Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC
- Age at study initiation: approximately 63 days
- Weight at study initiation: male 310-396g, female 212-225g
- Fasting period before study: No
- Housing: Individually in wire mesh cages. Follwoing positive evidence of mating females transferred to plastic maternity cages with nesting material and bedding
- Diet : Certified Rodent LabDiet 5002 (PMI Nutrition International LLC), ad libitum (except during fasting prior to blood collection for clinical pathology for males as part of toxicological assessment)
- Water: Reverse osmosis-purified (on-site) water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 8 July to 29 August 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 10, 30, 120 mg/mL
- Amount of vehicle (if gavage): 5mL/kg
- Lot/batch no. (if required): ZT1301, 2AD0465, 2AE0415
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until positive evidence of mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidenceof mating were palced in plastic maternity cages.
- After successful mating each pregnant female was caged in a plastic maternity cages with nesting material and bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and resuspension homogeneity of the test substance in formulation following 10 days of refrigerated storage at concentrations of 1 and 200 mg/mL were established in a previous study so stability and resuspension homogeneity analyses were not conducted on this study.

Samples for homogeneity and concentration determinations were collected from the top, middle, and bottom strata of the first dosing formulations prepared during the study (study week 0), including the control group. In addition, samples for concentration analyses were collected from the middle stratum of each test substance formulation prepared during study weeks 1, 2, 3, and 7. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored refrigerated (2°C - 8°C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography method with flame ionization detection
Duration of treatment / exposure:
Males dosed during study days 0-30 (total of 31 doses)
Females with viable pups were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-47 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 40 or 52 doses. Females with total litter loss received 38 or 40 doses.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 12.5 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous 28-day toxicity study in which severe toxicity was noted at 1000 mg/kg/day, including mortality, moribundity, adverse clinical signs, mean body weight losses, and reduced food consumption during the first week of treatment, which resulted in early termination of the 1000 mg/kg/day group on study day 7 and the subsequent early termination of the study on study day 13

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and approximately 1 hour after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males weekly. Females weekly until positive evidence of mating then gestational days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4

FOOD CONSUMPTION:
Males - weekly
Females - Weekly until positive evidence of mating then gestational days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.
- Food consumption not recorded for paired animals during mating period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animals/day and g food/kg body weight/day: Yes


OTHER:
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After up to 31 doses
- Maternal animals: Lactation day 4 (after 39-47 doses)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively
Postmortem examinations (offspring):
None - discarded without examination
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, pre coital intervals, and FOB data values were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth, and postnatal survival were subjected to the Kruskal Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Reproductive indices:
Male and female fertility, male and female copulation index.
Number of former implantation sites and corpora lutea.
Number of pups/litter size
Offspring viability indices:
Mean litter size, postnatal survival between birth and postnatal day 0 or 4, postnatal survival for all other intervals

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings of clear material around the mouth were noted in the 150 and 600 mg/kg/day groups in a dose related manner at approximately 1 hour following dose administration. In addition, red material around the mouth was noted for the 600 mg/kg/day group at approximately 1 hour following dose administration and salivation prior to dosing was noted in this group . The aforementioned clinical findings generally occurred throughout the treatment period and were considered test substance related. Other clinical findings noted in the test substance treated groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All males and females survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related, significant (p<0.01) mean body weight loss during study days 0-7 and lower (not statistically significant) mean body weight gain during study days 7-13 were noted for males in the 600 mg/kg/day group. As a result, mean body weight gains in this group were significantly (p<0.01) lower than the control group when the pre-mating (study days 0-13) and entire treatment (study days 0-27) periods were evaluated and mean body weights in this group were 10.3% to 12.3% lower than the control group during study days 7-30; the differences in mean body weight were significant (p<0.01 or p<0.05). Mean body weight gains in this group were similar to the control group during study days 13-30. Mean body weights and body weight gains for males in the 50 and 150 mg/kg/day groups were unaffected by test substance administration throughout the treatment period. Differences from the control group were slight and not statistically significant.

Mean body weights and body weight gains for treated females were unaffected by test substance administration throughout the pre-mating period, gestation and lactation periods. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption, evaluated as g/animal/day and g/kg/day, for males in the 600 mg/kg/day group was significantly (p<0.01 or p<0.05) lower than the control group during study days 0-7, corresponding to a mean body weight loss noted for these males during this period. Mean food consumption for these males was generally similar to the control group throughout the remainder of the treatment and post-treatment evaluation periods. Mean food consumption for males in the 50 and 150 mg/kg/day groups was unaffected by test substance administration throughout the treatment period. Differences from the control group were slight and not statistically significant.

