Reaction mass of (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene and (2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene and (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

Repeated dose toxicity: oral

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: see 'Remark'

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. Although data provided have a report year after 2008, the study was performed to fulfill needs required by government regulators and/or for product stewardship purposes. DuPont’s stewardship principle states that “We will adhere to the highest standards for the safe operation of facilities and the protection of our environment, our employees, our customers and the people of the communities in which we do business”. The study was carried out in accordance with our internal Product Stewardship standard which is part of the American Chemical Council’s “Responsible Care Program”. This study was not performed to fulfill an information requirement under REACH, but since the test data were already available they were provided as part of the REACH submission.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Activated partial thromboplastin time (APTT), and urea were not examined as required by the guideline.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Remarks:

The study was conducted according to guideline in effect at time of study conduct.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Remarks:

The study was conducted according to guideline in effect at time of study conduct.)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene; (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene; (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

EC Number:

700-755-2

Molecular formula:

C8H3F13O

IUPAC Name:

(2E)-1,1,1,2,3,4,5,5,6,6,7,7,7-tridecafluoro-4-methoxyhept-2-ene; (3E)-1,1,1,2,2,3,4,5,6,6,7,7,7-tridecafluoro-5-methoxyhept-3-ene; (3E)-1,1,1,2,2,4,5,5,6,6,7,7,7-tridecafluoro-3-methoxyhept-3-ene

Details on test material:

- Purity: 99.2%

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: group mean was 281.8 to 296.1 g (males); 204.2 to 215.6 g (females)
- Housing: Individually in solid bottom caging with bedding and nestlets as enrichment
- Diet (e.g. ad libitum): ad libitum, except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION: The test substance was dosed undiluted. The amount of test substance each animal received was based on the most recently collected body weight and the density of the test substance. Control animals were dosed with deionized water at the same volume as the high-dose group.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

95 days (males)
96 days (females)

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500, 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

15/sex at 0 and 1000 mg/kg (10/sex/dose for main study; 5/sex/dose for recovery)
10/sex at 250 and 500 mg/kg

Control animals:

other: treated with deionized water

Details on study design:

- Dose selection rationale: In an acute oral toxicity study conducted in rats, no deaths and no clinical signs were observed when the test substance was dosed neat at single oral gavage doses up to 5000 mg/kg. In the repeated dose toxicity study, 4 groups of male and female Crl:CD(SD) rats (10/sex/group) were dosed by oral gavage (neat test substance) for at least 28 days at 0 (deionized water), 250, 500, or 1000 mg/kg/day. The test substance was not able to be solubilised or suspended in any appropriate vehicle. Therefore, it was dosed neat for the repeated dose toxicity study. No test substance-related deaths occurred and there were no test substance-related effects on any of the in-life parameters assessed. There were no adverse, treatment-related changes in haematology, coagulation parameters, or clinical chemistry. A test substance-related increase in absolute and/or relative liver weights was observed in all male and female dose groups. A test substance-related increase in absolute and relative kidney weights was observed in all male dose groups. In 1000 mg/kg/day males, the liver weight increase was associated with a small increase in the incidence of minimal centrilobular hypertrophy, but was not associated with any clinical or microscopic evidence of liver toxicity. No correlative clinical or microscopic pathology was associated with liver weight effects in other dose groups or the kidney weights in any dose group. The no-observed-adverse-effect level (NOAEL) in the 28-day study was 1000 mg/kg/day. Dosages for the present study, 0, 250, 500 and 1000 mg/kg/day, were selected based on the results of the acute oral toxicity study, the 28-day repeated dose toxicity study, and the limitations of dose volume when the test substance was dosed neat.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A check for acute clinical signs of toxicity was conducted approximately 2 hours post-dosing. Detailed clinical observations were conducted on Test day 0 and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Test day 0 and weekly thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and Week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Study week 14 (main study) and Study week 18 (recovery)
- Anaesthetic used for blood collection: Yes (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: Study week 14 (main study) and Study week 18 (recovery)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table No. 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Study week 14 (main study) and Study week 18 (recovery)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table No. 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pretest and Week 13
- Dose groups that were examined: all
- Battery of functions tested: Sensory function, grip strength, and motor activity (MA)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 3)
HISTOPATHOLOGY: Yes (see Table 3)

All tissues collected from male and female rats in the control (0 mg/kg/day) and high-dose (1000 mg/kg/day) groups were processed to slides and evaluated microscopically. Gross lesions from all rats were also processed and examined. Based on the microscopic findings and organ weights of the control and high-dose groups, potential target organs were also evaluated for both sexes in the low- and mid-dose groups in the main study and all recovery rats. In this study, male and female livers, kidneys, and adrenal glands were microscopically examined from all rats.

