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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Remarks:
conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Remarks:
conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
other: U.S. EPA, OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Health Effects Test Guidelines
Deviations:
no
Remarks:
conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
other: U.S. EPA, OPPTS 870.3050: Repeated Dose 28-Day Oral Toxicity Study in Rodents, Health Effects Test Guidelines
Deviations:
no
Remarks:
conducted according to guideline in effect at time of study conduct
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 28 wt%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 66 days
- Weight at study initiation: 298.0-401.6 grams (males) and 186.3-253.9 grams (females)
- Housing: Solid-bottom cages with bedding and Nestlets(TM) as enrichment.
- Diet (e.g. ad libitum): ad libitum, except when fasted for clinical pathology
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): approximate 12-hour light/dark cycle
- Air Changes: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionised water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations of the test substance were prepared daily and stored at room temperature until used. Once room temperature storage stability was established, formulations were prepared twice weekly and stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Formulation Sampling: Near the beginning of the study, dose formulations were sampled and analysed for verification of concentration, homogeneity, and stability. Near the middle and end of the study, dose formulations were sampled for an additional concentration verification check.

Method of Analysis: Analysis was performed by high-performance liquid chromatography (HPLC) with mass spectrometry detection (LC/MS). Samples were submitted for analysis shortly after preparation. At the time of analysis, the samples were diluted with an appropriate solvent.

Analyses results showed the test substance was at targeted concentrations and stable under the conditions of the study.
Duration of treatment / exposure:
28-day subset: 28 consecutive days. 28-day subset with one month recovery: 29 days and then not dosed for the remainder of the recovery period. Reproductive subset: 30 days prior to and during cohabitation. Male rats, and female rats showing no evidence of copulation: until the day before sacrifice. Females showing evidence of copulation: until day 3 postpartum.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 50, 200 mg/kg
Basis:
other: actual ingested corrected for purity
No. of animals per sex per dose:
20 animals/sex/dose (including 10 animals/sex/group for the reproductive subset and 5/sex/group for both the 28-day and 28-day with recovery subset animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previous repeated-dose oral toxicity 7-day gavage study in rats groups of five rats/sex/group were administered the test substance for at least 7 consecutive days at either 0 (control), 30, 500, or 1000 mg/kg/day. At 1000 mg/kg/day, degeneration/atrophy of the nasal olfactory epithelium was present in male and female rats; this effect was not observed at 30 or 500 mg/kg/day. Based on these results, dosages of 0, 10, 50, and 200 mg/kg/day were selected for the current study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side examinations to detect moribund or dead animals and abnormal behaviour and/or appearance among animals were conducted at least once daily during quarantine and prior to study start, and twice daily thereafter. An additional cage-side evaluation was conducted daily at approximately 2-3 hours post dosing to detect acute clinical signs of systemic toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At every weighting (excluding weights on days of necropsy).

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing on test day 0 and weekly during premating and postmating; gestation days (GD) 0, 7, 14, and 20 and lactation days (LD) 0 and 4 and at neurobehavioral evaluations and scheduled sacrifice

FOOD CONSUMPTION:
- Food consumption for each animal determined (g/food/animal/day): Yes; Weekly during premating; GD 0, 7, 14, and 20; and LD 0 and 4. Food consumption was not measured during the postmating period for males, and for females that did not mate.

FOOD EFFICIENCY:
- Calculated as g weight gained/g food consumed: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Test day 29-30
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: 5/sex/group from the reproductive subset and 5/sex/group from the 28-day with recovery subset
- Parameters in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Test day 29-30
- Animals fasted: Yes
- How many animals: 5/sex/group from the reproductive subset and 5/sex/group from the 28-day with recovery subset
- Parameters in table No. 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (refer to Section 7.9.1, DI.K1.Neuro.R.D-19570-1422 for additional details).

OTHER: Coagulation
- Time schedule for collection: during week 4 and during week 6 and 8 for males and females, respectively, from the reproductive subset.
- How many animals: 5/sex/group
- Anaesthetic used: Yes; isoflurane

For additional details regarding observations and examinations of reproductive parameters refer to Section 7.8.1 (DI.K1.28Day.Gav.1Gen.R.D-19570-1422.KD) and 7.8.2 (DI.K1.D.RD.Dev.R.D-19570-1422.KD)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)

Gross examinations were performed on all adult rats. The gross examination included examination of external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The uteri of all cohabited P1 females were examined for the presence and number of implantation sites and corpora lutea. Reproductive organs from P1 rats with suspected impaired reproductive performance (e.g., failure to mate, conceive, sire, or deliver healthy offspring; or effects in oestrous cyclicity or sperm parameters) were evaluated from all groups. Relative organ weights (percent of terminal body weight; percent of brain weight) were calculated. Male accessory sex gland weights were calculated by addition of the prostate and seminal vesicles with coagulating glands (including fluids). Terminal body weights determined just prior to necropsy were used in the assessment of organ weight changes. All tissues collected from animals in the high dose and control groups were further processed and examined microscopically. Gross lesions from animals in the low and intermediate dose groups were also processed to slides and evaluated microscopically. In addition, the following organs with potential treatment-related changes in 28-day, 28-day recovery, and/or P1 animals were processed from the low and intermediate dose groups and examined as follows:
• Noses from all 28-day, 28-day recovery, and P1 male and female groups were processed and examined microscopically; however, for the 28-day recovery group, only noses from the control and high dose group animals were examined.
• Liver and kidneys from all 28-day, 28-day recovery, and P1 male groups were processed and examined microscopically, however liver was only examined from the control and high dose animals.
Statistics:
See Table 4. Significance was judged at p < 0.05. Separate analyses were performed on the parental data collected for each sex. For litter parameters, the proportion of affected pups per litter or the litter mean was used as the experimental unit for statistical evaluation. For each parameter analysed with a trend test the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. For litter parameters, the proportion of affected foetuses per litter or the litter mean was used as the experimental unit for statistical evaluation.

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):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One female from the 10 mg/kg/day group in the reproductive subset was euthanized prior to scheduled sacrifice, on study day 41 (GD 8). Microscopically it was determined that the cause of moribundity for this animal was malignant lymphoma, which was not considered to be related to test-substance administration.

FOOD EFFICIENCY
In male rats dosed with 200 mg/kg/day of the test substance, there was a statistically significant decrease in mean food efficiency over test day intervals 0-7 (11% lower than controls), 7-14 (19% lower than controls), and 14-21 (13% lower than controls). However, since there were no effects on mean daily food consumption or mean body weights, and since the effects were similar to controls over test day intervals 21-28 and over the cumulative period of test substance administration (test days 0-28), the differences in food efficiency values were considered spurious.

HAEMATOLOGY
Mean corpuscular haemoglobin (MCH) was minimally lower (statistically significant) in male rats dosed with 200 mg/kg/day (3.6% below the control). However, there were no correlative changes in mean corpuscular haemoglobin concentration, the more biologically relevant index of red cell haemoglobin, and no changes in any red cell mass parameter (haemoglobin, red cell count, and haematocrit). Therefore, the minimal decrease in MCH in male rats was considered to be unrelated to treatment and nonadverse.
Prothrombin time (PT) was statistically significantly longer (6.7% longer than the control) in female rats dosed with 50 mg/kg/day following 4 weeks of treatment, but a similar difference in PT was not observed at higher doses, and therefore was considered to be unrelated to treatment and nonadverse.

CLINICAL CHEMISTRY
Aspartate aminotransferase (AST) was statistically significantly higher in male rats dosed with 200 mg/kg/day. The difference in AST in this group relative to controls was minimal (less that 1.5 fold higher than control), was not associated with changes in related clinical chemistry parameters (alanine aminotransferase, sorbitol dehydrogenase), and was not associated with correlative test substance-related microscopic changes (such as liver or muscle injury) in this group. In addition, no statistically significant changes in AST were observed in female rats at this dose level. Therefore, the higher mean AST value in the 200 mg/kg/day male group was likely unrelated to treatment and was considered to be nonadverse.
Similarly, minimally higher potassium (K) in female rats dosed with 50 mg/kg/day following 4 weeks of treatment was not considered to be treatment related because it did not occur in a dose-related manner.

NEUROBEHAVIOUR
During the baseline evaluation, one observation of significantly higher (p<0.05) duration of movement occurred during the 4th 10-minute interval, when females in the 200 mg/kg/day group were compared with controls. This difference, observed prior to test-substance administration, was not relevant to the interpretation of the study data.
During the week 4 evaluation, males in the 200 mg/kg/day group were observed with significantly lower (p<0.05) duration of movement in the 2nd and 4th 10-minute intervals, while females in the same group were observed with significantly higher (p<0.05) duration of movement in the 1st 10-minute interval, compared with controls. The differences were transient and were not reflected in significant differences in total duration of movement over 60 minutes. The lower values in males were not consistent with the higher values in females, and there were no corroborative differences in the number of ambulatory movements or any other neurobehavioral parameter. Therefore, the differences in duration of movement were not considered test substance-related.

ORGAN WEIGHTS
28-Day Subset: Statistically significant organ weight changes were an increase in heart percent of body weight (8.9%) in the 200 mg/kg/day males and an increase in spleen absolute (17.1%) and percent of body weight (16.8%) in females at 50 mg/kg/day. Neither of these weight differences was considered treatment-related as the heart weight difference was minimal, with no associated microscopic findings. The spleen weight differences were also relatively minimal and were not dose-related.
Reproductive Subset: Treatment-related organ weight changes occurred in the kidneys of males; a statistically significant increase in mean absolute (15%), percent of brain (15%), and percent of body weight (18.6%) was present at 200 mg/kg/day. This change was associated with an increase in hyaline droplets in kidney tubules. Other statistically significant organ weight differences in males occurred in the spleen and included decreased absolute and percent of brain weight at 200 mg/kg/day (both parameters decreased 14.5%) and decreased percent of both brain and body weight at 50 mg/kg/day (decreased by 12.1% and 14.6%, respectively). These changes in spleen weights were relatively minimal and were not associated with a microscopic finding, and thus were not considered treatment-related. In females, ovary weight parameters were increased at 200 mg/kg/day (to approximately 15% above controls) This difference in ovary weights was not associated with microscopic findings and was not considered treatment related. Other statistically significant organ weight differences in females included an increase in heart percent of brain weight (11%) at 50 mg/kg/day and a decrease in kidney percent of brain weight (9.6%) at 10 mg/kg/day. Neither of these changes were considered treatment-related as they were minimal and were not dose-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
28-Day Subset: In the kidneys of males, there was an increase in hyaline droplets in cortical tubules (primarily the proximal tubules) of mild severity at 200 mg/kg/day and minimal severity at 50 mg/kg/day. The droplets were variably sized, round, densely eosinophilic, and were consistent with alpha2u globulin, a urinary protein. Accumulation of hyaline droplets composed of alpha2u globin in renal tubules is a common finding in male rats following exposure to many different compounds and is not considered relevant to human risk assessment. Minimal to mild degeneration/atrophy of olfactory epithelium was present in the nose of 2/5 animals in both the male and female groups administered 200 mg/kg/day and in 1/5 females in the 50 mg/kg/day group.
Reproductive Subset: In the kidneys of P1 males, there was an increase in hyaline droplets in cortical tubules (primarily the proximal tubules) of mild severity at 200 mg/kg/day and minimal severity at 50 mg/kg/day. These changes were consistent with those observed in the 28-day subset. Degeneration/atrophy of nasal olfactory epithelium was present in 3/10 and 2/10 rats in the 50 and 200 mg/kg/day female groups, respectively. Lesions were minimal to mild except for one of the two affected rats in the 200 mg/kg/day group in which the lesions was severe. Although not clearly dose-related across the affected groups, these changes were consistent with those observed in the animals from the 28-day subset and were likely test substance-related. In males, single occurrences of usually minimal olfactory degeneration/atrophy were observed in all groups, including controls, and were thus considered to be incidental findings.
28-Day With Recovery Subset: Mild chronic progressive nephropathy was noted in two males at 200 mg/kg/day; one of these also had minimal granular casts. This slight increase in severity of chronic progressive nephropathy, as well as the presence of casts in one animal, may be residual lesions indicative of cell damage secondary to the hyaline droplets. Nasal olfactory lesions observed at the end of the dosing period were reversible, as there were no test substance-related changes in the nose in the 200 mg/kg/day recovery group. Minimal focal degeneration/atrophy of olfactory epithelium was noted in a single male control and in a single female 200 mg/kg/day animal. These were considered a normal background finding.

