Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Remarks:
Reproductive organs toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 May 2016 - 24 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of treatment, the animals were 5 to 6 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 252 g (range: 198 g to 314 g) and the females had a mean body weight of 189 g (range: 163 g to 210 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes from the same sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 11 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 13 June 2016 to 24 October 2016.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: suspension in the vehicle.

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in the vehicle
- Concentration in vehicle: 10, 50 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
A sample was taken from control and test item dose formulations and analyzed using the validated method.
The test item concentrations in the administered dose formulations analyzed in Weeks 1, 4, 8 and 11 remained within an acceptable range of -10.2% to -4.1% when compared to the nominal values (± 15% of the nominal concentrations).
No test item was detected in the control dose formulation.

Duration of treatment / exposure:
13 weeks followed by a 6-week treatment-free period
Frequency of treatment:
Daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 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:
15 animals per sex per dose for groups 1 and 4 (0 and 1000 mg/kg/day)
10 animals per sex group 2 (50 mg/kg/day)
11 males and 10 females for group 3 (250 mg/kg/day).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous study performed in the same species.
In this study, male and female Sprague-Dawley rats received m/p DIOL daily by gavage at 100, 300 or 1000 mg/kg/day for 4 weeks.
At 1000 mg/kg/day, in-life observations were limited to ptyalism in all animals and transient piloerection and/or staggering gait in 3/5 males and 1/5 females. There were no toxicologically relevant effects on functional observation battery, motor activity, body weight, body weight change, food consumption, hematology or macroscopic post-mortem parameters. Blood biochemical findings (higher cholesterol level in both sexes, higher protein and albumin levels and lower bile acid level in males) were considered to be non-adverse as well as organ weight changes (higher liver and kidney weights in both sexes and higher adrenal weights in males) and microscopic findings (hepatocellular hypertrophy, renal tubular dilatation and renal infiltrate of mononuclear cells in both sexes and renal hyaline droplets and increased vaculation in adrenals in males). Adverse increased tubular basophilia suggesting previous chronic cell damage and increased cell turnover were noted in kidneys of 3/5 males. Therefore, the No Observed Adverse Effect Level (NOAEL) was considered to be 300 mg/kg/day in males and 1000 mg/kg/day in females.
Thus, based on these available data, the selected dose levels of the study were: 50, 250 and 1000 mg/kg/day.

- Rationale for animal assignment: computerized randomization procedure.
Positive control:
no (not required)
Observations and examinations performed and frequency:
MORTALITY / MORBIDITY: Yes
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment and treatment-free periods.

CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then at least once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule:
The body weight of each animal was recorded:
- once before the beginning of the treatment period,
- on the first day of treatment,
- at least once a week until the end of the study.

FOOD CONSUMPTION: Yes
- Time schedule: the quantity of food consumed by the animals in each cage was recorded at least once a week, over a 7-day period, during the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule:
Ophthalmological examinations were performed:
- on all animals, before the beginning of the treatment period,
- on all control- and high-dose animals on one occasion at the end of the treatment period.
As no relevant changes were detected in the treated animals at the end of the treatment period, these examinations were not extended to groups 2 and 3 and to recovery animals at the end of the treatment-free period.

NEUROBEHAVIOURAL EXAMINATION: Yes
Time schedule: all main animals were evaluated once in Week 12.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

HEMATOLOGY: Yes
Peripheral blood
- Time schedule: The parameters were determined for all surviving animals sacrificed at the end of the treatment period.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

CLINICAL CHEMISTRY: Yes
- Time schedule: The parameters were determined for all surviving animals sacrificed at the end of the treatment period.
In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.

URINALYSIS: Yes
- Time schedule: the parameters were determined for all surviving animals sacrificed at the end of the treatment period.
In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the treatment or treatment-free period, after at least 14 hours fasting, all surviving animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and after removal of the left epididymis for the sperm analysis (see § Seminology) were euthanized by exsanguination.
A complete macroscopic post-mortem examination was performed on all animals.

ORGAN WEIGHTS: Yes
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment-free period.
The ratio of organ weight to body weight (recorded immediately after euthanasia) was calculated.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for all animals euthanized prematurely,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period,
- immunostained kidneys from control and high-dose males (groups 1 and 4) sacrificed at the end of the treatment period in order to characterize the tubular hyaline droplets.
In addition testicular staging was performed for control and high-dose males (main animals from groups 1 and 4).
Other examinations:
BONE MARROW
- Time schedule: two bone marrow smears were prepared from the femoral bone (at necropsy) of each animal sacrificed on completion of the treatment or treatment-free period.
As no relevant abnormalities were observed during the hematological investigations, the bone marrow differential cell count was not determined and smears were archived.

