (g/kg) / spec.gravity (g/mL). Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No chemical analyses of dose preparations was conducted since the test substance was dosed without the use of a vehicle.

Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
other: Control animals received water (Elix) with the same dose volume as Group 4 animals.
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 14-day oral range finding study with Dioctyl fumarate by daily gavage in the rat (Project 501491: Groups of 3 female Wistar Han rats were treated at 0, 500 and 1000 mg/kg body weight (undiluted test substance). No toxicologically relevant changes were noted in any of the parameters examined (mortality, clinical appearance, body weights, food intake, macroscopy and liver and kidney weights)).
Positive control:
Not required.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were conducted for all animals immediately after dosing (based on the absence of a peak effect in occurrence of clinical signs in the dose range finding study (project 501491). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION:
- Time schedule for examinations: Weekly.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: examined by means of an ophthalmoscope (Heine Beta 200):
At Pre-test: All animals (including spare animals)
At Week 13: Group 1 and 4 animals. Since no treatment-related ophthalmologic findings were noted in Group 1 and 4 animals in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: During week 12 of treatment, at no specific time point, but within a similar time period after dosing.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines

OTHER:
Additional slides of the testes of all males of Groups 1 and 4 were examined for staging of spermatogenesis.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. Incidental findings noted for control and/or treated animals included scabs, scales, a wound and/or alopecia on various body areas, bleeding fissures on the ear, and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these signs were not considered toxicologically relevant.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically significant changes in body weights and body weight gain were noted. The statistically significantly higher body weight gain of females at 1000 mg/kg on Days 8 and 22 remained within the range considered normal for rats of this age and strain, and reduced weight gain would be expected in case of toxicity.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicologically significant changes in food consumption before or after correction for body weight were noted. Any statistically significant changes in food consumption before or after correction for body weight were considered not toxicologically relevant, since these were slight and transient in nature, and/or occurred in the absence of a dose-related trend.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings consisted of focal corneal opacity or oedema, pinpoint corneal opacities and haemorrhage from the hyaloid vessel during pre-test and of retinal congestion at Week 13. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant changes in haematological parameters were noted. Any statistically significant changes in haematological parameters were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain. These changes consisted of higher white blood cell counts (WBC) and lower relative neutrophil counts in males at 1000 mg/kg, lower mean corpuscular haemoglobin (MCH) in males at 300 and 1000 mg/kg, lower mean corpuscular haemoglobin concentration (MCHC) in males at 1000 mg/kg, and lower haematocrit level in females at 300 and 1000 mg/kg.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically significant changes in clinical biochemistry parameters were noted. The higher total protein and potassium levels in males at 1000 mg/kg were slight in nature and remained within the range considered normal for rats of this age and strain. The lower aspartate aminotransferase activity (ASAT) of females at 300 mg/kg showed no dose-related trend and remained within the range considered normal for rats of this age and strain, and the opposite effect (i.e. an increase) would be expected in case of target organ toxicity. No toxicological relevance was therefore ascribed to these changes.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
− higher absolute and relative liver weights at 1000 mg/kg (both sexes; relative weights were approximately 26 and 18% higher than controls for males and females respectively) and 300 mg/kg (females; relative weight was approximately 12% higher than controls). Relative liver weights were also higher for males at 100 and 300 mg/kg (relative weight was approximately 8 and 11% higher than controls respectively).
− higher absolute and relative kidney weights at 1000 mg/kg (both sexes; relative weights were approximately 21 and 17% higher than controls for males and females respectively). Females at 300 mg/kg also had higher relative kidney weights (relative weight was approximately 13% higher than controls).
The higher absolute and relative thyroid weights at 1000 mg/kg (females) was minor in nature and occurred in the absence of treatment-related morphological changes. The significantly lower seminal vesicle weight and higher relative adrenal weight (males) at 100 mg/kg occurred in the absence of a dose-related trend. These changes were therefore not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations. The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related microscopic findings present in Group 4 (1000 mg/kg):
- Kidneys (males): Cortical hyaline droplets were slightly increased in incidence and severity in males of Group 4 (1000 mg/kg: 1: minimal, 6: slight, 3: moderate). The severity grades of minimal and slight, recorded in Group 1 (control: 2 minimal, 2: slight), Group 2 (100 mg/kg: 5: minimal, 3: slight) and Group 3 (300 mg/kg: 5: minimal, 4: slight) are considered to be within background and are frequently recorded in untreated male rats of this age and strain.
- Liver (males): Centrilobular hypertrophy of the hepatocytes was recorded in 5/10 males of Group 4 at a minimal degree.
- Spleen (females): A slightly increased mean severity of hemosiderin pigment was recorded in females of Group 4. This was recorded in 10/10 females (6: slight, 4: moderate, mean severity: 2.4). A background level of hemosiderin pigment was recorded in females of Group 1 (control; 3: minimal, 6: slight, 1: moderate), Group 2 (100 mg/kg; 4: minimal, 6: slight) and Group 3 (300 mg/kg; 2: minimal, 8: slight), mean severities respectively 1.8, 1.6 and 1.8.
All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal for all males examined.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Wistar rats were treated with dioctyl fumarate for 13 weeks by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg.

The slightly increased incidence and severity of hyaline droplets in the male kidneys at 1000 mg/kg were considered to represent alpha2uglobulin (Ref. 1). Hyaline droplets are a specific male rat response and the hyaline droplets recorded in this study were not accompanied by indicators of tubular damage and were therefore not considered an adverse finding in this treatment group. Kidney weights were higher at 1000 mg/kg in males and females (relative weights were approximately 21 and 17% higher than controls, respectively). At 300 mg/kg, relative kidney weight of females was approximately 13% higher than controls. These changes in kidney weight were considered not toxicologically relevant since these occurred in the absence of toxicologically relevant morphological or biochemical lesions.

A significant increase in liver weights was noted at 1000 mg/kg in both sexes (approximately 26 and 18% higher than controls for males and females, respectively). Only for males, a minimal centrilobular hypertrophy was recorded in the liver at 1000 mg/kg which was considered adaptive in nature and was within physiological limits. At 300 mg/kg (both sexes) and 100 mg/kg (males), the increase in (relative) liver weight was less pronounced, and typically around 10% higher than controls. Since there were no significant morphological or biochemical changes that would support these higher liver weights, these were considered not toxicologically relevant.

The increase in severity of hemosiderin pigment in the female spleen at 1000 mg/kg was not considered to be adverse given the minimal nature of this finding, and absence of relevant changes in red blood cell parameters.

Overall, none of the above findings were considered to be detrimental to organ system function or vitality of the animals.

No toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations and macroscopic examination).

No adverse effects were observed in the reproductive organs examined in this study. Spermatogenic staging profiles were normal for all males examined.

Ref. 1: Alden, CL and Frith Urinary,15, pp 315-387. In: Handbook of Toxicologic Pathology Eds.Haschek WM and Rousseaux CG. Academic Press,. 1991.

Applicant's summary and conclusion

Conclusions:
In this OECD 408 study a No Observed Adverse Effect Level (NOAEL) for Dioctyl Fumarate of at least 1000 mg/kg was established.
No toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations and macroscopic examination).
No adverse effects were observed in the reproductive organs examined in this study. Spermatogenic staging profiles were normal for all males examined.
Executive summary:

Guidelines

The study was based on the following guidelines.

- EC No 440/2008, B.26 Repeated Dose (90 days) Toxicity (oral), 2008.

- OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

- OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

- Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331

No.7), METI (No. 5) and MOE (No. 110331009)

Rationale for dose levels

Based on the results of a 14-day range finding study (Project 501491; See APPENDIX 4), the dose levels for this 90-day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg.

Study outline

The test substance was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Evaluated parameters

The following parameters were evaluated: clinical signs daily; functional observation tests in week 12 body weight and food consumption weekly; ophthalmoscopy at pretest and in week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results

The slightly increased incidence and severity of hyaline droplets in the male kidneys at 1000 mg/kg were considered to represent alpha2uglobulin. Hyaline droplets are a specific male rat response and the hyaline droplets recorded in this study were not accompanied by indicators of tubular damage and were therefore not considered an adverse finding in this treatment group. Kidney weights were higher at 1000 mg/kg in males and females (relative weights were approximately 21 and 17% higher than controls, respectively). At 300 mg/kg, relative kidney weight of females was approximately 13% higher than controls. These changes in kidney weight were considered not toxicologically relevant since these occurred in the absence of toxicologically relevant morphological or biochemical lesions.

A significant increase in liver weights was noted at 1000 mg/kg in both sexes (approximately 26 and 18% higher than controls for males and females, respectively). Only for males, a minimal centrilobular hypertrophy was recorded in the liver at 1000 mg/kg which was considered adaptive in nature and was within physiological limits. At 300 mg/kg (both sexes) and 100 mg/kg (males), the increase in (relative) liver weight was less pronounced, and typically around 10% higher than controls. Since there were no significant morphological or biochemical changes that would support these higher liver weights, these were considered not toxicologically relevant.

The increase in severity of hemosiderin pigment in the female spleen at 1000 mg/kg was not considered to be adverse given the minimal nature of this finding, and absence of relevant changes in red blood cell parameters.

Overall, none of the above findings were considered to be detrimental to organ system function or vitality of the animals.

No toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations and macroscopic examination).

No adverse effects were observed in the reproductive organs examined in this study. Spermatogenic staging profiles were normal for all males examined.

Conclusion

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for Dioctyl fumarate of at least 1000 mg/kg was established.