Statistics:

See Table 4 below

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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:
Main Study
There were no test substance-related clinical signs in males and females at any dose level. Clinical signs observed during the test period included hair loss, abnormal gait, dehydration, ear twitch, enopthalmus, misshapen tail, and high posture. None of these clinical signs were considered test substance-related, as there were no dose response, observed in only few animals, few intervals, or incidences were higher in control rats.

Recovery
There were no test substance-related deaths. One control group male was found dead on test day 85. Gross and microscopic pathology revealed the cause of death to be aspiration pneumonia. The remaining rats designated for recovery (4-5/sex/dose) were sacrificed by design approximately one-month after 90-days of dosing (test day 125).

BODY WEIGHT AND WEIGHT GAIN: There were no test substance-related effects on body weight parameters in male and female rats at any dose level in Main Study or Recovery animals. The statistically significant changes noted in the body weight parameters were considered spurious and not test substance-related because: 1) there was no dose response for the changes seen in body weights or body weight gains; 2) changes were not consistent between males and females - decrease in males vs. increase in females compared to controls; and 3) there were no changes noted in the 28-day study with this compound at the same dose levels. Mean body weights in males at 250 mg/kg/day were statistically significantly lower compared to control throughout the exposure period including prior to dosing on day 0. At 500 mg/kg/day, the statistical significance for lower body weights was noted from day 70 to end of the dosing period. However, body weights were not affected in the 1000 mg/kg/day group animals in any of the weekly intervals. Mean body weight on test day 91 in the 250, 500, and 1000 mg/kg/day groups were 85%, 90%, and 93%, respectively, of control. Mean body weights in females at 250 mg/kg/day were statistically significantly higher throughout the exposure period except on days 14 and 21. Body weights were not affected in the 500 or 1000 mg/kg/day group animals in any of the weekly intervals. Mean body weight on test day 91 in the 250, 500, and 1000 mg/kg/day groups were 111%, 103%, and 102%, respectively, of control. Mean body weight gains in males at 250 mg/kg/day were statistically significantly lower in many weekly intervals. Sporadic statistical significance was noted in 500 and 1000 mg/kg/day groups. Overall (day 0-91) mean body weight gains in the 250, 500, and 1000 mg/kg/day groups were 76%, 83%, and 87% (statistically significant), respectively, of control. Mean body weight gains in females at 250 mg/kg/day were generally higher, but statistical significance was not noted in any of the weekly intervals. There were no statistically significant changes in the 500 or 1000 mg/kg/day groups in any of the weekly intervals except days 21-27 and 112-119 at 1000 mg/kg/day. Overall (day 0-91) mean body weight gains in the 250, 500, and 1000 mg/kg/day groups were 123%, 104%, and 98% (not statistically significant), respectively, of control.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): There were no test substance-related effects on food intake parameters in male and female rats at any dose level in Main Study or Recovery animals. The statistically significant changes noted in the food intake parameters were considered spurious and not test substance-related, as they were sporadic or there was no dose dependency. Mean food consumption in males at 250 mg/kg/day were statistically significantly lower compared to control throughout the exposure period except day 42-56. There were no statistically significant changes in the 500 or 1000 mg/kg/day groups except day 14-21. Overall (day 0-91) mean food consumption in the 250, 500, and 1000 mg/kg/day groups were 87%, 95%, and 98%, respectively. There were no statistically significant changes in food consumption in females at any dose level except the increases seen on days 35-42 and 0-91 in the 250 mg/kg/day group. Overall (day 0-91) mean food consumption in the 250, 500, and 1000 mg/kg/day groups were 108%, 102%, and 100%, respectively.