Mean food consumption for treated females was unaffected by test substance administration throughout the pre-mating period, gestation and lactation periods. Differences from the control group were slight.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related alterations in hematology parameters included significantly (p<0.05) lower mean hemoglobin concentration and lower mean corpuscular hemoglobin concentration (MCHC) in the 600 mg/kg/day group males. In addition, significantly (p<0.01 or p<0.05) lower mean hemoglobin concentration, hematocrit, mean corpuscular hemoglobin (MCH), and MCHC were observed in the 600 mg/kg/day group females. All changes were of small magnitude and were not associated with gross observations, organ weight changes, or microscopic observations, and were considered nonadverse. The prothrombin time was lower in the 600 mg/kg/day group males. This change was in a direction of no known toxicologic significance.
Test substance-related changes in hematology and coagulation parameters persisted at the recovery necropsy in the 600 mg/kg/day group males and females. Significantly (p<0.01 or p<0.05) lower mean hemoglobin concentration, lower mean hematocrit, lower MCH, lower MCHC, higher mean absolute reticulocyte count, higher mean percent reticulocytes, and higher mean red cell distribution width (RDW) were observed in the 600 mg/kg/day group males at the recovery necropsy. Significantly (p<0.01 or p<0.05) higher mean hematocrit, lower MCHC, lower mean absolute monocyte count, and lower mean hemoglobin distribution width (HDW) were observed in the 600 mg/kg/day group females. The changes were of small magnitude, not associated with gross observations, organ weight changes, or microscopic observations, and were considered nonadverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related changes in serum chemistry values occurred in the 600 mg/kg/day group males and females. Significantly (p<0.01 or p<0.05) higher mean serum total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), and cholesterol levels were observed in the 600 mg/kg/day group males. Significantly (p<0.01) higher mean serum globulins, total protein, GGT, and cholesterol levels were observed in the 600 mg/kg/day group females. The changes in bilirubin, ALT, AST, and GGT levels were associated with absolute and relative liver weight changes and microscopic observations in the liver, and were considered adverse.
There were no other test substance-related effects on serum chemistry parameters. However, significantly (p<0.01) lower mean serum alkaline phosphatase (ALP) was observed in the 600 mg/kg/day group females. This change was in a direction of no known toxicological significance.
At the recovery necropsy, significant (p<0.05 or p<0.01) differences were noted when the control and test substance-treated groups were compared. Higher mean serum albumin, higher mean serum A/G ratio, lower mean serum creatinine, lower glucose levels, and lower AST levels were observed in the 600 mg/kg/day group males. Higher serum ALP level was observed in the 600 mg/kg/day group females. Changes were of small magnitude, the values fell within the WIL Research historical control database reference range, and the changes were considered a result of individual animal variation rather than test substance administration and were indicative of recovery.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Home cage, handling, open field, sensory, neuromuscular and physiological parameters were unaffected by test substance administration.
Significant (p<0.001), test substance-related increases in mean cumulative total and ambulatory counts were noted for males in the 600 mg/kg/day group. Males in this group exhibited slower habituation over the entire test session. Locomotor activity patterns (total activity as well as ambulatory activity counts) for males in the 50 and 150 mg/kg/day groups and toxicology phase females at all dosage levels were unaffected by test substance administration when evaluated at study week 4; values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51 60 minutes) and the overall 60 minute test session values were comparable to the concurrent control values and/or the WIL historical control data, with the following exception. A significant (p≤0.045) increase in mean total counts during 51-60 minutes and decrease in mean ambulatory counts during 21-30 minutes were noted for females in the 600 mg/kg/day group; however, these results were transient and discordant. Other differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred for males in the 50 and 150 mg/kg/day groups and toxicology phase females at all dosage levels when evaluated at study week 4.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were noted in the liver of the 600 mg/kg/day group males and females and the kidneys, adrenals, and pituitary of the 600 mg/kg/day group males.