Effect levels

Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: Histopathologic effects in the noses and kidneys of male rats at 200 mg/kg/day and in the noses of female rats at 50 and 200 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (systemic toxicity)= 10 mg/kg/day
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The objective of the study was to evaluate the potential repeated-dose and reproductive toxicity of the test substance when administered by gavage to male and female rats during premating, cohabitation, gestation, and lactation up through day 3. Groups of 20 Crl:CD(SD) rats were dosed with vehicle (deionised water) containing 0, 10, 50, or 200 mg/kg/day test substance during the treatment period. The dose solutions were sampled and analysed during the course of the study and were confirmed to be at targeted concentrations and stable under the conditions of the study.
Following 28 days of dosing, five rats/sex/group were selected and euthanized to collect data to satisfy the 28-day subchronic toxicity, clinical pathology, and histologic endpoints. Five remaining rats/sex/group were selected for a 28-day recovery group, and test substance administration was ceased, on test day 29, for the remainder of the recovery period. For the remaining ten rats/sex/group, test substance administration was continued until one day prior to scheduled euthanasia. These rats were co-housed within their respective treatment groups to produce F1 litters. Dams were allowed to deliver and rear their offspring until postnatal day 4.
Clinical observations, body weight, and food consumption were determined at least weekly throughout the study. Clinical pathology evaluations were conducted for five rats/sex/group from the reproductive subset and five rats/sex/group from the 28-day with recovery subset (prior to cessation of test substance administration) on test day 29-30 (haematology and clinical chemistry), and then again at terminal sacrifice for rats from the reproductive subset (coagulation; test day 45 for males and 57-61 for females). Rats selected for haematology and clinical chemistry evaluations were fasted overnight prior to blood collection. Clinical pathology evaluations were not conducted on the 28-day with recovery rats at the end of the recovery period since there were no effects during treatment. Neurobehavioral evaluations were conducted during acclimation (baseline) and on test days 27-28 on five rats/sex/group from the reproductive subset and five rats/sex/group from the 28-day with recovery subset. Litter examinations (live, dead, or missing pups, individual pup weights, clinical observations) were determined at birth and on postnatal day (PND) 4. Gross postmortem examinations were performed on selected rats and selected organs were weighed and/or retained for histopathological examination. There were no test substance related deaths or clinical observations noted during the study. No test substance-related effects on body weight or nutritional parameters were observed. There were no test substance-related effects on neurobehavioral endpoints, clinical pathology, reproductive performance, or effects on offspring at any concentration tested.
Test substance-related histopathologic changes occurred at 50 and 200 mg/kg/day, and consisted of effects in the kidneys of male rats and in the nose of both male and female rats. Increased hyaline droplets consistent with alpha2u globulin were noted at 50 and 200 mg/kg/day in the cortical tubules of males after 28 days of administration, and were also observed in the P1 males after 45 days of test substance administration. Increased hyaline droplet accumulation was not present in the recovery males. However, the presence of a slight increase in severity of chronic progressive nephropathy in two recovery males, one with minimal granular casts, suggests that the increased droplets at 200 mg/kg/day were adverse. Kidney effects at 50 mg/kg/day were considered non-adverse. Low incidences of degeneration/atrophy of nasal olfactory epithelium were present in the 28-day and/or P1 groups at 200 mg/kg/day in male rats, and at 50 and 200 mg/kg/day in female rats. Olfactory lesions were reversible as no test substance-related changes were present in the nose of the 200 mg/kg/day recovery groups.
Under the conditions of this study, the no-observed adverse effect level (NOAEL) for systemic toxicity was 10 mg/kg/day based on histopathologic effects that were observed in the noses and kidneys of male rats at 200 mg/kg/day and in the noses of female rats at 50 and 200 mg/kg/day.