FOOD EFFICIENCY: In males, statistically significant changes in food efficiency were observed sporadically during dosing period and for overall (0-91) dosing period at all dose levels. Overall (day 0-91) mean food efficiency in the 250, 500, and 1000 mg/kg/day groups were 87%, 87%, and 88%, respectively. There were no statistically significant changes in food efficiency in females at any dose level except on days 21-27 and 112-119 in the 1000 mg/kg/day group. Overall (day 0-91) mean food consumption in the 250, 500, and 1000 mg/kg/day groups were 120%, 102%, and 98%, respectively.

HAEMATOLOGY: There were no adverse or test substance-related changes in group mean haematology parameters in male or female rats at test day 95/96. The following statistically significant changes in mean haematology parameters were not considered to be test substance-related or adverse:
• In 1000 mg/kg/day male rats, slightly lower (4% less than controls) haemoglobin (HGB) and higher (7.5% higher than controls) red cell distribution width (RDW) were present at the end of the exposure period. These changes were not associated with changes in red blood cell count (RBC) or hematocrit (HCT), and no histological changes suggestive of an effect on the erythron were observed at any of the doses tested. In addition, there were no test substance-related effects on these parameters in female rats. Therefore, the slight differences in HGB and RDW in 1000 mg/kg/day male rats relative to controls were not considered to be test substance-related.
• Mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were slightly lower (approximately 5% percent or less below controls) in all treated male groups. However, these changes were not associated with correlative changes in red cell mass parameters or with changes in mean corpuscular haemoglobin concentration (MCHC), and were poorly correlated with dose across the groups. Therefore, these slight differences in MCV and MCH were also not considered to be test substance-related.

The following statistically significant changes in group mean haematology parameters at test day 96 (females) or following the 1-month recovery were considered to be unrelated to treatment (and non-adverse) because the change did not occur in a dose-related pattern or occurred only after 1-month of recovery:
• Lower MCHC in the 1000 mg/kg/day male recovery group
• Lower RBC and MCV in the 250 mg/kg/day female group at test day 96
• Lower RBC, HGB and HCT in the 1000 mg/kg/day female recovery group
• Higher RDW and absolute reticulocyte count (ARET) in the 250 and 1000 mg/kg/day female groups at test day 96 (no statistically significant change in these parameters in the 500 mg/kg/day female group at test day 96).

There were no treatment-related changes in coagulation parameters at test day 95 (males) or test day 96 (females). A statistically significant, minimal shorter (6% less than control) prothrombin time (PT) in the 250 mg/kg/day female group at test day 96 was not dose related and thus was considered to be spurious.

CLINICAL CHEMISTRY: Cholesterol (CHOL) was mildly increased in male rats dosed with 500 or 1000 mg/kg/day (46% and 74% above control, respectively) at test day 95. Following 1-month recovery, cholesterol was statistically higher (26% above control) but values were well-within historical control range for similarly aged rats (44-109 mg/kg), and thus the higher cholesterol observed at the end of the exposure period was considered reversible. These changes in cholesterol were considered to be treatment-related but of uncertain biological significance. Similar changes were not observed in female rats and there were no changes in serum triglyceride in any treated male or female groups.

The following statistically significant changes in mean clinical chemistry parameters in male and female rats at test day 95/96 were considered to be unrelated to test substance administration and/or non-adverse:
• Minimally higher total protein (TP), characterized by minimally higher group means for both albumin (ALB) and globulin (GLOB), were present in all treated male groups. These differences did not occur in a dose-related manner across the doses tested. No statistically significant changes in any protein parameters were present in the 1000 mg/kg/day male recovery groups. Therefore, these changes in protein parameters were likely unrelated to treatment and, based on their minimal nature, were considered to be non-adverse.
• Chloride (CL) was slightly lower in male rats dosed with 500 or 1000 mg/kg/day (approximately 2% below control). Individual animal values for CL in these groups were within the historical control range for similarly aged rats (96.4-104.6 mmol/L), while CL values in 3/10 rats in the control group were near or above the historical range. Therefore, this minimal change in chloride was considered to be unrelated to treatment and non-adverse. After 1-month recovery the group mean value for CL in male rats dosed with 1000 mg/kg/day was similar to the control group.
• Creatinine (CREA) was minimally lower in female rats dosed with 1000 mg/kg/day (14% lower than control). This change was not associated with changes in other kidney-related end points in females (BUN, kidney histopathology) and the direction of change (decreased rather than increased), has no toxicological significance. Therefore, this change was considered to be unrelated to treatment and non-adverse. After 1-month recovery the group mean value for CREA in female rats dosed with 1000 mg/kg/day was similar to the control group.
• Potassium (K) was minimally higher in female rats dosed with 1000 mg/kg/day (6% above control). This difference was not associated with changes in other electrolytes and a similar change was not observed in males. Therefore, this minimal change in potassium was considered to be unrelated to treatment and non-adverse. After 1-month recovery the group mean value for K in male rats dosed with 1000 mg/kg/day was similar to the control group.

The following statistically significant changes in group mean clinical chemistry parameters at test day 96 (females) were considered to be unrelated to treatment, as the changes did not occur in a dose-related pattern:
• Higher cholesterol (CHOL) in the 250 mg/kg/day female group.
• Higher globulin (GLOB) in the 250 or 500 mg/kg/day female groups (no statistically significant differences relative to controls in the 1000 mg/kg/day females).

URINALYSIS: Urine micro-total protein was increased in male rats at 250, 500 and 1000 mg/kg (83%, 186% and 253% above the control, respectively). These increases were statistically significant at 500 and 1000 mg/kg/day. Urine micro-total protein values in individual animals were mostly correlated to higher (grade 2) severity grade for chronic progressive nephropathy (CPN) in these groups. Therefore, these changes were considered test substance-related and adverse. Finely granular and coarsely granular casts were also observed, during microscopic examination of the sediment, in all treated male groups. The incidence of granular casts was 0/10, 3/10, 2/10 and 1/10 in male rats at 0, 250, 500 and 1000 mg/kg/day, respectively. Although these incidences were not dose related, they were considered to be test substance-related based on the changes observed in renal histopathology and urine micro-total protein. Changes in these urinalysis parameters were reversible, as values were similar to controls following the one-month recovery period. One animal in the 1000 mg/kg/day recovery group had high urine protein (630 mg/dL) and microscopic observation of granular casts which were not considered to be test substance-related, as this animal had pyelonephritis that was not considered to be test substance-related. In males, group mean urine volume was increased at 500 and 1000 mg/kg/day (variable statistical significance; 71% and 100% above the control, respectively). Likewise, specific gravity was decreased in these same groups (1% below the control). The increased urine volume and decreased specific gravity may also be test substance-related based on the changes noted above. However, in both groups mean values for urine volume and specific gravity were influenced by one rat in each group, whose urine volume was 2- to 8-fold higher than other individual values within each group. For rats in the 500 and 1000 mg/kg/day group, 9/9 and 7/9, respectively, of the remaining individual values for urine volume were within the historical control range (4.1-23 mL) for similarly aged rats. All individual specific gravity values within these groups for were within the historical control range (1.015-1.053) with the exception of the 2 animals previously mentioned. Therefore, relationship to treatment is uncertain for these parameters. After 1-month recovery, mean urine volume and specific gravity were similar to the control mean. There were no test substance-related changes in urinalysis in female rats at any dose.

ORGAN WEIGHTS:
Main Study
After 90 days of dosing, there were increases in mean liver weight parameters in all dose groups (≥250 mg/kg/day) in both sexes. Mean kidney weight parameters were increased in males only at doses ≥500 mg/kg/day. In males, mean absolute liver weights were increased 22%, 29%, and 45% in the 250, 500, and 1000 mg/kg/day dose groups, respectively, as compared to the control value. Mean relative values (% brain weight and % body weight) were also increased at all dose levels, and all differences were statistically significant. In females, mean absolute liver weights were increased 25%, 17%, and 21% in the 250, 500, and 1000 mg/kg/day dose groups, respectively, as compared to the control value. Mean relative values (% brain weight and % body weight) were also increased at all dose levels. All differences were statistically significant except for the relative liver weight (% brain weight) of the 500 mg/kg/day females, which was, nonetheless, increased 11% over the control value. The test substance-related increase in liver weights correlated with an increase in centrilobular hepatocellular hypertrophy in all but the low-dose (250 mg/kg/day) females. This suggests that the increased liver weights were a consequence of the non-adverse pharmacological induction of hepatic enzymes. See Table below for additional information.

