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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-15 to 2012-07-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented GLP study performed according to OECD Guideline 422. However, dose formulations were not analyzed.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
no analytical verification of the dose is performed
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation : liquid
- Appearance : colourless to yellow brown
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: # 1D403

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from humidity and light

OTHER SPECIFICS:
- Name of test material (as cited in study report): Jeffcat DMDEE
- Physical state: Liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Age at study initiation: 10 - 11 weeks old
- Weight at study initiation: males: 324.4 - 341.5 grams; females: 204.3 - 217.3 grams; satellite males: 336.0 - 371.0 grams; satellite females: 207.4 - 208.0 grams
- Fasting period before study: no
- Housing: Each animal was housed individually, except during cohabitation. After acclimation, one male was placed into each female cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually. The rats were housed in polypropylene cages (41x34x19 cm) with wire mesh tops and bedding material (wood shavings). Clean cages were provided twice weekly for all animals. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-22.6°C
- Humidity (%): 40.5-69.9%
- Air changes (per hr): 10-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For each dosage group, the appropriate amount of Jeffcat DMDEE was weighed into a pre-calibrated beaker. The vehicle (deionized water) was added in sufficient quantity to achieve the desired concentration. Each solution was stirred and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was similarly dispended for administration to control animals. The prepared solutions were stored at room temperature.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Parental animals (males and females) were treated with a volume of 4 mL/kg bw, starting at 10-11 weeks old and ending when the animals were euthanized. Satellite animals (5 animals per sex of the control and 5 animals per sex of the high dose group) were 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 were not mated and, consequently, were not used for assessment of reproduction/developmental toxicity.
Frequency of treatment:
7-day per-week-basis
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 in the main study
5 animals per sex of the control group and 5 animals per sex of the high dose group for the satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels (in mg/kg body weight/day) were selected following consultation with the Sponsor.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
All animals underwent 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.

BODY WEIGHT: Yes
Males were weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were 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).

FOOD CONSUMPTION
Food consumption was determined on the same day of body weight determination during premating and lactation periods, except on day 0. During the gestation period food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After the mating period, food consumption of males was determined weekly. Food consumption was not determined for exposed animals during the mating period.

HAEMATOLOGY: Yes
The following parameters were examined:
red blood cell count, hemoglobin, hematocrit, platelets, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total white blood cell count, differential leukocyte count, band neutrophils, monocytes, segmented neutrophils, lymphocytes, eosinophils, basophils
Clotting parameters: prothrombin time, activated partial thromboplastin time.

Hematology determinations were performed on 5 parental animals/sex/group, randomly selected from each group and on satellite animals from control and high dose group. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture).

CLINICAL CHEMISTRY: Yes
Clinical chemistry parameters:
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, glucose, total cholesterol, urea nitrogen, creatinine, sodium, potassium, calcium, globulin, albumin/globulin ratio.

Clinical chemistry determinations were performed on 5 parental animals/sex/group, randomly selected from each group and on satellite animals from control and high dose group. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture). The clinical chemistry determination was performed in an accredited laboratory - Sabin - DF.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery:
Sensory reactivity to stimuli and motor activity assessment were performed in 5 animals/sex/group. For males these evaluations were performed at the end of the dosing period before scheduled necropsy, and for females, these evaluations were performed during the lactation period. The following parameters were 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 mobility and gait pattern in an open field;
E - Abnormal clinical signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth

OTHER:
Organ weights: At scheduled necropsy, testes and epididymides of all males were weighed.
Organ weights were obtained for the following organs from 5 animals/sex/group:
liver, kidneys, adrenals, thymus, spleen, brain, heart
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At termination, all parental animals were examined macroscopically for any abnormalities or pathological changes. The animals were euthanized in a carbon dioxide chamber. The number of implantation sites and corpora lutea were recorded. The animals were disposed in biological garbage and then incinerated. All pups were grossly examined for abnormalities of the oral, thoracic and abdominal cavities.

HISTOPATHOLOGY: Yes
At the scheduled necropsy, the following organs of all animals were preserved:
testes, epididymides, ovaries, prostate, semincal vesicle and coagulating gland, bulbourethral gland, organs showing alterations

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

Full histopathology of the preserved organs and tissues listed above were performed in highest dose and control animals. The examination of the thymus was extended to females of other dosage groups and satellite females, because changes were observed in the highest dose group.
Statistics:
Quantitative variables such as body weights, food consumption and organ weights were analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance is detected, or by the non-parametric test of Kruskal-Willis, according to the results of tests for normaility and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means were carried out using Fisher's Exact Test or the Chi-Square Test. The level of significance was set at 5%.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Two females and one male at 300 mg/kg/day presented clinical signs between treatment days 25 to 41. These clinical signs were variable in nature, transitory, occurred in only a few animals, and were not observed in the high dose group satellite animals. The significance of these findings is not clear, but they are unlikely to be specific to the test article.
Mortality:
no mortality observed
Description (incidence):
The test substance did not cause mortality during the study
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were unaffected at all dose levels
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected at all dose levels.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean total protein, globulin and albumin in males exposed to 150 and 300 mg/kg/day were statistically significant lower with a dose related trend, while potassium in females at 300 mg/kg/day was also higher than control. However, these findings were not observed in any other high dose groups, either study or satellite and are unlikely to be specific to the test article. Decreased diffuse lymphocyte cellularity in thymus and thymus involution was observed in females exposed to 150 and 300 mg/kg/day, respectively. In both cases, these lesions were correlated with a lower thymus weight. These findings were not observed in treated and satellite males or satellite females. It is important to note that for all treated females, including the control group females, thymus weights were substantially decreased in comparison to the satellite control group. This is most likely due to the animals' physiological response to pregnancy, and not test article related.

Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the functional observational battery, hyperreflexia was observed in three females at the 300 mg/kg/day dose level. The incidence of this finding was not statistically significant when compared to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Kidney weights in males at 300 mg/kg/day was statistically significantly higher (+21%) when compared to the control group. Relative to body weight, although the difference was higher (+31.2%), it was not statistically significant, although relative to brain weight, kidney weights were statistically significant again (+20.8%). These changes were not dose related and no macroscopic or microscopic lesions were found, this finding was not considered to be test item related.
A statistically significant increase was observed in liver, kidneys, spleen and thymus weights of treated satellite males when compared to the control group. Relative to body weight, only liver and kidney weights were statistically significant, but relative to brain weight, the difference in these four organs was statistically significant again. Despite differences, no supporting macroscopic or microscopic lesions were noted in these organs, and these findings were not considered to be treatment-related.
Mean thymus weight was statistically significant lower in females exposed to 300 mg/kg/day (-39.5%) compared to the control group. Also, a statistically significant decrease was observed in thymus weights relative to body weight (-33%) and relative to brain weight (-39.1%). It is important to note that for all treated females, including the control group females, terminal thymus weights were substantially decreased in comparison to the female satellite control group and the treated and satellite males. This observation is most likely due to the female animals' physiological response to, and following, pregnancy, and is not considered test article related.
Finally, the differences found in liver and kidney weights in treated satellite females were not considered test item related because they were moderate in magnitude and there were no supporting macroscopic or microscopic lesions observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were observed upon necropsy in male or female rats
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Decreased diffuse lymphocyte cellularity in thymus was observed in five females exposed to 150 mg/kg/day (animals 71, 74, 76, 77 and 82), while in females at 300 mg/kg/day was noted a thymus involution in rats 95, 98, 102, 104 and 106. These findings were not observed in treated and satellite males or satellite females, terminal thymus weights were substantially decreased in comparison to the female satellite control group. This observation is most likelly due to the animals' physiological response to, and following, pregnancy, and is not considered test article related.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was 300 mg/kg/day for males and females. The substance is therefore not classified as STOT RE.