MONITORING OF ESTROUS CYCLE: Yes
- Time schedule: the estrous cycle stage was determined for each female to be sacrificed at the end of the treatment period, for all females, daily for 21 consecutive days before the end of the treatment period.

THYROID HORMONES: Yes
- Time schedule: an additional blood sample was taken from all surviving animals sacrificed at the end of the treatment and treatment-free periods.
The levels of thyroid hormones T3 and T4 and thyroid stimulating hormone were not determined as there was no indication of an effect on the pituitary-thyroid axis.

SEMINOLOGY: Yes
- Time schedule: at the end of the treatment period.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
cf. Table 1
At 1000 mg/kg/day, 5/15 females had staggering gait for one or 2 days during the first week of the treatment period, together with hypoactivity in two of the females or hypotonia and dyspnea in another one. This last female showed staggering gait, hypoactivity, slight tremors and half-closed eyes a few minutes to 3 hours after administration, and abdominal breathing on several occasions for 1 to 9 days in Weeks 10, 11, 12 and/or 14.
Hyperactivity was noted in 4/15 females from Day 48 to Day 50. Piloerection was observed in 3/15 females from Day 65 to Day 76 or from Day 81 to Day 84 or 85.
These clinical signs were attributed to the test item treatment.

Ptyalism was transiently observed in all control and test item-treated groups in a dose-related manner, from the first week of treatment in group 4 males. This non-adverse finding, commonly reported when a test item is administered by gavage, was attributed to the taste of the formulation.

Reflux at administration noted in 1/10 females given 250 mg/kg/day and in 1/14 males and 2/15 females given 1000 mg/kg/day on one occasion was not considered to be test item treatment related. This sign, commonly observed when a test item is administered by gavage, was considered not to be of toxicological importance.

The other clinical signs recorded during the study, i.e. alopecia, scabs, soiled head, thinning of hair, bent tail, nodosities, chromodacryorrhea, chromorhynorrhea, dyspnea, reddish discharge, loud breathing and/or hypoactivity were of isolated occurrence, observed both in control and test item treated animals and/or with no dose-relationship. They were therefore considered to be unrelated to the test item treatment.

Test item treatment-related clinical signs were no longer observed during the treatment-free period. The piloerection noted in 1/5 control males was considered to be incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Control group, 0 mg/kg/day
One male was sacrificed prematurely for humane reasons on Day 78 (Week 12). Prior to sacrifice, hypoactivity, loss of balance, dyspnea, bent/tilted head, hyperextension of the cervical vertebrae, decreased grasping reflex and half-closed eyes were observed. A body weight loss (-37 g) was recorded between Days 71 and 78. At pathology, brain necrosis was observed.

Intermediate-dose group, 250 mg/kg/day
One male was sacrificed prematurely for humane reasons on Day 5 (Week 1). Prior to sacrifice, the animal was isolated in a cage without sawdust because signs of poor clinical condition were noted (exteriorized penis and swollen penis). Appropriate medical care, as defined by the Veterinarian, was given as follows: the penis was replaced in the sheath without success under isoflurane gas anesthesia after application of ice and vaseline. At pathology, preputial abscess was observed.

High-dose group, 1000 mg/kg/day
One male was sacrificed prematurely for humane reasons on Day 78 (Week 12). Hypoactivity, abdominal and loud breathing, nasal discharge, dyspnea, chromorhynorrhea, soiled mouth and swollen neck were observed on the day of sacrifice. Ptyalism (from Day 8), reflux at administration (on Day 25), chromorrhynorrhea (from Day 49 to Day 52) and alopecia on one forelimb (from Day 71 to Day 75) were also noted. A body weight loss (-47 g) was recorded between Days 71 and 78. At pathology, dosing-related lesions were noted.

These deaths were considered to be unrelated to m/p DIOL.
There were no other unscheduled deaths during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
cf. Table 2
At 1000 mg/kg/day in males, when compared with controls, statistically significantly lower mean body weight gain was recorded throughout the treatment period (-13% vs. controls) leading to a statistically significantly lower mean body weight from Day 36 until the end of the treatment period (-9% vs. controls on Day 91). The differences from controls were particularly relevant over the second month. This effect was attributed to the test item treatment. During the recovery period, a statistically significantly higher mean body weight gain was observed in males previously treated at 1000 mg/kg/day, leading to a terminal mean body weight similar to the control animals.
In females, an opposite trend (also statistically significant) was observed mainly during the first month (+20%, p<0.01). Consequently, the mean body weight was not affected at the end of the treatment period. The variations observed in the body weight evolution of females during the whole study period were considered to be of no toxicological importance.

