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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-19 to 2012-11-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

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 was performed
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Details described in study specific records
Specific details on test material used for the study:
- Name of test material (as cited in study report): JEFFCAT DPA
- Physical state: Liquid
- Lot/batch No.: # 1D516
- pH: 11.7
- Storage of the test item:
The samples were stored in a specific room (Test Item Storage Room), at room temperature, protected from humidity and light;
A sub-sample of the test item (Archival of Test Item) was preserved in the same condition as the main sample;
The remaining amount of test item was disposed according to the Sponsor's instruction.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorias Ltda-DF
- Age at study initiation: 10 - 11 weeks old
- 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 (41 x 34 x 19 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: Upon receipt from the supplier, animals were placed into cages (1 animal/cage/sex) examined and acclimated for 7 days. All animals were observed for morbidity and mortality. At the end of this period, the animals were weighed and a detailed clinical examination was performed. Animals not selected were euthanized by inhalation of carbon dioxide and then exsanguinated. Only animals with body weights within ± 20% from the group mean body weight and with no abnormal signs were used in this study. In addition, all animals used in the study had a regular estrous cycle.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 23.6 °C
- Humidity (%): 40.7-70.0%
- Air changes (per hr): ambient room air will be changed 10-20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test solution was prepared daily and was used within 2 hours after preparation

For each dosage group, the appropriate amount of the test item 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 be similarly dispensed for administration to control animals. The prepared solutions were stored at room temperature.

VEHICLE:
- Amount of vehicle (if gavage): The volume administered each day was 4 mL/kg body weight.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Parental animals (males and females) were treated 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 days per week basis
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
nominal ingested
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
nominal ingested
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
nominal ingested
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
nominal ingested
No. of animals per sex per dose:
12 animals/sex/dose group (including vehicle group) + 5 animals/sex/satellite vehicle group + 5 animals/sex/500 mg/kg/day satellite group
Control animals:
yes, concurrent vehicle
Details on study design:
The following doses were chosen for this study:
- 10 mg/kg body weight/day as the expected dose which causes no signs;
- 100 mg/kg body weight/day as the intermediate dose level;
- 500 mg/kg body weight/day as the expected dose which causes signs of systemic toxicity, but not death or severe suffering.

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.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
During premating, mating, gestation and lactation period, the animals were observed twice a day for morbidity, mortality and clinical observation for overt signs of ill health on working days and once a day on weekend and public holidays. 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 were necropsied. Weak or moribund animals were euthanized and necropsied. The carcasses were disposed in biological garbage and then incinerated.

BODY WEIGHT:
Males were weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were weighed on the 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 weighing of litters (on days 0 and 4 postnatal).

FOOD CONSUMPTION:
Food consumption was determined on the same day of body weight determination (except on day 0) during premating and lactation periods. During 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 during mating period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Hematology and clinical chemistry determinations were performed on 5 parental animals/sex/group, randomly selected from each group. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture). The following parameters were measured:
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, activity partial thromboplastin time

CLINICAL CHEMISTRY: Yes
Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, glucose, total cholesterol, urea nitrogen, creatinine, sodium, potassium, calcium, globulin, albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Sensory reactivity to stimuli and motor activity assessment were performed in 5 animals/sex/group. In males, these tests will be done at the end of the dosing period, before scheduled necropsy, and in females during lactation. The following parameters were assessed:
A - Autonomic functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection, respiration;
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
Sacrifice and pathology:
- Gross necropsy
At termination all parental animals were examined macroscopically for any abnormalities or pathological changes. The animals were disposed in biological garbage and then incinerated.
For all animals a core list of organs was collected, weighed and preserved in 10% neutral formalin during necropsy.
Paired organs were weighed together.
- Histopathology:
At scheduled necropsy, the following organs of all animals were 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 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), peripheral nerve (sciatic), spinal cord (cervical, midthoracic and lumbar sections); spleen, stomach (glandular and non-glandular), trachea, pancreas, thymus, thyroid/parathyroid, urinary bladder, uterus and all gross lesions.
Full histopathology of the preserved organs and tissues listed were performed in the highest dose and control animals. These examinations not extended to animals of other dosage groups, because treatment-related changes were not observed in the high dose group.
Other examinations:
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
Statistics:
Quantitative variables such as body weights, food consumption and organ weights have been analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance was detected, or by non-parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means have been carried out using the Fischer'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, treatment-related
Description (incidence and severity):
Female No 105 presented respiratory sounds at day 6 of the test item administration; while female No 104 did not show any clinical sign and female No 116 had moderate dyspnea, piloerection and exophthalmos on day 7. Male No 109, at the same dose level, was euthanized for humane reason on day 8 of treatment, after severe signs of dyspnea, respiratory sounds, apathy and rhinodacryorrhea. Necropsy and histopathological examination of animals No 104, 105 and 109 showed pulmonary congestion and edema as result of bronchoaspiration by gavage error.

