Itaconic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Itaconic acid

EC Number:

202-599-6

EC Name:

Itaconic acid

Cas Number:

97-65-4

Molecular formula:

C5H6O4

IUPAC Name:

2-methylidenebutanedioic acid

Test material form:

other: solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

After a premating period of 2 weeks, females will be cohabited with an assigned male (1 female:1 male) from the same dose level until evidence of copulation is observed, or either 3 estrous periods or 2 weeks have elapsed. Care will be taken to avoid sibling mating. Vaginal smears will be collected daily during mating period and examined for the presence of sperm. Day 0 of gestation is defined as the day a sperm is found in the vaginal smear. Females that show no evidence of copulation will be assumed pregnant at the end of the mating period, and housed accordingly. Males will be killed after a minimum dosing period of 28 days has been completed.

Duration of treatment / exposure:

The test solution or vehicle will be administered daily, by gavage, on a 7-day-week-basis.

Frequency of treatment:

The test solution or vehicle will be administered daily, by gavage, on a 7-day-week-basis.

Details on study schedule:

1. Treatment
The test solution or vehicle will be administered daily, by gavage, on a 7-day-week-basis. Individual doses will be calculated using the most recent body weight. The oral route of administration has been selected for this study since this is a potential route of human exposure and by gavage because it is an accurate and reliable method for dosing. The volume administered each day will not exceed 1 mL/100g body weight. A constant dosage-volume of 4 ml/kg will be used.
Parental animals (male and females) will be treated starting at 10-12 weeks old and ending when the animals are euthanized. Satellite animals (5 animals per sex of the control and 5 animals per sex of the high dose group) will be kept for 14 days after the scheduled necropsy of parental animals without treatment, for observation of reversibility, persistence or delayed occurrence of toxic effects. Satellite animals will not be mated and, consequently will not use for assessment of reproduction/developmental toxicity.

2. Experimental Procedure

2.1. Premating
At the end of acclimation period, animals will be randomly assigned to the experimental groups, housed and the treatment started.

2.2. Mating
After a premating period of 2 weeks, females will be cohabited with an assigned male (1 female:1 male) from the same dose level until evidence of copulation is observed, or either 3 estrous periods or 2 weeks have elapsed. Care will be taken to avoid sibling mating. Vaginal smears will be collected daily during mating period and examined for the presence of sperm. Day 0 of gestation is defined as the day a sperm is found in the vaginal smear. Females that show no evidence of copulation will be assumed pregnant at the end of the mating period, and housed accordingly. Males will be killed after a minimum dosing period of 28 days has been completed.

2.3. Gestation
During gestation period all females will be housed individually in the cages. Females without copulation date will be housed in individual cages at the end of the mating period. Females showing no evidence of copulation will be killed 24-26 days after the last day of mating period.

2.4. Lactation
The day when delivery is completed will be designated day 0 of lactation (postnatal day 0). On day 0 of lactation the number of alive and dead pups/sex will be recorded. Dams with offspring will be killed on day 4 postnatal, or shortly thereafter. All pups will be killed at day 4 postnatal or shortly thereafter.

3. Observations of Parental Animals
3.1. Clinical Signs
Clinical examination will be performed weekly on the animals.
All animals will undergo a daily clinical observation for overt signs of ill health. These include, but are not limited to, changes in skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous system, motor activity and behavioral patterns. Animals found dead will be necropsied. Weak or moribund animals will be euthanized and necropsied. The animals will be disposed in biological garbage and then incinerated.

3.2. Functional Observational Battery (FOB)
Sensory reactivity to stimuli, assessment of grip strength, and motor activity assessment will be performed in 5 animals/sex/group. On males will be performed at the end of dosing period, before scheduled necropsy, and on females during lactation, before scheduled necropsy. The following parameters will be assessed:
A – Autonomic Functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection, respiration, urination and defecation.
B – Reactivity and Sensitivity: sensor motor responses to approach tactile and tail flick.
C – Excitability: reactions to handling and behavior in an open field.
D – Gait and Sensor Motor Coordination: degree of morbidity, gait pattern in an open field, aerial righting reflex and landing foot splay.
E – Abnormal Clinical Signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth.

3.3. Body Weights
Males will be weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females will be weighed on first day of dosing and once a week during premating and mating periods, on days 0, 7, 14 and 20 of gestation, and during lactation on the same days as the weighing of litters (on days 0 and 4 postnatal).

3.4. Food Consumption
Food consumption will be determined on the same day of body weight determination during premating and lactation periods. During gestation period food consumption will be determined on days 3, 6, 9, 12, 15, 18 and 20. After mating period, food consumption of males will be determined weekly. Food consumption will not be determined during mating period.