At 250 mg/kg/day in males, when compared with controls, lower mean body weight gain was occasionally observed over the first month (-15 to -18% vs. controls in Weeks 1, 2 and 3). No relevant effects were noted on the body weight or body weight gain of females during the study period.
At 50 mg/kg/day, no relevant effects were noted on body weight or body weight gain in males or females during the study period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No relevant effects were observed on food consumption in test item-treated animals during the treatment period.
At 250 mg/kg/day in males and at 1000 mg/kg/day in males and females, statistically significantly higher mean food consumption, when compared with controls, was noted on rare occasions. As these differences were of isolated occurrence, they were considered to be of no toxicological importance.
Mean food consumption was slightly lower in previously test item-treated females during the treatment-free period. This was due to the high mean food intake recorded in controls between Days 105 and 112 (29.1 g/animal/day) that most probably resulted from spillage in the second cage (33.7 g/animal/day).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings were observed at the end of the treatment period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item treatment-related effects on the hematology parameters were observed at the end of the treatment period.

Statistically significant differences between controls and males given 1000 mg/kg/day (i.e. white blood cell count: 13.80 G/L vs. 17.59 G/L in controls; lymphocyte count: 10.29 G/L vs. 14.24 G/L in controls) were considered to be of no toxicological importance as they resulted from mean control values in the upper range of historical control data and since values remained within the range of the historical control data (white blood cell count: 6.08 to 17.50 G/L and lymphocyte count: 5.12 to 15.30 G/L).
The other statistically significant differences between control and test item-treated animals, namely in red blood cell and reticulocyte counts (females given 50 mg/kg/day) were considered to be of no toxicological importance as they were of low magnitude and noted with no dose-relationship.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
cf. Table 3
At 1000 mg/kg/day and when compared with mean control values, statistically significant blood biochemistry changes of minor toxicological importance and still within the range of the Historical Control Data for most of them, were observed:
- lower mean potassium and chloride levels in males (-9% and -1%, respectively),
- higher mean urea level in males (+15%),
- higher mean creatinine level in females (+9%),
- lower mean glucose level in females (-17%),
- higher mean cholesterol level in females (+41%) along with higher mean triglyceride level (+114%).

At 250 mg/kg/day and when compared with mean control values, statistically significantly, higher mean cholesterol level, but within the range of the Historical Control Data, was also noted in females (+23%).

These findings were noted in a context of histological changes in the liver of both sexes and in the kidneys of males.

The other statistically significant differences observed between control and test item-treated animals, namely in the glucose (males at 250 mg/kg/day) and total bilirubin (females at 1000 mg/kg/day) levels were within the range of physiological values or incidental and not test item-related, as they were noted with no dose-relationship or without any relationship to microscopic findings.
Test item treatment-related effects on the blood biochemistry parameters were no longer observed at the end of the treatment-free period. The higher mean albumin level observed in previously test item treated males was considered not to be test item treatment-related as this difference was not observed at the end of the treatment period and was within the range of physiological values.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
cf. Table 4
At 1000 mg/kg/day and when compared with mean control values, statistically significant urinalysis changes of minor toxicological importance were observed::
- lower mean pH value was recorded in males. This value was very close to the lower limit of the Historical Control Data,
- higher mean volume was also observed in females and higher mean specific gravity was noted in males. These differences did not correlate with changes in specific gravity in females.

At 250 mg/kg/day and when compared with mean control values, statistically significant lower mean pH values, but within the range of the Historical Control Data, was also noted in males.

Test item treatment-related effects on the urinary parameters were no longer observed at the end of the treatment-free period.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
cf. Table 5
Higher mean numbers of horizontal movements and rearing were noted in males given 50 or 1000 mg/kg/day and were considered to be of no toxicological importance as they were of low magnitude and not dose related.
No differences from controls were noted in the motor activity of test item-treated females.

Higher mean landing foot splay values were recorded in females given 50, 250 or 1000 mg/kg/day (79, 95 and 80 mm vs. 68 mm in controls). This finding was considered not to be test item-related as it was of low magnitude and not dose-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
cf. Table 6
At the end of the treatment period
The following treatment-related increases in absolute or relative weights were present at the end of the treatment period:
- adrenals (males and females receiving 1000 mg/kg/day, correlating with cortical vacuolation at microscopic examination in males),
- kidney (males receiving 250 mg/kg/day or above, correlating with hyaline droplets),
- liver (males receiving 250 mg/kg/day or above and females receiving 1000 mg/kg/day, correlating with hepatocyte hypertrophy).