Male rats No 83, 86, 87, 92 and 94, exposed to 500 mg/kg/day presented moderate dyspnea and respiratory sounds between days 9 and 31 of test item administration; while males No 91 and 93 were noted with respiratory sounds on day 30 and 30-31, respectively. At the same dose level, male No 86 had piloerection and rhinodacryorrhea from day 13 to day 17. In females No 97, 101, and 102 was noted respiratory sounds from day 17 to day 27 of test item administration and also on day 43 of the test item administration (animal 97). Male No 92 and females No 102 and 106 had rhinodacryorrhea on days 10, 35 and 44 of treatment respectively. Finally, from day 17 to day 25 female No 97 showed moderate dyspnea; while female No 106 presented piloerection and slight apathy on days 43 and 44 of treatment.

Although these clinical signs were moderate in magnitude, they were sporadically observed throughout the dosing period in the test animals and were transitory in nature. Due to the extremely corrosive nature of the test substance to animal tissues and the dosing volumes used in the dosing of the high dose animals, it is likely that these respiratory observations were artefacts from the gavage dosing procedure. While these observations could be considered test item-related, they are not considered to be toxicologically relevant and are of low concern in the absence of other treatment related effects.
Mortality:
mortality observed, treatment-related
Description (incidence):
The test substance did not cause treatment-related mortality during the study. However, 3 females (No 104, 105 and 116) at 500 mg/kg/day were found dead on days 13, 7 and 7 of treatment, respectively.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No biologically or statistically significant changes were observed in body weight of treated males compared to the control group. Nevertheless, statistically higher body weight gain was observed in male rats exposed to 100 mg/kg/day from day 0 to day 7 (+ 1.8%); while at 500 mg/kg/day the difference was + 302.6%, from day 14 to day 21. Despite changes, these did not affect statistically overall body weight gain (from day 0 to day 28), although it was slightly lower in treated males when compared to the control group, but without a clear dose response relationship (-7.5% low, -8.3% mid and -8.2% high dose), and thus, were not considered related to treatment with test item.
During treatment and recovery period, mean body weight of treated satellite males was biologically and statistically unaffected by the treatment. However, overall body weight gain was statistically lower (-27%) during the treatment period (from day 0 to day 35); while in the recovery period, although without statistical significance overall body weight gain (from day 0 to day 14) was higher than control (+ 165.5%). In spite of these differences, these findings were not considered biologically relevant because in both cases body weight was unaffected by the treatment with the test item.
Statistically lower mean body weights were observed in female rats at 500 mg/kg/day on days 7 (-15.1%), 14 (-14.1%) and 20 (-19.3%) of the gestation period and on days 0 (-19.4%) and 4 (-17.5%) of the lactation period. Despite differences, these were not considered to be dose related, because at the study initiation the mean body weight of this group was lower than control (-7.7%) and then the differences during the gestation and lactation period were slight (<12%) and without a dose response relationship. During all treatment periods, the mean body weight gain of treated females was lower when compared to the control group, however, a dose related trend was not observed and these differences only were statistically significant at 500 mg/kg/day, affecting overall body weight gain (-34.1%) during the gestation period (from day 0 to day 20). Because body weight gain of treated females was erratic and without a dose related trend, these changes were not considered to be test item related.
At different observation intervals, body weight and body weight gain of treated satellite females showed statistically significant differences; however, these changes began at the study initiation when body weight of treated satellite females was lower than control (-17.6%) and accordingly it affected overall body weight and body weight gain during treatment and recovery period. Accordingly, changes observed in body weight and body weight gain were not considered to be test item related.
At different observation intervals, body weight and body weight gain of treated satellite females showed statistically significant differences; however, these changes began at the study initiation when body weight of treated satellite females was lower than control (-17.6%) and accordingly it did affect overall body weight and body weight gain during treatment and recovery period.
Accordingly, changes observed in body weight and body weight gain were not considered to be test item related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No biologically or statistically significant changes were observed in food consumption of treated males when compared to the control group.
Statistically significant lower food consumption was observed in females exposed to 500 mg/kg/day on days 0 to 7 (-13.5%) and on days 0 to 14 (-11.3%) of the premating period. During the gestation period, statistically lower food consumption was observed on days 3 to 6 (-14.7%) and on days 18 to 20 (-5%) at 500 mg/kg/day; while at 100 mg/kg/day statistically lower food consumption was noted on days 9 to 12 (-6.4%) and on days 15 to 18 (-0.6%) compared to the control group. These differences were small in magnitude and not dose related and thus, were not considered to be test item related.
At treatment initiation (from day 0 to day 7) food consumption of treated satellite females was statistically lower (-28.4%) compared to the control group, affecting statistically overall food consumption (from day 0 to day 35) -12.1%. Because the difference was very slight, it was not considered to be treatment-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant higher red blood cell count (+1.7%) was observed in male rats at 10 mg/kg/day; while male rats exposed to 500 mg/kg/day showed statistically lower mean corpuscular hemoglobin concentration (-3.2%). Because the differences were very small in magnitude and without a dose related trend, these findings were not considered to be treatment related.
Females at 500 mg/kg/day displayed statistically lower mean corpuscular values (-5.3%) and mean corpuscular hemoglobin (-7%) and females exposed to 10 mg/kg/day had statistically lower mean corpuscular hemoglobin (-4%). These changes were isolated and very small in magnitude and therefore, not considered toxicologically relevant. In treated satellite females, statistically significant lower platelets (-22%) was observed, but this value was only slightly lower than Bioagri's Historical Data Base (553.5-696.9) and is without toxicological significance.
In male rats exposed to 10 mg/kg/day was noted statistically lower lymphocytes (-0.8%), while at 500 mg/kg/day was observed statistically higher monocytes (+120%) and eosinophils (+40%). Despite these differences, in all cases they occurred without a dose response relationship and are not considered to be related to treatment with the test item.
Statistically significant lower segmented neutrophils were observed in females exposed to 500 mg/kg/day (-57.5%) with a dose related trend; however, the values are within of Bioagri's Historical Data Base (325.4-1217) and therefore was considered as not toxicologically relevant in absence of the control group, but without a dose response relationship and then not considered to be test item related. Statistically lower segmented neutrophils (-46.6%), monocytes (-16.7%) and eosinophils (-2.6%) were observed in treated satellite females, but these values are within Bioagri's Historical Data Base and are without toxicological significance.
No biologically or statistically significant changes were observed in clotting parameters of treated males and females compared to the control group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant higher sodium (+10%) at 500 mg/kg/day and aspartate aminotransferase at 100 and 500 mg/kg/day (+6.4% and 3.6%, respectively) were observed in male rats when compared to the control group. These differences were considered as not biologically relevant, because in both cases were small in magnitude and a dose response relationship has not been observed;
Treated satellite males showed statistically higher alanine aminotransferase (+35.2%) but within laboratories' historical database and not considered to be treatment related.
In females exposed to 500 mg/kg/day statistically significant higher glucose (+44.5%) and albumin/globulin ratio (+14.3%) was noted compared to the control group. However, in the first case no dose related and in the second one in small magnitude and therefore not considered to be test item related. A very small difference in mean sodium was noted in females at 10 mg/kg/day (+0.4%) compared to the control group, although it was considered to be an incidental finding.
Statistically significant higher urea nitrogen (+59.1%), total protein (+68.6%), globulin (+63.2%), albumin (+75%), calcium (+57.4%), cholesterol (+112.3%) and potassium (+63.8%) were observed in treated satellite females. The changes, while statistically significant, were not observed in any other high dose group (male or female) and were not supported by other pathology findings. Also, the values were within the laboratories' historical database and these findings were not considered to be biologically relevant, and not test item related.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery:
Male No 83 at 500 mg/kg/day presented moderate dyspnea and respiratory sounds; while female No 97 at the same dose level had respiratory sounds. Despite that these findings were observed at the highest dose level, they occurred only in two animals, without statistical significance. No other findings related to the sensory reactivity to stimuli and motor activity assessment was noted.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Adrenal weight was statistically lower in males exposed to 500 mg/kg/day (-24.3%) compared to the control group. Relative to body weight, although without statistical significance, adrenal weight was lower than control (-13.6%), being statistically lower relative to brain weight (22.7%). Despite differences, these were considered as not biologically significant, because they were moderate in magnitude and also no macroscopic or microscopic lesions were observed. Also, heart weight relative to body weight was statistically higher at 100 mg/kg/day compared to the control group (+18.6%), although it was considered incidental.
Treated satellite males showed statistically higher kidney weights relative to brain weight (+31.8%) when compared to the control group. However, in absence of statistical significance in kidney weight and kidney weights relative to body weight and because no macroscopic or microscopic lesions were observed, this finding was not considered to be treatment related.
Statistically lower heart weight was noted in female rats at 10 and 500 mg/kg/day (-18.6% and -20.4%, respectively) compared to the control group. However, no statistical differences were observed in heart weight relative to body weight, and relative to brain weight only were observed statistical significance at 10 mg/kg/day. In addition, at 500 mg/kg/day spleen and brain weights were statistically lower than control (-34.9% and -7.3%, respectively). Nevertheless, relative to body weight, liver and brain weights were statistically higher (+12.5% and +19.8%, respectively). Finally, relative to brain weight spleen and brain weights were statistically lower (-29.5% and -7.3%, respectively) when compared to the control group. Because in all cases the differences were moderate in magnitude and without a dose response relationship, this finding was not considered related to treatment with the test item.
Statistically significant lower heart (-20.4%), kidney (-33%) and brain (-6.3%) weights were observed in treated satellite females when compared to the control group. Nevertheless, relative to body weight, these organs did not show statistical significant and relative to brain weight only was observed in kidney weight (-28.4%). Also, in these animals, adrenal weights, relative to body weight (+29%) and relative to brain weight (+22.8%) was statistically significant compared to the control group. Despite changes observed, these were not considered to be treatment related, because were moderate in magnitude and no macroscopic or microscopic lesions were observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related findings were observed upon necropsy in male or female rats. Animals No 104, 105 and 109 exposed to 500 mg/kg/day, found dead or euthanized for human reasons during the study showed macroscopic lesions or bronchoaspiration, compatible with gavage error and confirmed during histopathological examination.
Although pulmonary emphysema (bilateral), in female No 116 at 500 mg/kg/day was observed, this lesion was not found in other treated animals. Despite clinical signs observed in both male and female rats exposed to 500 mg/kg/day during treatment period, no macroscopic lesions were found, except in male No 83, where pulmonary congestion was observed.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related microscopic changes were noted in male or female rats.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Male systemic toxicity, maternal systemic toxicity

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

In this study, the only findings considered to be treatment-related were certain clinical signs observed only in male and female rats at high dose level. While these findings were likely attributable to the dosing of the test article, they were not considered to be of toxicological significance.

Although these clinical signs were moderate in magnitude, they were sporadically observed throughout the dosing period in the test animals and were transitory in nature. Due to the extremely corrosive nature of the test substance to animal tissues and the dosing volumes used in the dosing of the high dose animals, it is likely that these respiratory observations were artefacts from the gavage dosing procedure. While these observations could be considered test item-related, they are not considered to be toxicologically relevant and were of low concern in the absence of other findings related to male sytemic toxicity, maternal systemic toxicity, and embryo-fetal toxicity.

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 greater than 500 mg/kg/day for male systemic toxicity and maternal systemic toxicity.
Executive summary:

The test substance was tested in rats according to OECD 422 guideline. The substance was dosed at 0, 10, 100, 500 mg/kg/day. No treatment-related mortality during the study was reported. Most male and female rats exposed to 500 mg/kg/day presented respiratory clinical signs between treatment days 6 to 44. Although these clinical signs were moderate in magnitude, they were sporadically observed throughout the dosing period in the test animals and were transitory in nature. Due to the extremely corrosive nature of the test substance to animal tissues and the dosing volumes used in the dosing of the high dose animals, it is likely that these respiratory observations were artefacts from the gavage dosing procedure. While these observations could be considered test item-related, they are not considered to be toxicologically relevant and are of low concern in the absence of other findings related to male systemic toxicity, maternal systemic toxicity, and embryo-fetal toxicity.

No treatment-related changes in body weight and body weight gain, food consumption, haematology, clinical chemistry, neurobehavioural changes, organ weight, macroscopic and microscopical (histophatological) evaluation of organs were reported.

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was greater than 500 mg/kg/day for male systemic toxicity and maternal systemic toxicity. For risk assessment purposes, 500 mg/kg bw/day is selected as NOAEL value.