Minimal to mild hepatocellular hypertrophy was observed in the liver of all dose groups. In the males, the distribution was diffuse. A centrilobular pattern was apparent in the females. In addition to hepatocellular hypertrophy, increased numbers of bile duct profiles were observed in the 600 mg/kg/day group males and the 150 and 600 mg/kg/day group females (bile duct hyperplasia). In the 600 mg/kg/day group only, the bile ducts often contained brown pigment and were surrounded by few concentric layers of fibrous connective tissue (peribiliary fibrosis). Under polarised light some pigment accumulations show birefringence, but this finding was not consistent in size or between animals. Microscopic observations in the liver correlated to the higher absolute liver weight and higher liver weight relative to body weight and brain weight in the males and females. Hepatocellular hypertrophy was considered adaptive and nonadverse. Biliary hyperplasia with peribiliary fibrosis and brown pigment were considered adverse in the 600 mg/kg/day group males and females.

Minimal to mild increased concentrations of eosinophilic proteinaceous intracytoplasmic and intraluminal droplets (hyaline droplets) were observed in the proximal convoluted tubules of the kidneys in treated males. Hyaline droplets are considered a normal finding in male rats; however, the aggregation and condensation pattern of the droplets was more prominent in the test substance-treated rats. This progressed to hyaline droplet nephropathy in 1 of 4 in the 50 mg/kg/day group males, 4 of 7 in the 150 mg/kg/day group males and 3 of the 7 males in the 600 mg/kg/day group with hyaline droplets. Hyaline droplet nephropathy was characterized by dilated tubules with flattened epithelium in the inner stripe of the outer medulla. Tubules contained lightly eosinophilic granular cell debris (granular casts). The combination of granular casts and hyaline droplets was characteristic of hyaline droplet nephropathy (Hard, 2008). Immunohistochemical staining for alpha-2u globulin of the kidneys of males was negative. Hyaline droplet nephropathy was considered an adverse finding in the males in all dose groups.

Minimal to mild adrenal cortical atrophy was observed in treated males. Atrophy was characterized by decreased overall size. Adrenal cortical atrophy correlated to the lower absolute adrenal gland weight and lower adrenal gland relative to brain weight observed at the primary necropsy in the 600 mg/kg/day group males. This finding was not associated with gross observations and was considered nonadverse.

Minimal to mild vacuolation of the pituitary was observed in treated males. Pituitary vacuolation was not associated with gross observation or organ weight changes, and was considered nonadverse.

Moderate decreased corpora lutea were observed in the ovaries in one 600 mg/kg/day group female. This finding only occurred in 1 female, can occur sporadically in rats, and the relationship to test substance administration was unlikely.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. Males that did not sire a litter numbered 1, 0, 1, and 2 in the control, 50, 150, and 600 mg/kg/day groups, respectively. Females that had evidence of mating but did not deliver numbered 1, 0, 0, and 1 in the same respective groups. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value; the difference from the control group was slight and not statistically significant.
Mean gestation lengths in treated groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive Tox
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The general physical condition of F1 pups in the 50 and 150 mg/kg/day groups were unaffected by test substance administration. Pups that were found dead in the 50 and 150 mg/kg/day groups numbered 7 and 2, respectively. One, 2, and 0 pups in the control, 50, and 150 mg/kg/day groups, respectively, were missing and presumed to have been cannibalized.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Postnatal survival in the 600 mg/kg/day group was lower than the control group during PND 0-1 and 1-4 and from birth to PND 4. Although not statistically significantly different from the concurrent control group, the values of postnatal survival were below the minimum mean values in the WIL historical control data. These results were primarily due to 2 total litter losses noted on PND 1 and 3, respectively, and were considered test substance-related and adverse. Postnatal survival, the number of pups born, live litter size, and percentage of males at birth in the 50 and 150 mg/kg/day groups were unaffected by test substance administration.
Higher numbers of pups found dead (19 pups) and missing (7 pups) were noted in the 600 mg/kg/day group compared to the control group (11 pups found dead and 1 pup missing). This was primarily due to 2 test substance related total litter losses. In addition, increased incidences of pups with cool and pale bodies were noted.
The numbers of pups (litters) found dead during PND 0-4 numbered 11(3), 7(4), 2(2), and 19(4) in the control, 50, 150, and 600 mg/kg/day groups, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower F1 pup birth weights were noted for male and female pups in the 600 mg/kg/day group. Slightly lower mean pup body weight gains were noted for these pups during PND 1-4, resulting in mean pup body weights that were 9.4% (males) and 7.0% (females) lower than the control group on PND 4. Although the mean body weight gain for females in the 600 mg/kg/day group (2.8 g) was higher than that noted in the 150 mg/kg/day group (2.7 g), the value at 600 mg/kg/day was slightly skewed by 1 litter with a high mean body weight gain; with this litter excluded, mean body weight gain during PND 1-4 for females in the 600 mg/kg/day group was 2.4 g. These results were considered test substance-related and adverse.
Mean male and female pup body weights and body weight changes in the 50 and 150 mg/kg/day groups were unaffected by test substance administration during PND 1-4.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One pup in the 150 mg/kg bw/day group had the malformation ectopia cordis; however, this finding was not noted in the 600 mg/kg bw/day group. In addition, one pup in the control group had the variation anophthalmia. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

As a result of the lower number of implantation sites noted for females in the 600 mg/kg/day group the mean number of pups born and live litter size in this group were lower (not statistically significant) than the control group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Total litter loses, effects on postnatal survival, pup body weights and pup clinical findings at 600 mg/kg/day.

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1 - Reproductive performance

 

Dosage Level (mg/kg/day)

WIL HCa

Parameter

0

50

150

600

Mean (Range)

Male Mating Index

100.0

100.0

90.0

90.0

96.8 (84.0-100.0)

Female Mating Index

100.0

100.0

90.0

90.0

98.2 (86.7-100.0)

Male Fertility Index

90.0

100.0

90.0

80.0

91.1 (60.0-100.0)

Female Fertility Index

90.0

100.0

90.0

80.0

93.2 (60.0-100.0)

Male Copulation Index

90.0

100.0

100.0

88.9

94.4 (71.4-100.0)

Female Conception Index

90.0

100.0

100.0

88.9

95.0 (65.2-100.0)

Pre-Coital Interval (days)

3.1

2.4

3.3

2.3

2.9 (1.8-4.8)

a= WIL historical control data
None significantly different from control group.

Table 2 - Organ Weights

Test Substance-Related Organ Weight Changes at Necropsy

Parameter

Direction and magnitude of change

Dosage level (mg/kg/day)

Sex

 

 

 

 

Liver

  Absolute

  Relative to body weight

  Relative to brain weight

 

  Absolute

  Relative to body weight

  Relative to brain weight

 

↑18.6%*/21.2%**/40.5%**

↑11.1%*/18.7%**/61.9%**

↑14.8%/22.0%**/46.0%**

 

↑15.2%*/30.6%**

↑13.8%**/40.6%**

↑14.5%**/33.0%**

 

50/150/600

50/150/600

50/150/600

 

150/600

150/600

150/600

 

M

M

M

 

F

F

F

 

Adrenal Gland

  Absolute

  Relative to brain weight

 

↓25.9%**

↓23.6%**

 

600

600

 

M

M

* =   Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

Table 3 - Microscopic Findings

Incidence of Selected Histopathologic Findings at the Primary Necropsy

 

Males

Females

Dosage (mg/kg/day):

0

50

150

600

0

50

150

600

 

 

 

 

 

 

 

 

 

Livera

10

10

10

10

10

10

10

10

  Fibrosis, Peribiliary

0

0

0

3

0

0

0

1

      Minimal

-

-

-

1

-

-

-

1

      Mild

-

-

-

2

-

-

-

0

  Hyperplasia, Bile Duct

0

0

0

4

0

0

2

1

      Minimal

-

-

-

1

-

-

2

0

      Mild

-

-

-

3

-

-

0

1

  Hypertrophy, Hepatocellular

0

3

8

10

0

6

10

10

      Minimal

-

3

4

2

-

6

10

5

      Mild

-

0

4

8

-

0

0

5

  Pigment, Brown

0

0

0

5

0

0

0

1

      Minimal

-

-

-

3

-

-

-

0

      Mild

-

-

-

2

-

-

-

1

 

 

 

 

 

 

 

 

 

Kidneya

10

10

10

10

10

0

0

10

  Droplet, Hyaline

0

4

7

7

0

-

-

0

      Minimal

-

3

4

3

-

-

-

-

      Mild

 

1

3

4

-

-

-

-

  Nephropathy, Hyaline Droplet

0

1

4

3

0

-

-

0

      Minimal

-

1

3

2

-

-

-

-

      Mild

-

0

1

1

-

-

-

-

 

 

 

 

 

 

 

 

 

Adrenal Cortexa

10

10

10

10

10

0

0

10

  Atrophy

0

1

2

6

0

-

-

0

      Minimal

-

1

2

5

-

-

-

-

      Mild

-

0

0

1

-

-

-

-

 

 

 

 

 

 

 

 

 

Pituitarya

10

10

10

10

10

0

0

10

  Vacuolation, Cytoplasmic

0

4

6

8

0

-

-

0

      Minimal

-

4

6

5

-

-

-

-

      Mild

-

0

0

3

-

-

-

-

a Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
Based on the lack of adverse effects on any reproductive parameter evaluated, a dosage level of 600 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats.
Executive summary:

In a well-conducted, GLP compliant, OECD 422 study (reliability score 1), all animals survived to their scheduled euthanasia. Clinical findings noted in the test substance treated groups included clear material around the mouth noted in a dose-related manner for the 150 and 600 mg/kg/day groups, red material around the mouth for the 600 mg/kg/day group at approximately 1 hour following dose administration, and salivation prior to dosing in this group. These findings were considered test substance-related.

Test substance-related mean body weight losses or reduced mean body weight gains were generally noted for males throughout the treatment period, resulting in reductions in mean body weights. Corresponding reductions in mean food consumption were noted for males in the 600 mg/kg/day group during study days 0-7. Mean body weights, body weight gains, and food consumption for males in the 50 and 150 mg/kg/day groups and for females at all dosage levels throughout the study, were unaffected by test substance administration.

A reduction (not statistically significant) in the mean number of implantation sites was noted in the 600 mg/kg of body weight/day group. This value (13.0 sites) was attributed to 1 female (no. 17852) with only 3 implantation sites that delivered 2 pups; the value for this group with this female excluded (14.4 sites) was similar to the WIL historical control data mean of 14.7 sites. Therefore, these results were not attributed to test substance administration. The mean numbers of implantation sites in the 50 and 150 mg/kg of body weight/day groups and mean numbers of unaccounted-for sites and corpora lutea in the 50, 150, and 600 mg/kg of body weight/day groups were unaffected by test substance administration.

Test substance-related organ weight alteration at necropsy included higher absolute and relative (to final body weight and brain weight) liver weights for treated males and females in the 150 and 600 mg/kg/day groups and lower absolute and relative to brain weight adrenal gland weights for males in the 600 mg/kg/day group. Test substance-related macroscopic findings were limited to pale kidneys for 1 male in the 600 mg/kg/day group; this finding corresponded to microscopic findings of hyaline droplet nephropathy for this male.

Bile duct hyperplasia, peribiliary fibrosis, and brown pigment in the bile duct were noted microscopically for males and females in the 600 mg/kg/day group; bile duct hyperplasia was also noted for females in the 150 mg/kg/day group and were considered adverse. Examination of the brown pigment by polarized light revealed red birefringence with “Maltese cross” formation, consistent with porphyrin pigment.  In addition, hepatocellular hypertrophy was noted in the liver of males and females in the 50, 150, and 600 mg/kg/day groups at the primary necropsy; this finding was considered nonadverse. 

 

Microscopic findings of hyaline droplets (nonadverse) were noted in the kidney for males at all dosage levels at the primary necropsy. This finding progressed to hyaline droplet nephropathy (adverse) at all dosage levels. Immunohistochemistry for alpha-2u globulins was positive in the male rats with changes in distribution and morphology of the positive staining consistent with alpha-2u globulin hyaline droplet nephropathy. The hyaline droplet nephropathy persisted at the recovery necropsy. Alpha-2u globulin nephropathy is a male rat specific finding and renal effects induced in the male rats are unlikely to occur in humans.Adrenal cortical atrophy and cytoplasmic vacuolation of the pituitary were noted for males at all dosage levels at the primary necropsy; these findings were considered nonadverse. The adrenal cortical atrophy corresponded to lower absolute and relative adrenal gland weights for males in the 600 mg/kg/day group at the primary necropsy. The cytoplasmic vacuolation observed in the pituitary at the primary necropsy wasobserved at the recovery necropsy with decreased incidence and severity, indicating a trend toward recovery. Adrenal cortical changes were not observed at the recovery necropsy.