At the end of the treatment-free period
There were no organ weight changes related to administration of the test item at the end of the treatment-free period.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period
There were no m/p DIOL-related macroscopic observations.
None of the macroscopic findings were m/p DIOL effects because they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.

At the end of the treatment-free period
There were no m/p DIOL-related macroscopic observations.
None of the macroscopic findings were m/p DIOL effects because they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
cf. Table 7
At the end of the treatment period
The following treatment-related microscopic observations were present at the end of the treatment period:
- liver (centrilobular hepatocyte hypertrophy, single cell necrosis, and bile duct fibrosis in males and females receiving 250 mg/kg/day m/p DIOL or above),
- adrenals (cortical vacuolation in males receiving 1000 mg/kg/day),
- kidneys (hyaline droplets in cortical tubular epithelium, tubular basophilia, hyaline casts in males receiving 50 mg/kg/day or above). Hyaline droplets in the kidney were shown to be a2u-globulin protein by immunochemistry,
- female reproductive tract (ovarian luteal cysts, decrease in incidence of dilated uterine lumen, and vaginal mucification, squamous metaplasia and/or epithelial vacuolation in females receiving 250 mg/kg/day or above).
Hepatocyte hypertrophy was characterised by diffuse and/or periportal enlargement of hepatocytes. Single cell necrosis was characterised by cells with condensed, deeply eosinophilic cytoplasm in contrast to adjacent normal hepatocytes and pyknotic or fragmented nuclear material, without associated inflammation.
Adrenal vacuolation was characterised by macrovesiculation and microvesiculation of cortical cells.
Hyaline droplets are a normal occurrence in younger male rats and were only diagnosed where there was an increase relative to controls. These droplets were shown to be a2u-globulin protein by immunochemical staining. Tubular basophilia and hyaline casts are minor degenerative changes known to be secondary to a2u-globulin protein nephropathy.
Ovarian luteal cysts are larger than a normal corpus luteum and lined by one to several layers of large polygonal luteinized cells.
The remaining microscopic findings were not treatment-related because they were consistent with spontaneously occurring findings described in the literature, the findings were distributed randomly among groups, or their appearance was similar to findings found in controls.

At the end of the treatment-free period
Complete reversibility from microscopic observations seen at main necropsy occurred in liver (hypertrophy, bile duct fibrosis), kidneys (hyaline casts), and female reproductive tract (all findings). Partial reversibility occurred in the liver (single cell necrosis), adrenals (cortical vacuolation), kidneys (hyaline droplets, tubular basophilia). Treatment-related microscopic observations at recovery necropsy that were not present at main necropsy occurred in kidneys (granular casts).
Granular casts in the kidneys are minor degenerative changes known to be secondary to a2u-globulin protein nephropathy.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cycle
cf Table 8
There were no statistically significant test item-related effects on mean length of the estrous cycle or the mean number of cycles. A trend towards an increase in the mean length of diestrus was observed in females given 1000 mg/kg/day at the end of the treatment period. This was due to three females for which the diestrus period represented 7 to 14 days and lasted between 5 to 9 consecutive days.
This was considered to be of minor importance in the absence of statistical significance and in absence of adverse pathology findings in the female reproductive tract, and since these variations were no longer observed at the end of the treatment-free period.

Seminology
cf Table 9
No test item treatment-related effects were noted on testicular sperm count or on epididymal sperm count, motility or morphology.
The slight, not statistically significant, in the testicular sperm count recorded in males given 250 or 1000 mg/kg/day resulted from higher mean control values due to two isolated males. Therefore these differences were considered not to be treatment-related.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: excluding the adverse lesions seen in male kidneys (a2u-globulin protein nephropathy-related findings) which are specific to male rats and with no relevance for human risk assessment
Critical effects observed:
no

Table 1: Clinical signs

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Hyperactivity

-

-

-

-

-

-

-

4

Hypoactivity

-

1

-

-

-

-

-

3

Hypotonia

-

-

-

-

-

-

-

1

Piloerection

-

-

-

-

-

-

-

3

Half-closed eyes

-

-

-

-

-

-

    -

    1

Staggering gait

-

-

-

-

-

-

-

5

Tremors

-

-

-

-

-

-

-

1

Abdominal or loud breathing

-

1

-

-

-

-

-

1

Dyspnea

-

1

-

-

-

-

-

1

Total affected animals

0/14

1/10

0/10

0/14

0/15

0/10

0/10

8/15

Ptyalism

7

10

10

14

1

5

9

15

Reflux at administration

-

-

-

1

-

-

1

2

Total affected animals

7/14

10/10

10/10

14/14

1/15

5/10

9/10

15/15

-: no clinical signs.