3.5. Observations of the Offspring
Live pups will be counted, sexed and weighed on days 0 and 4 postnatal.


3.6. Clinical Pathology
Hematology and clinical chemistry determinations will be performed on 5 parental animals/sex/group, randomly selected from each group, on the day of scheduled necropsy. The animals will be fasted overnight and anesthetized by CO2 prior to blood collection (cardiac punction). The following parameters will be measured:

a. Hematology Parameters:

1. Red Blood Cell Count (RBC)
2. Hemoglobin (Hb)
3. Hematocrit (Hct)
4. Platelets (PLT)
5. Mean Corpuscular Volume (MCV)
6. Mean Corpuscular Hemoglobin (MCH)
7. Mean Corpuscular Hemoglobin Concentration (MCHC)
8. Total White Blood Cell Count (WBC)
9. Differential Leukocyte Count

b. Clotting Parameters:

1. Prothrombin Time (PT)
2. Activity Partial Thromboplastin Time (APTT)


c. Clinical Chemistry Parameters:

1. Aspartate Aminotransferase (AST)
2. Alanine Aminotransferase (ALT)
3. Alkaline Phosphatase (ALP)
4. Total Protein (TP)
5. Albumin (ALB)
6. Glucose (GLU)
7. Total Cholesterol (CHOL)
8. Urea Nitrogen (BUN)
9. Creatinine (CRE)
10. Sodium (Na)
11. Potassium (K)
12. Calcium (Ca)

4. Postmortem Evaluation
4.1. Gross Necropsy
At termination, or if unscheduled death or humane euthanasia occur during the study, all parental animals will be examined macroscopically for any structural abnormalities or pathological changes. The animals will be euthanized in a carbon dioxide chamber. The number of implantation sites and corpora lutea will be recorded. Animals found dead or sacrificed moribund during the study will be evaluated by gross examination as soon as possible after death. If the necropsy can not be performed immediately, the animal will be kept in a refrigerator until the examination is performed. Then, the animals will be discarded in biological garbage and incinerated.
All pups will be grossly examined for abnormalities of the thoracic and abdominal cavities.

4.2. Organ Weights
At scheduled necropsy, testes and epididymides of all males will be weighed.
Organ weights will be obtained for the following organs from 5 animals/sex/group:
- liver;
- kidneys;
- adrenals;
- thymus;
- spleen;
- brain;
- heart.

4.3. Histopathology
At scheduled necropsy, the following organs of all animals will be preserved:
- testes;
- epididymides;
- ovaries;
- prostate;
- seminal vesicle and coagulating gland;
- bulbourethral gland;
- organs showing alterations.

The following organs and tissues of 5 animals/sex/group will be preserved:
- adrenals (right and left);
- bone marrow (femur);
- brain (cerebrum, cerebellum and pons);
- heart;
- intestine (duodenum, jejunum, ileum – including Peyer’s patches, colon, rectum/anus);
- kidneys (right and left);
- liver (3 lobes);
- lungs;
- lymph nodes (mesenteric and submaxillary);
- peripheral nerve (sciatic);
- spinal cord (cervical, midthoracic and lumbar sections);
- spleen;
- stomach (glandular and non-glandular);
- trachea;
- thymus;
- thyroid;
- urinary bladder;
- uterus;
- all gross lesions.

Full histopathology of the preserved organs and tissues listed above will be performed in highest dose and control animals. These examinations will be extended to animals of other dosage groups and satellites, if treatment-related changes are observed in the highest dose group. All gross lesions found in other groups will be examined.

Doses / concentrations

Remarks:

Doses / Concentrations:
150, 300 and 600 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

The experimental design for the study is as follows:



Main Study Satellite Dose Level
Group No. of animals No. of animals (mg/kg body weight/day)
Male Female Male Female
1 12 12 5 5 Corn oil
2 12 12 - - 150
3 12 12 - - 300
4 12 12 5 5 600

Control animals:

yes, concurrent vehicle

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Generation not specified (migrated information)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Evaluation of Pups and Litter

1. Pups Clinical and Mortality

No clinical signs or mortality occurred in pups from treated females.

2. Pup Body Weight Data

Differences on pup body weight were observed between treated and control group, statistically significant higher at day 0 postnatal at low dose and at day 4 postnatal in all treated groups. These changes were related with a lower number of pups in all treated group, then considered no Test Item associated.

3. Macroscopic Observations of Pups

No macroscopic finding was observed in pups.

Applicant's summary and conclusion

Conclusions:

Itaconic Acid caused mortality and clinical signs of toxicity during the study in three animals at high dose; besides one female of this group was sacrificed by animal welfare and confirmed aborting. No effects on body weight, body weight gain and food consumption were observed. No effects were observed in functional observational battery, hematology and clotting parameters, clinical chemistry parameters and organ weight. The most common macroscopic and microscopic lesions were congestion and hemorrhagic in some organs in either males or females. In all cases these findings were considered Test Item related with toxicological significance. All treated females showed lower implantation sites, corpora lutea and pups, however, these findings were no dose related and then was considered as no clear Test Item related at low and mid dose.

Executive summary:

Accordingly, in the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test item Itaconic Acid in Wistar rats was 300 mg/kg/day for males and females and 300 mg/kg/day for maternal-embryo-fetal toxicity.