In males, kidney weights were considered increased at 500 and 1000 mg/kg/day. Mean absolute kidney weights were increased 10%, and 29% in the 500 and 1000 mg/kg/day dose groups, respectively, as compared to the control value. Mean relative values (% brain weight and % body weight) were also increased at least 10% at these doses, although not all differences were statistically significant. A statistically significant increase in the mean relative kidney weight (% body weight) in the 250 mg/kg/day males was not considered test substance-related because of the large, apparently spurious, decrease in the mean terminal body weight at this dose. In females, mean kidney weight parameters were generally similar in all groups and any differences were not interpreted to be test substance-related. Statistically significant increases in mean kidney weight values in the low- and mid-dose groups lacked a dose response, and, in the low dose rats, reflected a higher mean terminal body weight than in the control rats. The increase in male kidney weights in the 500 and 1000 mg/kg/day dose groups correlated with a slight increase in the severity of microscopic CPN at these dose levels. Considering the hepatocellular hypertrophy observed at these doses, it is also possible that non-adverse renal tubular enzyme induction contributed to the increased kidney weights. Exposure to xenobiotics commonly induces hepatic metabolic enzymes, and less commonly renal tubular metabolic enzymes, in laboratory animals. Although microscopic hypertrophy of hepatocytes or renal tubular epithelium may occur in enzyme induction, small increases in mean organ weight parameters are often not associated with a morphological change. See Table below for additional information.

All other individual and mean organ weight differences were considered to be spurious and unrelated to test substance administration. Several organ weight parameters in the low- and intermediate dose groups, in both sexes, were statistically increased or decreased when compared to the respective control values. However, these differences neither demonstrate a dose response nor had a gross or microscopic correlate that suggested a test substance-related effect. Highly variable, apparently spurious, mean terminal weight values, such as a decrease in the low-dose males and an increase in the low-dose females contributed to highly variable organ weight data. A small (11%), statistically significant, increase in the mean testes weight of the 1000 mg/kg/day males, as compared to the control males, was not considered test substance-related since there was no microscopic correlate and mean relative testes weight values did not demonstrate a dose response.

Recovery

At the recovery sacrifice, there was an increase in mean liver weight parameters in the 1000 mg/kg/day dose group in both sexes. Mean kidney weight parameters were comparable to control group values. In males, the mean absolute liver weight in the 1000 mg/kg/day dose group was 16% greater than the control group value. Mean relative values (% brain weight and % body weight) were also increased, although only the increase in mean % body weight value was statistically significant. In females, the mean absolute liver weight in the 1000 mg/kg/day dose group was 12% greater than the control group value. Mean relative values were similarly increased, although none were statistically significant. The increases in mean liver weight parameters in the high-dose male and female recovery rats were substantially less than those observed in the main study. The decrease in liver weight effect over the one-month recovery period correlates with the lack of hepatocellular hypertrophy in these same rats. See Table below for additional information.

All other individual and mean organ weight differences in the recovery rats were considered to be spurious and unrelated to test substance administration. Specifically, kidney weight parameters in the high-dose males and females were similar to the control values. A 17%, statistically significant, increase in the mean absolute accessory sex organ weight of the 1000 mg/kg/day recovery males, as compared to control, was not interpreted to be test substance-related because this effect was not observed in the main study, and was not associated with any gross or microscopic finding.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Main Study
Test substance-related microscopic findings included minimal to mild, non-adverse hepatocellular hypertrophy in the livers of males (≥250 mg/kg/day) and females (≥500 mg/kg/day) and an adverse increase in the severity of chronic progressive nephropathy (CPN) in the kidneys of males (≥250 mg/kg/day). In males, hepatocellular hypertrophy was observed in all groups receiving the test compound with an incidence of 0/10, 3/10, 6/10, and 8/10 in the 0, 250, 500, and 1000 mg/kg/day dose groups, respectively. The hypertrophy was graded minimal (grade 1 of 4) in all rats, except for one high-dose rat, which was graded mild (grade 2). In females, hepatocellular hypertrophy was observed only in the mid- and high-dose groups. The group incidences were 0/10, 0/10, 1/10, and 4/10 in the 0, 250, 500, and 1000 mg/kg/day dose groups, respectively, and all were graded minimal. The hepatocellular hypertrophy observed in this study was predominantly centrilobular. It was characterized by an increase in the size of hepatocytes due to an increase in cytoplasmic volume. In both sexes, the hypertrophy correlated with the increase in liver weight parameters. At all doses, the hepatocellular hypertrophy was attributed to non-adverse metabolic enzyme induction. See Table below for additional information.
In males, CPN was observed in all groups, including controls, and the incidence was only slightly dose related: 7/10, 8/10, 10/10, and 10/10 in the 0, 250, 500, and 1000 mg/kg/day dose groups, respectively. However, while all lesions were graded minimal to mild, there was a dose-related increase in the incidence of mild lesions (1/10, 4/10, 6/10, and 9/10, respectively). In females, the incidence and severity of CPN were not dose related, and therefore were not considered test substance-related. CPN is a common background lesion in control rats with a higher incidence and severity in males than females. Minimal lesions (grade 1 of 4) generally consist of a few tubules (per kidney section) with regions of regenerative hyperplasia (“basophilic tubules”) and variable thickening of the tubular basement membrane. Mild lesions (grade 2 of 4) include more affected tubules, with the onset of tubular dilatation (with eosinophilic tubular fluid), epithelial degeneration, and atrophy. Moderate to severe lesions (grades 3 and 4, respectively) are characterized by increased tubular degeneration with interstitial fibrosis and inflammation. In the current study, the increased severity of CPN in male rats correlated with an increase in mean urinary total protein at all dose levels. The marginal increase in CPN severity observed in the 250 mg/kg/day males was also considered test substance-related since it correlated with a marginal increase in urine total protein and appeared to represent the lower end of a dose-related trend. The increase in CPN severity was interpreted to be an adverse test substance-related effect. See Table below for additional information.

All other microscopic observations in the 90-day main study were consistent with normal background lesions in rats of this age and strain. The adrenal glands, which were examined at all dose levels in order to clarify the incidence of microvesiculation at the high dose. The microvesiculation was determined not to be test substance-related.

Recovery
There were no test substance-related microscopic findings in the recovery rats. Microscopic evaluation of the recovery rat tissues was limited to the male and female livers, kidneys, and adrenal glands.

Hepatocellular hypertrophy, which was observed in both sexes following the 90-day dosing period, was not observed in any of the 1000 mg/kg/day males or females following the 30-day recovery period. See Table below for additional information.

Chronic progressive nephropathy, which had increased severity in males of all dose groups in the main study, was not considered increased in the 1000 mg/kg/day males or females following the 30-day recovery period. Although the small number of rats in the recovery groups cannot verify complete recovery of the mild CPN observed in 9/10 1000 mg/kg/day males after 90 days of exposure, the low incidence of mild CPN (1/5) indicates that the CPN is reversible. See Table below for additional information.

Effect levels

1

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable

Remarks:

no NOAEL identified

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed at highest dose tested

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 

Main Study: Mean Absolute and Relative Liver Weights in Male and Female Rats
	Group:	1	2	3	4
	Dose (mg/kg/day)	0	250	500	1000
Male (No. of rats)		(10)	(10)	(10)	(10)
Mean final body weight (g)		596.0	500.8*	528.1*	549.1
Liver
absolute weight (g)		15.956	19.424*^a	20.553*^a	23.066*^a
liver weight/brain weight x 100		740.023	892.465*^a	982.239*^a	1039.749*^a
liver weight/body weight x 100		2.668	3.882*^a	3.900*^a	4.196*^a
Female (No. of rats)		(10)	(10)	(10)	(10)
Mean final body weight (g)		278.0	310.5*	288.4	291.1
Liver
absolute weight (g)		7.314	9.127*^a	8.535*^a	8.833*^a
liver weight/brain weight x 100		388.899	461.755*^a	432.315^a	449.899*^a
liver weight/body weight x 100		2.639	2.947*^a	2.953*^a	3.041*^a
^*Statistically significant (Dunnett 2-sided p<0.05), compared to Group 1 (control) value. ^a Testsubstance related differences, compared to Group 1.

 

Main Study: Mean Absolute and Relative Kidney Weights in Male and Female Rats
	Group:	1	2	3	4
	Dose (mg/kg/day)	0	250	500	1000
Male (No. of rats)		(10)	(10)	(10)	(10)
Mean final body weight (g)		596.0	500.8*	528.1*	549.1
Kidneys
absolute weight (g)		3.412	3.460	3.765^a	4.409*^a
weight/brain weight x 100		158.287	158.866	179.136^a	198.344*^a
weight/body weight x 100		0.573	0.694*	0.711*^a	0.801*^a
Female (No. of rats)		(10)	(10)	(10)	(10)
Mean final body weight (g)		278.0	310.5*	288.4	291.1
Kidneys
absolute weight (g)		1.828	2.190*	2.053*	2.017
weight/brain weight x 100		97.287	110.728*	103.992	102.741
weight/body weight x 100		0.660	0.708	0.712	0.695
^*Statistically significant (Dunnett 2-sided p<0.05), compared to Group 1 (control) value. ^a Test substance related differences, compared to Group 1.

 

Main Study: Incidence and Severity of Hepatocellular Hypertrophy in the Livers of Male and Female Rats
	Group:	1	2	3	4
	Dose (mg/kg/day)	0	250	500	1000
Male
Liver (n)		(10)	(10)	(10)	(10)
Hypertrophy, hepatocellular*		0	3^a	6^a	8^a
Minimal		-	3	6	7
Mild		-	-	-	1
Female
Liver (n)		(10)	(10)	(10)	(10)
Hypertrophy, hepatocellular*		0	0	1	4
Minimal		-	-	1	4
^*Hypertrophy, when present, was graded on a 4-point scale: minimal, mild, moderate, severe. (n) number of rats with liver examined. ^a Test substance related differences, compared to Group 1.

 

Main Study: Incidence and Severity of Chronic Progressive Nephropathy (CPN) in the Kidneys of Male and Female Rats
	Group:	1	2	3	4
	Dose (mg/kg/day)	0	250	500	1000
Male
Kidneys (n)		(10)	(10)	(10)	(10)
Chronic Progressive Nephropathy^*		7	8	10	10
Minimal		6	4	4	1
Mild		1	4	6	9
Female
Liver (n)		(10)	(10)	(10)	(10)
Chronic Progressive Nephropathy^*		1	1	2	0
Minimal		1	1	2	0
^*Chronic Progressive Nephropathy (CPN), when present, was graded on a 4-point scale: minimal, mild, moderate, severe. (n) number of rats with kidneys examined. ^a Test substance related differences, compared to Group 1.

 

Recovery: Mean Absolute and Relative Liver Weights in Male and Female Rats
	Group:	1	4
	Dose (mg/kg/day)	0	1000
Male (No. of rats)		(5)	(5)
Mean final body weight (g)		622.3	590.7
Liver
absolute weight (g)		15.001	17.457^a
liver weight/brain weight x 100		685.684	766.643^a
liver weight/body weight x 100		2.413	2.963*^a
Female (No. of rats)		(5)	(5)
Mean final body weight (g)		303.9	304.8
Liver
absolute weight (g)		7.534	8.466^a
liver weight/brain weight x 100		375.832	420.614^a
liver weight/body weight x 100		2.488	2.780^a
^*Statistically significant (Dunnett 2-sided p<0.05), compared to Group 1 (control) value. ^a Test substance related differences, compared to Group 1.

 

Main Study: Incidence and Severity of Hepatocellular Hypertrophy in the Livers of Male and Female Rats
	Group:	1	4
	Dose (mg/kg/day)	0	1000
Male
Liver (n)		(4)	(5)
Hypertrophy, hepatocellular^#		0	0
Female
Liver (n)		(5)	(5)
Hypertrophy, hepatocellular^#		0	0
^#Hepatocellular hypertrophy was not observed in any recovery rats. (n) number of rats with liver examined.

 

Recovery: Incidence and Severity of Chronic Progressive Nephropathy (CPN) in the Kidneys of Male and Female Rats
	Group:	1	4
	Dose (mg/kg/day)	0	1000
Male
Kidneys (n)		(4)	(5)
Chronic Progressive Nephropathy^*		3	4
Minimal		3	3
Mild		0	1
Female
Liver (n)		(5)	(5)
Chronic Progressive Nephropathy^*		0	0
^*Chronic Progressive Nephropathy (CPN), when present, was graded on a 4-point scale: minimal, mild, moderate, severe. (n) number of rats with kidneys examined.

 

Applicant's summary and conclusion

Conclusions:

NOAEL (males): Not determined based on slight increase in severity, but not incidence, of CPN and associated clinical pathology changes at all dose levels.
NOAEL (females) = 1000 mg/kg (highest dose tested)

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

The objective of this study was to assess the potential subchronic toxicity of the test substance in rats. Four groups of young adult male and female Crl:CD(SD) rats (15/sex/group in control and high dose groups and 10/sex/group in the mid- and low-dose groups) were administered by oral gavage daily doses that contained 0, 250, 500 or 1000 mg/kg/day for approximately 90 days. Main study animals (10 rats/sex/group) were sacrificed at the end of dosing period (males on day 95 and females on day 96) and 1-month recovery animals (5/sex in control and high dose groups) were sacrificed 1 month after the end of the treatment (test day 125). Body weights, food consumption, and detailed clinical observations were evaluated weekly until sacrifice. The animals were examined daily after dosing for acute clinical signs of systemic toxicity. Neurobehavioural evaluations were conducted during pretest and weeks 13. Clinical pathology endpoints were evaluated at necropsy. At scheduled necropsy, the rats were sacrificed and given a gross and microscopic pathological examination.

There were no test substance-related effects on any in-life parameters, ophthalmological observations, or neurobehavioural evaluations at any dose level tested in males or females. Test substance-related increase in urine micro-total protein and granular cast were noted in males of all dose groups at the end of the treatment period. Since these changes correlated to chronic progressive nephropathy (CPN), they were considered adverse. Test substance-related increases in cholesterol were observed in the 500 and 1000 mg/kg/day male groups. The changes in urine micro-total protein and cholesterol were reversible following the one-month recovery period. There were no test substance-related clinical pathology changes in female rats at any of the doses evaluated. Test substance-related adverse changes were noted in certain anatomic pathology parameters. Increased liver weights were observed in both sexes at all doses and correlated with minimal to mild centrilobular hepatocellular hypertrophy at all doses except the 250 mg/kg/day females at the end of the treatment period. Liver weight changes and hepatocellular hypertrophy were consistent with metabolic enzyme induction by a xenobiotic. Therefore, these changes were considered test substance-related, but non-adverse. Kidney weights were increased in males at doses ≥500 mg/kg/day. Severity, but not incidence, of CPN was increased in males at all doses, including a marginal effect at 250 mg/kg/day compared to control. The increased incidence of CPN was considered adverse. In females, there were no test substance-related kidney weight changes or CPN. All these effects were reversible following a one-month recovery period.

Under the conditions of the study, the no-observed-adverse-effect-level (NOAEL) for the test substance was not determined for male rats based on a slight increase in severity, but not incidence, of CPN and associated clinical pathology changes at all dose levels. However, the NOAEL for female rats was 1000 mg/kg/day based on the lack of adverse effects on in-life parameters and clinical or anatomic pathology parameters. CPN is a common spontaneous age-related renal disease occurring in high incidence in male rats of strains that are commonly used in preclinical toxicology studies. Although usually considered as a disease of the aging rat, CPN is detected in male rat kidney at least as early as 2 months of age and can confound the interpretation of toxicology studies. Because the biology and pathology of rat CPN are not similar to human nephropathies and there is no clear counterpart of rat CPN in humans, CPN is regarded as a rodent-specific disease. Therefore, chemically induced exacerbation of CPN in a safety evaluation study in rats is not likely relevant to humans.