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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
Study period:
01 Mar 2018 - 16 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Remarks:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar- Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed on August 2019
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in
the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at
appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han), outbred, SPF-Quality
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 d
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single housing
Duration of treatment / exposure:
males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 days after delivery,
up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.
Frequency of treatment:
daily (7d/week)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substance was orally (gavage) applied at concentrations of 0, 557 and 1855 mg/kg body weight per day for 14 days to four animals per test group and sex. No treatment related alterations were observed in any of the observed parameters at 1855 mg/kg body weight (high dose) dose level. Hence, this short-term repeated dose oral toxicity study was performed at similar dose levels.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: First day of treatment (prior to dosing) and weekly thereafter (after dosing)
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Females which delivered on PND 14-16, females which failed to deliver post-coitum Day 25 or 26 (with evidence of mating) or 25 days after the last day of the mating period (without evidence of mating), dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing

- Organs examined:
Selected animals: Tissues identified in table 5 (see any other information on materials and methods) with the exception of animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue,
Females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina,
Females with total litter loss: Mammary gland,
Remaining animals: Gross lesions/masses.

OTHER: Hematology, coagulation, clinical chemistry, thyroid hormones (see any other information on materials and methods)





Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No (exception: if no macroscopic visible implantation sites were present)
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
Gestation index, postimplantation survival index, live birth index, viability index, lactation index (for detailed formulas see "Any other information on materials and methods"
Historical control data:
Historical control data to allow comparison with concurrent controls were provided by the test facility for the following parameters: Maternal body weight gain (period 2015- end 2017), clinical chemistry (period 2015- end 2017), thyroid hormone analyses (period 2015- end 2017), post-implantation survival index (period 2015 - May 2018)


Clinical signs:
no effects observed
Description (incidence and severity):
Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its
time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity
Mortality:
no mortality observed
Description (incidence):
One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was
apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related irregular surface was observed in the (fore)stomach in 2/10 females treated at 375 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the (fore)stomach (2/5females). Squamous cell hyperplasia was present in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.





Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125
and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- No deficiencies in maternal care were observed.
- Litter size was not considered affected by treatment. Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
> 375 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse developmental effects observed up to and including the highest dose tested.
Fetal body weight changes:
not examined
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Dead pubs at the first litter check: 11 in the control group and 1 daed pub in the exposure group 40 mg/kg bw/d
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
One litter each was affected in the control, 125 and 375 mg/kg bw/d group (one dead pub), representing 1, 1.2 and 1% of living pubs
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- The post-implantation survival index, the live birth index, viability index and lactation index were not affected by the treatment.
- No clinical signs occurred among pups that were considered to be related to treatment. For the pup of female no. 71 (375 mg/kg) who was missing on Day 4, less milk in the
stomach and a lean appearance were noted on Day 1. Alopecia of the whole body was noted for several pups of the control, 40 and 375 mg/kg groups (not toxicological relevant as within the range considered normal)
- No macroscopic findings among the pubs
Dose descriptor:
NOAEL
Effect level:
> 375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse developmental effects observed up to and including the highest dose tested.
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1 Developmental indeces

Dose in mg/kg bw/d

0

40

125

375

Post-implantation survival index (%)

92

79

88

85

Live birth index (%)

90

99

100

100

Viability index (%)

99

100

99

99

Lactation index (%)

100

100

100

100

Table 2 Developmental data

Dose in mg/kg bw/d

0

40

125

375

Total number of offspring born

109

84

85

104

Total number of uterine implantation sites

118

106

97

123

Number of live offspring on day 1 after littering

98

83

85

104

Number of live offspring on day 4 (before culling)

97

83

84

103

Number of live offspring on day 4 (after culling)

63

65

56

70

Number of live offspring on day 13 after littering

63

65

56

70

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of developmental toxicity in in maternal animals and their offspring up to and including the tested dose of 375 mg/kg bw/d.

Therefore, the NOAEL for developmental toxicity was set to be at least 375 mg/kg bw/d.
Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

The following developmental parameters were determined: Number of implantation sites, gestation index and duration, parturition and maternal care, live birth index, viability index and lactation index, post-implantation survival index and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Gestation index and duration of gestation were not considered to be affected by treatment. Except for one female of the control group, all pregnant females had live offspring. The gestation indices were 89% for the control and 100% for the 40, 125 and 375 mg/kg groups, respectively. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125 and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range. Litter size was not considered affected by treatment. Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 90%, 99%, 100% and 100% for the control, 40, 125 and 375 mg/kg groups, respectively.

Eleven pups of the control group and one pup at 40 mg/kg were found dead at first litter check. Eight of these pups were of the control group female, which had a total litter loss. No toxicological relevance was attributed to these dead pups since the mortality incidence mainly occurred in the control group and did not show a dose-related trend. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 99%, 100%, 99% and 99% for the control, 40, 125 and 375 mg/kg groups, respectively. Three dead/missing pups were present throughout the study (one pub of the control, 125 and 375 mg/kg bw/d expsoure group), however, the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups. The postnatal pub deveopment was unaffected by the treatment.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no developmental toxicity was observed up to and including the highest tested dose of 375 mg/kg test item due to the abscence of respective adverse effects under the conditions of this study.

Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar - Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliance
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed on August 2019
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han), outbred, SPF-Quality
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 8, 25, 75 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Duration of treatment / exposure:
males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 days after delivery,
up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.
Frequency of treatment:
daily (7d/week)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item.


Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
F0 animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (F0 and pubs)

BODY WEIGHT: Yes
- Time schedule for examinations: F0: First day of treatment (prior to dosing) and weekly thereafter (after dosing); F1: Live pups were weighed individually on PND 1, 4, 7 and 13.
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

HAEMATOLOGY: Yes, F0 animals
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 1 were examined.

COAGULATION
Blood plasma of F0 animals was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 2 were examined.

OTHER:
Functional observational battery (FOB): Males only
- Functional tests were performed on the selected 5 males (F0) during Week 4 of treatment (after dosing, after completion of clinical observations)
- Examined parameters:
• Hearing ability
• Pubillary reflex
• Static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity

Estrous cycle:
- Daily vaginal lavage was performed for all females (F0) beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Female reproduction and delivery data
From the mating period onwards, the following parameters were recorded for each female (F0): male number paired with, mating date, confirmation of pregnancy and delivery day.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care

Thyroid hormones
- Time schedule for collection of blood: All F0 animals on scheduled necropsy, PND 14-16 and PND 4 for 2 pubs per litter
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males only ( mximum of 24 h)
- How many animals: All F0 animals, 2 pubs per litter on PND 4 and PND 4-16
- For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0- males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded
- Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16

OTHER:
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3 and 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 5 for collected tissue)
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected animals: Tissues identified in Text Table 5 (except animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.
Statistics:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Further observed clinical signs affecting the skin/fur (alopecia, scabs), the eye (Chromodacryorrhoea) and breathing (rales) were observed.
Mortality:
no mortality observed
Description (incidence):
One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend.

No treatment-related changes were noted in clinical chemistry parameters in females
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 375 mg/kg/day group males.
There were no other test item-related organ weight changes.
The significant relative prostate gland weight decrease and liver weight increase in the 40 mg/kg/day treated males was considered incidental and not related to treatment in absence of a dose-related trend.

for details see table 6
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related irregular surface was observed in the (fore)stomach in 2/10 males treated at 125 mg/kg/day and in 10/10 males and 2/10 females treated at 375 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the (fore)stomach of males and females and the liver and kidneys of males.

Stomach, squamous cell hyperplasia was present in 2/5 males starting at 125 mg/kg/day up to marked degree and in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach was present in males starting at 125 mg/kg/day at minimal degree.
Stomach, inflammation forestomach was present in males starting at 125 mg/kg/day up to moderate degree.

Liver, hepatocellular hypertrophy was present in males treated at 375 mg/kg/day at minimal degree. This correlated with the increased liver weight. Due to the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities.

Kidneys, an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg/day up to slight degree. The increased hyaline droplet accumulation in the male kidneys was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
- Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.
-Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects up to and including the highest tested dose
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

The various analyses confirmed

- Accuracy: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 6: Mean Percent Liver Weight Differences from Control Groups

Dose level in mg/kg bw/d

Males

Females

40

125

375

40

125

375

LIVER

 

absolute

13

4

31**

-7

-4

1

Relative to bodyweight

9*

5

25**

-6

-1

0

*: P < 0.05, **: P < 0.01

Table 7: Summary Test Item-Realted Microcopic Findings-(fore)stomach

Dose level in mg/kg bw/d

Males

Females

0

40

125

375

0

40

125

375

STOMACHa

5

5

5

10

5

5

5

5

Hyperplasia squamous cell

 

Slight

-

-

-

-

-

-

-

1

Moderate

-

-

2

6

-

-

-

1

Marked

-

-

-

4

-

-

-

-

Ulcer forestomach

 

minimal

-

-

1

2

-

-

-

-

Inflammation forestomach

 

Minimal

-

-

1

6

-

-

-

-

Moderate

-

-

1

-

-

-

-

-

a = Number of tissues examined from each group

Table 8 Summary Test Item-Related Microscopic Findings – Liver and Kidneys – Males

 

Males

Dose level in mg/kg bw/d

0

40

125

375

LIVERa

5

5

5

5

Hepatocellular hypertrophy

 

minimal

-

-

-

4

KIDNEYa

5

5

5

5

Hyaline droplet accumulation

 

minimal

1

1

2

3

slight

-

-

-

2

a= Number of tissues examined from each group

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) were established:
Parental local NOAEL: 40 mg/kg (based on findings in the (fore)stomach)
Parental systemic NOAEL: at least 375 mg/kg due to the absence of adverse toxicity in the study for both sexes.
Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg.

