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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
other: combined repeated dose and one generation reproduction toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-06-27 to 2014-11-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid and conclusive guideline study under GLP; Relevant and adequate for this endpoint

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Remarks:
Double compliance with guidelines OECD 408 and 415
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 415 (One Generation Reproduction Toxicity Study)
Deviations:
no
Remarks:
Double compliance with guidelines OECD 408 and 415
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl itaconate
EC Number:
210-519-6
EC Name:
Dimethyl itaconate
Cas Number:
617-52-7
Molecular formula:
C7H10O4
IUPAC Name:
1,4-dimethyl 2-methylidenebutanedioate
Test material form:
other: supercooled liquid
Details on test material:
- Name of test material (as cited in study report): Dimethyl itaconate
- Expiration date of the lot/batch: 2014-04-05
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: approximately seven to eight weeks old
- Weight at study initiation: 193 - 245 g (males), 144 - 180 g (females)
- Fasting period before study: no
- Housing: prior pairing phase all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-07-19 to: 2012-11-25

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: formulations were prepared fortnightly and stored at 4ºC in the dark (the formulations to be stable for at least sixteen days).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis Oil was successfully used on the preliminary study and the same vehicle was therefore employed in this main study
- Concentration in vehicle: 0, 12.5, 25, 87.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of DMI in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test item formulations were extracted with methanol to give a final, theoretical test item concentration of approximately 0.1 mg/mL. Standard solutions of test item were prepared in methanol at a nominal concentration of 0.1 mg/mL. For the homogeneity determinations, the 3.75 mg/mL test item formulations were assessed by visual inspection. The 250 mg/mL test item formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. The 3.75 mg/mL test item formulations were deemed homogeneous by visual inspection. The test item formulations were stable at 4° C in the dark for 16 days. The prepared formulations were within 5 % of the nominal concentration.
Duration of treatment / exposure:
approximately 17 weeks
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 100 and 350 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24 males and 24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a fourteen day dose range finding study, the investigation dosages of 500 and 750 mg/kg bw/day were associated with macroscopic stomach changes which were considered to preclude these dosages from any long term investigation of toxicity. No macroscopic changes were apparent in animals receiving 250 mg/kg bw/day during the fourteen day study. Therefore the low, intermediate and high dose levels were selected at 50, 100 and 350 mg/kg bw/day, respectively.
Positive control:
Not available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing during the working week. Animals were observed immediately before dosing soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 7, 14 and 21 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

FOOD EFFICIENCY:
- The ratio of body weight change/dietary intake: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily throughout the study (with the exception of the pairing phase)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pre-treatment and during Week 10 of the study.
- Dose groups that were examined: control and high dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 10 of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 males and 10 females from each test and control group
- Following parameters were examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic): Methylene blue stained slides were prepared but reticulocytes were not assessed

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 10 of the study
- Animals fasted: No
- How many animals: 10 males and 10 females from each test and control group
- Following parameters were examined: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Bile acids (see Deviations from Study Plan), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals for the first 10 weeks of the study.
- Dose groups that were examined: all animals of all groups for functional/behavioural toxicity and ten selected males and females from each dose level for functional performance tests.
- Battery of functions tested: sensory reactivity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary, Prostate, Seminal vesicles (with coagulating gland and fluids), Spleen, Testes, Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with oviducts and cervix))

HISTOPATHOLOGY: Yes (Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Colon, Caecum, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary tissue, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulating gland and fluids), Skin (hind limb), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach, Thyroid/parathyroid, Trachea, Testes, Tongue, Thymus, Urinary bladder, Uterus (with oviducts and cervix), Vagina)
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance: at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed usingBartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Transient post-dosing salivation was observed for both sexes at 350 mg/kg bw/day and, to a lesser extent, both sexes at 100 mg/kg bw/day. Similar postdosing salivation was observed for one animal on a single occasion at 50 mg/kg bw/day. Among females there were three unscheduled death, one at 50 mg/kg bw/day and two at 350 mg/kg bw/day but these were considered not to be related to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Transient post-dosing salivation was observed for both sexes at 350 mg/kg bw/day and, to a lesser extent, both sexes at 100 mg/kg bw/day. Similar postdosing salivation was observed for one animal on a single occasion at 50 mg/kg bw/day. Among females there were three unscheduled death, one at 50 mg/kg bw/day and two at 350 mg/kg bw/day but these were considered not to be related to treatment.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 350 mg/kg bw/day male water consumption was increased compared to control.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Raised limiting ridge, frequently accompanied by raised areas in the nonglandular region, was observed in the stomach of most adult males at 350 mg/kg bw/day and one male at 100 mg/kg bw/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Diffuse epithelial hyperplasia of the non-glandular region of the stomach was observed for both sexes at 350 mg/kg bw/day.
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Transient post-dosing salivation was observed for both sexes at 350 mg/kg bw/day and, to a lesser extent, both sexes at 100 mg/kg bw/day. Similar postdosing salivation was observed for one animal on a single occasion at 50 mg/kg bw/day. Among females there were three unscheduled death, one at 50 mg/kg bw/day and two at 350 mg/kg bw/day but these were considered not to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN
Body weight and body weight gain of both sexes, including for females during gestation and lactation, were unaffected by treatment at 50, 100 or 350 mg/kg bw/day.

FOOD CONSUMPTION
Food consumption of both sexes, including for females during gestation and lactation, were unaffected by treatment at 50, 100 or 350 mg/kg bw/day.

FOOD EFFICIENCY
Food conversion efficiency of both sexes was unaffected at these dosages.

WATER CONSUMPTION
At 350 mg/kg bw/day male water consumption was increased compared to control.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmic examinations at 350 mg/kg bw/day did not reveal any effect of treatment for either sex.

HAEMATOLOGY
There were no treatment related effects on haematology parameters for either sex at 50, 100 or 350 mg/kg bw/day.

CLINICAL CHEMISTRY
There were no treatment related effects on blood chemistry parameters for either sex at 50, 100 or 350 mg/kg bw/day.

URINALYSIS
Not examined

NEUROBEHAVIOUR
No obvious neurological effects of treatment in behavioural assessment, in functional performance tests and sensory reactivity assessment were apparent at 50, 100 or 350 mg/kg bw/day.

ORGAN WEIGHTS
There were no statistically significant differences in organ weight that were considered to be of toxicological significance at 50, 100 or 350 mg/kg bw/day.

GROSS PATHOLOGY
Raised limiting ridge, frequently accompanied by raised areas in the nonglandular region, was observed in the stomach of most adult males at 350 mg/kg bw/day and one male at 100 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Diffuse epithelial hyperplasia of the non-glandular region of the stomach was observed for both sexes at 350 mg/kg bw/day; this finding is adverse but represents a local irritant effect of the test item rather than systemic toxicity. No treatment related findings were observed at 50 or 100 mg/kg bw/day.

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
>= 350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects at highest dose tested
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects at highest dose tested
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Diffuse epithelial hyperplasia of the non-glandular region of the stomach for both sexes at 350 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment at 350 mg/kg bw/day was associated with adverse histopathological changes in the stomach but this was considered to represent an irritant effect of the test item rather than systemic toxicity. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be ≥350 mg/kg bw/day and the No Observed Effect Level (NOEL) was 100 mg/kg bw/day.
Executive summary:

The study was designed to investigate the effects of the test item on reproductive, pre-natal and post-natal development of the rat when administered through the reproductive cycle and to assess subchronic exposure of the test item to the rat. The study is considered to be compatible with both OECD TG 408 and 415.

The test item was administered by oral gavage to three groups, each of twenty-four male and twenty-four female Wistar Han™:RccHan™:WIST strain rats, for approximately seventeen weeks (and including for females 10 weeks pre-pairing, gestation and lactation phases) at dose levels of 50, 100 and 350 mg/kg bw/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Clinical signs, behavioural assessments, body weight change, food and water consumption and ophthalmic change were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group after 10 weeks of treatment, with females subsequently being allowed to litter and rear their offspring to Day 21 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights. During Week 10, extensive functional observations were performed on ten selected males and females from each dose group. Haematology and blood chemistry were also evaluated on ten selected males and females from each dose group. Adult males were terminated during Week 18 with surviving females and offspring on Day 21 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Following this assessment, histopathological evaluation of the stomach was extended to both sexes from the low and intermediate dosage groups.

Among females there were three unscheduled death, one at 50 mg/kg bw/day and two at 350 mg/kg bw/day but these were considered not to be related to treatment. Transient post-dosing salivation was observed for both sexes at 350 mg/kg bw/day and, to a lesser extent, both sexes at 100 mg/kg bw/day. Similar postdosing salivation was observed for one animal on a single occasion at 50 mg/kg bw/day. No obvious neurological effects of treatment in behavioural assessment, in functional performance tests and sensory reactivity assessment were apparent at 50, 100 or 350 mg/kg bw/day. Body weight and body weight gain of both sexes, including for females during gestation and lactation, were unaffected by treatment at 50, 100 or 350 mg/kg bw/day. Food consumption of both sexes, including for females during gestation and lactation, were unaffected by treatment at 50, 100 or 350 mg/kg bw/day. Food conversion efficiency of both sexes was also unaffected at these dosages. At 350 mg/kg bw/day male water consumption was increased compared to control. Ophthalmic examinations at 350 mg/kg bw/day did not reveal any effect of treatment for either sex. There were no treatment related effects on haematology parameters and blood chemistry parameters for either sex at 50, 100 or 350 mg/kg bw/day. Raised limiting ridge, frequently accompanied by raised areas in the nonglandular region, was observed in the stomach of most adult males at 350 mg/kg bw/day and one male at 100 mg/kg bw/day. There were no statistically significant differences in organ weight that were considered to be of toxicological significance at 50, 100 or 350 mg/kg bw/day. Diffuse epithelial hyperplasia of the non glandular region of the stomach was observed for both sexes at 350 mg/kg bw/day. No treatment related findings were observed at 50 or 100 mg/kg bw/day. In conclusion, treatment at 350 mg/kg bw/day was associated with adverse histopathological changes in the stomach but this was considered to represent an irritant effect of the test item rather than systemic toxicity. Therefore the Lowest Observed Adverse Effect Level (LOAEL) for local toxicity was considered to be 350 mg/kg bw/day and the related NOAEL, local to be 100 mg/kg bw/day. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be ≥350 mg/kg bw/day and the No Observed Effect Level (NOEL) was 100 mg/kg bw/day.