Table 2: Body weight

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Treatment period

Mean BW gain Days 1/29

+228

+208

+200*

+209

+75

+76

+81

+90**

Mean BW gain Days 29/57

+98

+95

+99

+79**

+37

+40

+33

+38

Mean BW gain Days 57/91

+58

+52

+49

+50

+21

+18

+17

+18

Mean BW gain Days 1/91

+383

+355

+349

+333*

+133

+134

+131

+146*

% from controls

-

-7

-9

-13

-

+1

-2

+10

Mean body weight on Day 1

251

252

267

243

187

188

192

189

Mean body weight on Day 91

633

606

616

576**

320

322

323

335

% from controls

-

-4

-3

-9

-

+1

+1

+5

Treatment-free period

Mean BW gain Days 91/133

+26

-

-

+53*

+18

-

-

+2*

Mean BW on Day 133

665

-

-

636

351

-

-

345

% from controls

-

-

-

-4

-

-

-

-2

Statistically significant from controls: *: p<0.05, **: p<0.01, -: not applicable.

Table 3: Blood biochemistry

Sex

Male

Dose-level (mg/kg/day)

0

50

250

1000

End of treatment period

Potassium (mmol/L)

4.00

(3.56-4.65)

4.06

3.82

3.63**

Chloride (mmol/L)

104.3

(103.9-105.8)

104.7

103.5

102.8**

Glucose (mmol/L)

6.47

(5.17-9.06)

7.02

5.42*

5.82

Urea (mmol/L)

3.9

(2.6-4.1)

4.0

4.3

4.5**

End of treatment-free period

 

 

 

 

Potassium (mmol/L)

3.85

/

/

3.95

Chloride (mmol/L)

105.3

/

/

105.4

Glucose (mmol/L)

6.43

/

/

6.40

Urea (mmol/L)

5.5

/

/

5.3

Albumin (g/L)

35

 

 

37*

/: not applicable; statistically significantfrom controls:*: p < 0.05 and**: p < 0.01.

( ): minimum-maximum values from Historical Control Data (HCD).

In bold characters, the statistical changes which are outside the range of the HCD.

 

Sex

Female

Dose-level (mg/kg/day)

0

50

250

1000

End of treatment period

Glucose (mmol/L)

7.02

(5.19-8.04)

6.42

6.49

5.80*

Creatinine (µmol/L)

38.14

(28.28-36.87)

37.61

37.04

41.56*

Cholesterol (mmol/L)

1.90

(1.18-2.69)

2.21

2.34**

2.67**

Triglycerides (mmol/L)

0.43

(0.21-0.69)

0.47

0.52

0.92**

End of treatment-free period

 

 

 

 

Glucose (mmol/L)

6.54

/

/

5.98

Creatinine (µmol/L)

42.22

/

/

41.41

Cholesterol (mmol/L)

2.64

/

/

2.78

Triglycerides (mmol/L)

0.50

/

/

0.46

/: not applicable; statistically significantfrom controls:*: p < 0.05 and**: p < 0.01.

( ): minimum-maximum values from Historical Control Data (HCD).

In bold characters, the statistical changes which are outside the range of the HCD.

Table 5: Motor activity

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Horizontal movements

391

591

413

458

605

601

583

642

% from controls

-

+51

+6

+17

-

-1

-4

+6

Rearing

126

165

109

148

201

223

198

226

% from controls

-

+31

-13

+17

-

+11

-1

+12

-: not applicable.

 

Table 6: Organ weights

Mean absolute and relative organ weight changes
at the end of the treatment period

 

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

50

250

1000

50

250

1000

Exam. animals / Num. of animals

 10/10

10/11

9/10

10/10

10/10

10/10

- Final body weight

-4

-3

-11*

+2

+2

+4

- Adrenal glands 

. absolute

-6

+10

+18

+4

+6

+22**

. relative

-2

+14

+31*

+2

+4

+18*

- Kidneys  

. absolute

-1

+9*

+15**

+2

+3

+7*

. relative

+4

+13**

+28**

0

+1

+3

- Liver  

. absolute

-3

+9

+28**

+7

+10

+29**

. relative

+1

+13*

+43**

+4

+8

+24**

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Table 7: Microscopic examination

Incidence and severity of test item-related microscopic findings

at the end of the treatment period

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Liver

 

 

 

 

 

 

 

 

- No. Examined

10

10

11

10

10

10

10

10

- Hypertrophy; hepatocyte

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

6

7

-

-

-

4

Slight (grade 2)

-

-

-

2

-

-

-

-

- Necrosis; single cell

 

 

 

 

 

 

 

 

Minimal (grade 1)

1

1

7

7

1

-

1

2

- Fibrosis; bile duct

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

-

1

3

-

-

-

-

Adrenals

 

 

 

 

 

 

 

 

- No. Examined

10

10

11

10

10

1

0

10

- Vacuolation; cortex

 

 

 

 

 

 

 

 

Minimal (grade 1)

9

8

9

4

-

-

 

-

Slight (grade 2)

-

1

-

5

-

-

 

-

Kidneys

 

 

 

 

 

 

 

 

- No. Examined

10

10

11

10

10

0

0

10

- Hyaline droplets; tubular epithel.

 

 

 

 

 

 

 

 

Minimal (grade 1)

-

7

6

5

-

 

 

-

Slight (grade 2)

-

-

3

4

-

 

 

-

Moderate (grade3)

-

-

-

1

-

 

 

-

- Basophilia; tubule

 

 

 

 

 

 

 

 

Minimal (grade 1)

3

5

8

2

1

 

 

2

Slight (grade 2)

-

-

-

5

-

 

 

-

Moderate (grade3)

-

-

-

1

-

 

 

-

- Cast; hyaline

 

 

 

 

 

 

 

 

Minimal (grade 1)

1

2

4

5

-

 

 

-

-: no findings.

Bold:treatment-related.

(continued)

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Kidneys Immunostain

 

 

 

 

 

 

 

 

- No. Examined

9

0

0

10

0

0

0

0

- Pigment; brown

 

 

 

 

 

 

 

 

Minimal (grade 1)

9

 

 

-

 

 

 

 

Slight (grade 2)

-

 

 

3

 

 

 

 

Moderate (grade3)

-

 

 

7

 

 

 

 

Ovaries

 

 

 

 

 

 

 

 

- No. Examined

0

0

0

0

10

10

10

10

- Cyst; luteal

 

 

 

 

-

-

1

2

Uterus

 

 

 

 

 

 

 

 

- No. Examined

0

0

0

0

10

10

10

10

- Dilated lumen

 

 

 

 

 

 

 

 

Minimal (grade 1)

 

 

 

 

1

-

2

-

Slight (grade 2)

 

 

 

 

4

3

3

-

Moderate (grade3)

 

 

 

 

-

2

-

1

Vagina

 

 

 

 

 

 

 

 

- No. Examined

0

0

0

0

10

10

10

10

- Mucification; epithelium

 

 

 

 

 

 

 

 

Marked (grade 4)

 

 

 

 

-

-

-

1

- Metaplasia; squamous

 

 

 

 

 

 

 

 

Moderate (grade 3)

 

 

 

 

-

-

-

1

- Vacuolation; epithelium

 

 

 

 

 

 

 

 

Minimal (grade 2)

 

 

 

 

-

-

-

1

-: no findings.

Bold:treatment-related.

Incidence and severity of test item-related microscopic findings
at the end of the treatment-free period

 

Sex

Male

Female

Group

1

4

1

4

Dose-level (mg/kg/day)

0

1000

0

1000

Liver

 

 

 

 

- No. Examined

5

5

5

5

- Necrosis; single cell

 

 

 

 

Minimal (grade 1)

2

4

-

-

Adrenals

 

 

 

 

- No. Examined

5

5

0

0

- Vacuolation; cortex

 

 

 

 

Minimal (grade 1)

1

1

 

 

Slight (grade 2)

-

1

 

 

Kidneys

 

 

 

 

- No. Examined

5

5

0

0

- Hyaline droplets; tubular epithel.

 

 

 

 

Minimal (grade 1)

-

3

 

 

- Basophilia; tubule

 

 

 

 

Minimal (grade 1)

-

3

 

 

- Cast; hyaline

 

 

 

 

Minimal (grade 1)

3

3

 

 

- Cast; granular

 

 

 

 

Minimal (grade 1)

-

3

 

 

-: no findings      Bold= Treatment-related.

Table 8: Estrous cycle data

 

Dose-level (mg/kg/day)

0

50

250

1000

Treatment period

 

 

 

 

Number of cycles

3.9

4.1

4.2

3.7

Cycle length (days)

4.2

4.0

4.0

4.4

Number of females having a mean
average cycle of 4-5 days

10

6

9

6

Number of days of diestrus

6.0

6.4

5.1

6.5

Treatment-free period

 

 

 

 

Number of cycles

2.6

na

na

2.2

Cycle length (days)

4.1

na

na

4.3

Number of females having a mean
average cycle of 4-5 days

3

na

na

5

Number of days of diestrus

3.8

na

na

3.6

na: not applicable.

Table 9: Sperm analysis data

 

Dose-level (mg/kg/day)

0

50

250

1000

% of motile epididymal sperm

92.8

96.1

95.8

96.7

% of morphologically normal epididymal sperm

95.7

/

/

96.4

Mean number of epididymal sperm (106/cauda)

171.2

153.3

153.8

160.2

Mean number of epididymal sperm (106/g cauda)

500.8

479.0

458.6

479.2

Mean number of testicular sperm heads (106/g testis)

107.2

118.5

96.6

95.9

Daily sperm production rate (106/g testis/day)

17.6

19.4

15.9

15.7

/: not applicable.

 

Conclusions:
The toxicity of the test item was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose-levels of 50, 250 or 1000 mg/kg/day for 13 weeks followed by a 6-week treatment-free period.
At 1000 mg/kg/day, non-adverse treatment-related changes were restricted to ptyalism in both sexes, some sporadic clinical signs (staggering gait, hypoactivity, hypotonia, half-closed eyes, dyspnea, abdominal breathing, slight tremors, hyperactivity and/or piloerection) in several females and lower body weight and body weight gain in males. A slight increase in the duration of diestrus in females, lower potassium and chloride levels, and higher urea and cholesterol levels in males, lower glucose levels and higher creatinine levels in females, lower urinary pH values in both sexes and higher urine density gravity in males were also noted and considered non-adverse. At pathology, administration of m/p DIOL resulted in treatment-related findings in the liver (centrilobular hepatocyte hypertrophy, single cell necrosis, and bile duct fibrosis in both sexes), adrenals (cortical vacuolation in males), kidneys (adverse a2u-globulin protein nephropathy in males).
At 250 mg/kg/day, the treatment produced only non-adverse in-life (ptyalism in both sexes and slightly lower body weight gain in males throughout the first 3 weeks of the treatment period) and blood biochemistry changes (higher cholesterol and triglyceride levels in females). At pathology, administration of m/p DIOL resulted in treatment-related findings in the liver (centrilobular hepatocyte hypertrophy, single cell necrosis, and bile duct fibrosis in both sexes) and kidneys (adverse a2u-globulin protein nephropathy in males).
At 50 mg/kg/day, only ptyalism was noted. At pathology, treatment-related findings consisted of adverse a2-u-globulin nephropathy in males.
Executive summary:

The potential toxicity of m/p-DIOL was evaluated following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 6-week treatment-free period in order to evaluate the reversibility of any findings. This GLP study was carried out according to OECD test guideline No. 408 (21 September 1998). One group of 15 male and 15 female Sprague-Dawley rats was treated daily by the oral route (gavage) with the test item at the dose-level of 1000 mg/kg/day (group 4) for 13 weeks. Two other groups were treated with the test item at dose-levels of 50 (group 2: 10 males and 10 females) or 250 (group 3: 11 males and 10 females) mg/kg/day. One control group of 15 males and 15 females received the vehicle only (corn oil) under the same experimental conditions, and acted as a control group (group 1). A constant dosage volume of 5 mL/kg/day was used. At the end of the treatment period, the animals were sacrificed, except for the first five surviving group 1 and 4 animals per sex, which were kept for a 6-week treatment-free period.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 11 were determined using a High Performance Liquid Chromatography with UV detection analytical method. The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12. Body weight was recorded pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly. Ophthalmological examinations were performed on all animals before the beginning of the study and on control and high-dose animals at the end of the treatment period (Week 13). Hematology, blood biochemistry and urinary investigations were performed at the end of the treatment period (Week 13). Blood biochemistry and urinalysis parameters were also determined at the end of treatment-free period (Week 19). Additional blood samples were collected in Weeks 13 and 19 for possible analysis of thyroid hormone levels. The estrous cycle was determined over 21 or 14 consecutive days for all females at the end of the treatment or treatment-free period, respectively. At the end of the treatment period, seminology investigations (count and motility) were performed on all males, and morphology was determined in control and high-dose males. On completion of the treatment or treatment-free period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination (including testicular staging) was performed on designated tissues from control and high-dose animals sacrificed at the end of the treatment period and from animals that were euthanized prematurely, on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period and on kidney slides immunostained with an antibody for a2u-globulinprotein from all control and high-dose males sacrificed at the end of the treatment period. The liver from low- and intermediate-dose main animals and from recovery animals, adrenals and kidneys from low- and intermediate-dose main males and from recovery males, and ovaries, vagina and uterus from low- and intermediate-dose main females and from recovery females were also microscopically examined as changes were revealed in these organs at the end of the treatment period.

Actual concentrations of the test item in the dose formulations administered to the animals during the study remained within an acceptable range (-13.3% to -4.1%) compared to the nominal concentrations. There were no test item-related unscheduled deaths in any group. At 1000 mg/kg/day, non-adverse sporadic clinical signs were noted in 8/15 females (i.e. staggering gait, hypoactivity, hypotonia, half-closed eyes, dyspnea, abdominal breathing, slight tremors, hyperactivity and/or piloerection) during the treatment period. Ptyalism was observed with a dose-related incidence at all dose-levels. This sign, commonly observed when a test item is administered by gavage, was considered not to be adverse. The FOB results were unaffected by the test item treatment. Lower body weight gain was recorded in males given 250 mg/kg/day over the first 3 weeks of treatment (-15 to -18% vs. control) and in males given 1000 mg/kg/day throughout the treatment period (-13% vs. controls), leading to a minimally lower body weight on completion of the treatment period at 1000 mg/kg/day (-9% vs. controls). Reversibility of these differences was noted at the end of the treatment-free period. Food consumption was not affected by the test item treatment. No ophthalmology findings were observed at the end of the treatment period. A slight increase in the length of diestrus was observed in females given 1000 mg/kg/day at the end of the treatment period (6.5 days vs.6.0 days in controls). Variations were no longer observed at the end of the treatment-free period. The epididymal sperm motility and morphology, and the spermatozoa count were unaffected by the test item treatment. At hematology investigations, no test item-related effects were observed at the end of the treatment period. At blood biochemistry investigations, higher cholesterol was noted in females from 250 mg/kg/day. At the dose-level of 1000 mg/kg/day, lower glucose and higher creatinine levels were also noted in females, and lower potassium and chloride levels and higher urea level were recorded in males. All these changes were considered to be of minor toxicological importance as they were of minimal magnitude and within the range of the Historical Control Data for most of them. At urinary investigations, lower pH values were noted in males from 250 mg/kg/day and in females at 1000 mg/kg/day. At the dose-level of 1000 mg/kg/day, higher volume was recorded in females and higher density was noted in males. All these changes were considered to be of minor toxicological importance as they were isolated and/or within the range of the Historical Control Data. Reversibility of these blood biochemistry and urinalysis differences was noted at the end of the treatment-free period. There was a treatment-related statistically significant increase in absolute or relative weight of the following organs at the end of the treatment period: adrenals (males and females receiving 1000 mg/kg/day, correlating with cortical vacuolation at microscopic examination in males), kidney (males receiving 250 mg/kg/day or above, correlating with hyaline droplets), liver (males receiving 250 mg/kg/day or above and females receiving 1000 mg/kg/day, correlating with hepatocyte hypertrophy). There were no organ weight changes related to administration of the test item at the end of the treatment-free period. There were no test item-related necropsy findings. Test item related microscopic observations at the end of the treatment period consisted of the following: liver centrilobular hepatocyte hypertrophy, single cell necrosis, and bile duct fibrosis (males and females receiving 250 mg/kg/day m/p DIOL or above), adrenal cortical vacuolation (males receiving 1000 mg/kg/day), kidney hyaline droplets, shown to be a2u-globulin protein, in cortical tubular epithelium, tubular basophilia, hyaline casts (males receiving 50 mg/kg/day or above), female reproductive tract findings (ovarian luteal cysts, decrease in incidence of dilated uterine lumen, and vaginal mucification, squamous metaplasia and epithelial vacuolation in females receiving 250 mg/kg/day or above). Complete reversibility from microscopic observations seen at main necropsy occurred in liver (hypertrophy, bile duct fibrosis), kidneys (hyaline casts), and female reproductive tract (all findings). Partial reversibility occurred in the liver (single cell necrosis), adrenals (cortical vacuolation), kidneys (hyaline droplets, tubular basophilia). m/p DIOL related microscopic observations at recovery necropsy that were not present at main necropsy occurred in kidneys (granular casts). No microscopic changes were adverse except kidneys findings in males but which were without significance to humans. 

Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 1000 mg/kg/day in females and males if we exclude the adverse lesions seen in male kidneys (a2u-globulin protein nephropathy-related findings) which are specific to male rats and with no relevance for human risk assessment.

 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Duration of treatment / exposure:
13 weeks followed by a 6-week treatment-free period
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Control animals:
yes, concurrent vehicle

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Remarks:
Reproductive organs toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Applicant's summary and conclusion