These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females.

Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no systemic no-observed-adverse-effect level (NOAEL) were established under the conditions of this study. The Parental local NOAEL of 40 mg/kg is based on the findings in the (fore)stomach. Due to the absence of systemic toxicity up to and including the highest tested dose of 375 mg/kg bw/d no systemic NOAEL could be derived.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males were 10 weeks old, females were 13 weeks old
- Weight at study initiation: males weighed between 251 and 322 g, females weighed between 198 and 263 g
- Housing:
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type). Pups were housed with the dam.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: ad libitum, Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, Municipal tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): 36 to 60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was selected as vehicle, at request of the Sponsor and based on previously performed studies.
- Concentration in vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Once mating had occurred, the males and females were separated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-62 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
375 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item in rats and in an attempt to produce graded responses to the test item.

- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: No data
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

FUNCTIONAL TESTS
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 9-12). These tests were performed after dosing, after completion of clinical observations and arena observations, if applicable. The following tests were performed: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, spermatogenic profiling
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); Blood samples were collected from two of the surplus pups (if possible from one male and one female pup).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

Clinical Observations: Clinical observations were performed at least once daily for all pups. Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter.
Body Weights: Live pups were weighed individually on PND 1, 4, 7 and 13.
Anogenital Distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/Nipple Retention: All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25 or 26
Without evidence of mating: 25 days after the last day of the mating period
Females with total litter loss: Dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing.

All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

GROSS NECROPSY
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
see table 1 and 2

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
Selected animals: Tissues identified in Table 2 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina
Females with total litter loss: Mammary gland
Remaining animals: Gross lesions/masses

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Animals fasted: only males
- How many animals: 5/sex/group
- Parameters checked: Alanine aminotransferase (ALAT), Glucose, Aspartate aminotransferase (ASAT), Cholesterol, Alkaline Phosphatase (ALP), Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Urea, Calcium, Creatinine, Inorganic Phosphate (Inorg. Phos)
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
Statistics:
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
see table 5
Offspring viability indices:
see table 5

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No treatment-related mortalities occurred. One female of the control group was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to have been unaffected by treatment. Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters. A decreased number of reticulocytes was observed in females at 125 mg/kg bw/day, which was considered unrelated to administration of the test item due to absence of a dose-related trend response. Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend. No treatment-related changes were noted in clinical chemistry parameters in females.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg bw/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
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 item-related microscopic findings after treatment were noted in the (fore)stomach of males and females and the liver and kidneys of males:
Stomach, squamous cell hyperplasia: was present in 2/5 males starting at 125 mg/kg bw/day up to marked degree and in females at 375 mg/kg bw/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach: was present in males starting at 125 mg/kg bw/day at minimal degree.
Stomach, inflammation forestomach: was present in males starting at 125 mg/kg bw/day up to moderate degree.
Liver, hepatocellular hypertrophy: was present in males treated at 375 mg/kg bw/day at minimal degree. This correlated with the increased liver weight.
Kidneys: an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg bw/day up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.



Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating index was not considered to be affected by treatment. The mating indices were 90%, 100%, 90% and 100% for the control, 40, 125 and 375 mg/kg groups, respectively. Precoital time was not considered to be affected by treatment. The mean precoital times were 2.6, 2.9, 2.6 and 4.5 for the control, 40, 125 and 375 mg/kg groups, respectively. The (non-statistically) higher mean precoital time for group 375 mg/kg was considered unrelated to treatment as 8 females were mated within the first three days of mating and the median precoital time was 3 for all dose groups. Number of implantation sites was not considered to be affected by treatment. Fertility index was not considered to be affected by treatment.
The fertility indices were 100%, 90%, 78% and 90% for the control, 40, 125 and 375 mg/kg groups, respectively. A total of 4 females were not pregnant (one at dose 40 and 375 mg/kg and two females at dose 125 mg/kg). Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, as the incidences were within normal limits, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
For the pup of one female in group 375 mg/kg bw/day who was missing on Day 4, less milk in the stomach and a lean appearance were noted on Day 1. Alopecia of the whole body was noted for several pups of the control, 40 and 375 mg/kg groups. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 99%, 100%, 99% and 99% for the control, 40, 125 and 375 mg/kg groups, respectively. One pup of the control group (litter 48) was killed in extremis on Day 1 of lactation. One pup at 125 mg/kg and one pup at 375 mg/kg were missing on PND 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. Litter size was not considered affected by treatment.
Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were not considered to be affected by treatment
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 375 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups. Gestation index and duration of gestation were not considered to be affected by treatment. Except for one female (no. 49) of the control group, all pregnant females had live offspring. The gestation indices were 89% for the control and 100% for the 40, 125 and 375 mg/kg groups, respectively. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125 and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range.Sex ratio was not considered to be affected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion