Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
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:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
7-day study, with hepatic biochemical and pharmacokinetic evaluation
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 19.7% solids in water

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at receipt: approximately 10 weeks
- Housing: group housed in solid-bottom caging with bedding and enrichment
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted
- Water: tap water ad libitum.
- Acclimation period: quarantined for at least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Photoperiod: 12-hour light/dark cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on oral exposure:
PREPARATION OF TEST SUBSTANCE FORMULATION:

-Solvent used: deionized water
-Preparation frequency: Dosing preparations of the test substance were prepared as a batch and stored at room temperature until used.
-Adjusted for purity: Dose preparations were prepared with a correction for the sponsor-reported % solids.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method of Analysis: The dose preparations remaining after dosing were collected and analysed once during the dosing period. Dose preparations to be analysed were stored at room temperature for up to 7 days.

Concentration verification: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Test substance was shown to be homogeneous and at targeted concentrations.
Duration of treatment / exposure:
7 days (7 days for main study animals and one additional dose for pharmacokinetic animals)
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
500 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:
Control and all male dose groups consisted of 5 animals/sex. Female dose groups consisted of 8 animals/sex.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral LD50 in fasted female rats is greater than 5000 mg/kg (corrected for purity).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were conducted at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Time points for clinical observations on test days 1-6 were based on observations made on test day 0, but at least once at weighing and 2-4 hours after dosing each weekday or at weighing on the weekend and on test day 7. Acute clinical signs of systemic toxicity were recorded. At every weighing, each rat was individually handled and examined for abnormal behaviour and appearance. Acute clinical signs of systemic toxicity were recorded. All main study animals were checked twice daily for mortality/morbidity throughout the study. Metabolism animals were checked twice daily for mortality/morbidity throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: approximately 7 days after initiation of the study
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: approximately 7 days after initiation of the study
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: approximately 7 days after initiation of the study
- Metabolism cages used for collection of urine: no data
- Animals fasted: Yes
- Parameters checked in Table No. 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: GROSS PATHOLOGY
- Time schedule for collection of tissues: approximately 7 days after initiation of the study
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation and exsanguinated. Microscopic examination was performed on all tissues from all animals in the control and high-dose groups euthanized at the scheduled primary necropsy. Gross lesions were examined from animals in the low- and mid-dose groups euthanized at the primary necropsy and animals in the control and high-dose groups euthanized at the recovery necropsy. In addition, the liver was evaluated from animals in the low- and mid-dose groups euthanized at the primary necropsy, as well as animals in the control and high-dose group at the recovery necropsy.
Statistics:
See Table 4

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- No effects

BODY WEIGHT AND WEIGHT GAIN
- No effects

HAEMATOLOGY
- Haematology
A decrease red cell mass parameters (haemoglobin, red cell count, haematocrit), without an associated increase in reticulocytes, was present in the 500 mg/kg/day females. This finding did not occur in a dose-related manner as similar changes were not observed in the 1000 mg/kg/day female group, and no haematology changes were observed in males at any dose level. Therefore, the haematology changes in the 500 mg/kg/day female group likely represent spurious findings.
- Coagulation
The following statistically significant difference did not occur in a dose related manner and was not considered to be test substance-related: Prothrombin time (PT) was slightly prolonged in male rats at 30 mg/kg/day.

CLINICAL CHEMISTRY
- Clinical Chemistry
Serum bilirubin was increased in the 1000 mg/kg/day female group (45% above control; statistically significant). Also, minimal increases in alkaline phosphatase and/or bilirubin in some individual animals in the 1000 mg/kg/day male and female groups may indicate a very slight cholestatic effect in the liver. However, there were no correlative histopathological findings. So relationship to treatment and biological relevance is uncertain, but based on the minimal nature of these changes, they are not considered to be adverse under the conditions of this study.

Creatinine (CREA) was lower in the 1000 mg/kg/day female group (13% below control; statistically significant). A similar change was not observed in males. Biologically relevant changes in CREA generally occur as increases rather than decreases. Therefore, based on the minimal nature of the change, as well as the direction of the change (decreased rather than increased), this lower CREA in the 1000 mg/kg/day female group was considered to be unrelated to treatment and non-adverse. Creatinine (CREA) was higher in male rats exposed to 500 mg/kg/day; however, the difference did not occur in a dose related manner and was not considered to be test substance-related.

Cholesterol (CHOL) was lower in male rats exposed to 30 mg/kg/day; however, the difference did not occur in a dose related manner and was not considered to be test substance-related.

URINALYSIS
- Urine volume was minimally increased in 1000 mg/kg/day females, but this change was not associated with correlative changes in other kidney-related clinical pathology parameters or with microscopic changes in the kidneys and thus was considered non-adverse. pH was higher in male rats exposed to 500 mg/kg/day; however, the difference did not occur in a dose related manner and was not considered to be test substance-related.

ORGAN WEIGHTS
- Liver weight parameters were increased in male rats at 1000 mg/kg/day and in female rats at 500 and 1000 mg/kg/day. In the 1000 mg/kg/day male group, liver weight increases were minimal and only liver weight relative to body weight (increased 14% above control) was statistically significant. In females, liver weight increases in the affected groups were statistically significant for most parameters; liver weight relative to body weight was increased 20% and 22% above controls, in the 500 and 1000 mg/kg/day groups, respectively. These liver weight changes were not associated with test substance-related changes in liver-related clinical pathology parameters or with microscopic changes in the liver. These changes likely represent a physiologic response (liver enzyme induction) associated with metabolism of the test article and are considered test substance related but non-adverse.

Kidney weight relative to body weight was minimally but statistically higher (12% above control) in females administered 1000 mg/kg/day. Absolute kidney weight and kidney weight relative to brain weight were similarly higher compared to control, but the differences were not statistically significant. These kidney weight changes were not associated with correlative clinical pathology or microscopic changes, and no test article-related changes in kidney weights occurred in males. Therefore, these kidney weight changes in the 1000 mg/kg/day female group may represent spurious findings, but regardless of relationship to test article administration, are considered non-adverse.

All other statistically significant organ weight changes were considered to be unrelated to test substance administration since they did not occur in a dose-related manner. These included lower thymus weights in the 500 mg/kg/day male group, and higher epididymides weights in all treated male groups. The epididymal weight differences were likely the result of a low epididymal weight in one control animal.

GROSS PATHOLOGY
- There were no gross findings in any treated male or female group.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Degeneration and atrophy of olfactory epithelium was present in one animal each in the 500 and 1000 mg/kg/day male and female groups, as well as in one control female. The olfactory lesion in treated animals was generally more severe than that observed in the one control. Therefore, notwithstanding the absence of a clear dose response, the olfactory changes observed in low incidences in the 500 and 1000 mg/kg/day groups may represent a test article related effect. Olfactory degeneration and atrophy in the one affected males at 500 mg/kg/day was associated with hypertrophy and hyperplasia of the epithelial mucosa. This latter finding may be indicative of regurgitation.
Accentuation of the normal striatal pattern of the dentin of the incisor tooth (graded as “present”) was observed in 0/5, 0/5, 0/5, and 4/5 males in the 0, 30, 500, and 1000 mg/kg/day groups, respectively. In females, this change was observed in 0/5, 0/5, 1/5, and 4/5 animals in the 0, 30, 500, and 1000 mg/kg/day groups, respectively. Dental striations are known to occur following exposure to fluoride or fluoride-containing compounds and have been shown to represent hypermineralized regions in the dentin. In the current study, dentin striations were not associated with other microscopic changes in the teeth (such as altered enamel or effects on the enamel organ) or with clinical abnormalities in dentition. Therefore, under the conditions of this study, these changes were considered indicative of exposure to a fluoride-containing compound but non-adverse.
There were no other test article-related microscopic changes. Low incidences of minimal single cell lymphoid necrosis (apoptosis) of the thymus was observed in all male groups, including the control group, and in 2/5 females in the 1000 mg/kg/day group. Slight increases in the density of apoptotic lymphocytes is a common finding in rodents, and based on the similar incidences and minimal severity of this change in control and treated groups, and the lack of a clear dose-response, this observation was considered to be unrelated to exposure to the test article.

Effect levels

Dose descriptor:
NOEL
Effect level:
30 other: mg/kg bw/day (actual dose received, corrected for purity)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOEL = 30 mg/kg bw/day
Executive summary:

The objective of this study was to assess the potential repeated-dose toxicity of the test substance in rats. Four groups of male and female (nulliparous and non-pregnant) Crl:CD(SD) rats (5/sex/group for main study and 3 females/dose group for pharmacokinetic evaluation) were administered dose formulations of 0, 30, 500, and 1000 mg/kg/day for approximately 7 days (7 days for main study animals and one additional dose for pharmacokinetic animals). Test substance was shown to be homogeneous and at targeted concentrations. Body weights and clinical observations were evaluated daily. Clinical and anatomical pathology endpoints, hepatic biochemical and pharmacokinetic evaluations were determined at the end of the exposure period.

No test substance-related deaths, clinical signs, or effects on body weight were observed at any dose level. There were no adverse differences in coagulation, clinical chemistry or urinalysis parameters in male or female rats at any concentration tested. A decrease in red cell mass parameters (haemoglobin, red cell count, haematocrit) without an associated increase in reticulocytes was present in the 500 mg/kg/day females. This finding did not occur in a dose-related manner, similar changes were not observed in the 1000 mg/kg/day group, and no haematology changes were observed in males at any dose level. Therefore, this finding is likely unrelated to treatment. Administration of the test substance at concentrations of up to 1000 mg/kg/day for seven days was associated with low incidences of degeneration and atrophy of the nasal olfactory epithelium in males and females at 500 and 1000 mg/kg/day. These findings did not occur in a clear dose-related pattern but may represent a test substance related effect based on the increased severity of the changes compared to that which may be seen in control animals.
Other changes considered test substance-related but non-adverse included increased liver weights in males at 1000 mg/kg/day and in females at 500 and 1000 mg/kg/day, and increased kidney weights in females at 1000 mg/kg/day. These organ weight changes were not associated with correlative changes in related clinical pathology parameters or with microscopic changes in the respective organs and were thus considered non-adverse. Similarly, accentuation of the normal striatal pattern of the dentin of the incisor teeth in the 1000 mg/kg/day males and 500 and 1000 mg/kg/day females was not associated with effects on other tooth structures or with clinical abnormalities of the teeth and thus were also considered non-adverse.
There were no test substance-related pathology changes in males or females administered 30 mg/kg/day of the test substance.