Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Dec 2019 - 17 Jun 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Dec 2019 - 17 Jun 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
please refer to "Principles of methods if other than guideline" for details
Principles of method if other than guideline:
Deviations to OECD 422 (2016): no functional observations and motor acitivity measurements were performed at the end of treatment for female animals, for one male, the testis and epididymides were directly placed in formalin instead of first modified Davidson's fixative for a minimum of 24 h; no food consumption was recorded for one female during LD 1-4, no bw and nipple retention were determined on PND13 for pups in two of the litters of the control group, it could not be retrieved from the raw data if one of the females dosed at 300 mg/kg bw/day was dosed post coitum Day 5; the fasting period exceeded 24 h in 4 males (3-64 min exceedance) of the 100 mg/kg bw/day group
GLP compliance:
yes (incl. QA statement)
Remarks:
Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females: nulliparous and non-pregnant
- Age at study initiation: Males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: Males 261-355g, females 203-246g
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams.
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
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY
No known contaminants in the feed or water at levels that would interfere with the objectives of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 39-57
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
From 12 FEB 2020 to 16 APR 2020

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Trial preparations were performed to select the vehicle (corn oil) and to establish a suitable formulation procedure.
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Test item dosing formulations were kept at room temperature until 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: Trial preparations were performed at the Test Facility to select corn oil as the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analyses were performed using a validated analytical procedure (UPLC-MS based on ammonium adduct).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was ±10%.
- Stability analyses were performed previously in conjunction with the method development and validation study (Test Facility Study No. 20218716) and demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations encompassing those used in the present study.
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 50-56 days.
Females which failed to deliver were treated for 40-43 days.
Frequency of treatment:
Daily, 7 days per week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finding Study (Test Facility Reference No. 20218717) with oral gavage administration of Alcohols, C12-13, branched and linear, ethoxylated (2 EO) in rats, and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.
BODY WEIGHT: 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.
FOOD CONSUMPTION: 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.
CLINICAL CHEMISTRY AND HAEMATOLOGY: Blood of all F0-animals was collected on the day of scheduled necropsy. Samples were collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthasia (isoflurane).
SERUM HORMONES: Measurement of total T4 was conducted for F0-males.
NEUROBEHAVIOURAL EXAMINATION: 5 males during Week 4 of treatment and 5 females during the last week of lactation (i.e. PND 6-13) were assessed. Tests were performed after dosing, after completion of clinical observations. All dose groups were assessed. The battery of functions tested were: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY AND ORGAN WEIGHTS: All animals were subjected to a full post mortem examination and the following organs weighed; Brain, cervix, epididymis, adrenal, coagulation gland, parathyroid, prostate, seminal vesicle, thyroid, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus.

HISTOPATHOLOGY: Histopathology was conducted for the following tissues: Aorta, nasopharynx, bone marrow, femur, sternum, brain, cervix, epididymides, esophagus, eye, adrenal, coagulation gland, harderian, lacrimal, mammary, parathyroidc, pituitary, prostate, salivary, seminal vesicle, thyroid,
gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea urinary bladder uterus, vagina.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. 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):
No toxicologically relevant clinical signs were noted during daily detailed clinical observations.No findings were noted during the weekly arena observations in this study. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups starting in Week 4 or Week 2 of treatment, respectively, was considered not toxicologically relevant, taking into account the nature and in general limited severity of the effect and its time of occurrence (i.e. after dosing). This observation was considered to be a physiological response 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 low incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no mortalities that could be attributed to treatment with the test item.
One female of the control group was euthanized on lactation Day 2, as she had a total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered not affected by treatment. In males at 1000 mg/kg bw/day, body weight gain was decreased with statistical significance on Day 8 of pre-mating. In females at 1000 mg/kg bw/day, body weight gain was increased with statistical significance on Day 7 post-coitum and Day 7 of lactation. These and any remaining statistically significant changes were considered not toxicologically relevant as no trend was apparent regarding dose and duration of treatment and mean values were within normal ranges of biological variation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. In females at 1000 mg/kg bw/day, absolute and relative food consumption on Days 1-8 of pre-mating were slightly lower (0.86x of control for absolute food consumption, not statistically significant). This change was considered treatment-related. However, as complete recovery of food consumption was recorded during Days 8-15 of treatment, no toxicological relevance was attributed to this finding and it was therefore considered no-adverse.
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):
Hematology parameters were considered not affected by treatment with the test item in males up to 1000 mg/kg bw/day.
Decreased white blood cell (WBC) counts was seen in females at 1000 mg/kg bw/day (0.69x of control), caused by a slight decrease in neutrophils, lymphocytes and eosinophils. This change was considered treatment-related. Increased red blood cell distribution width (RDW) was seen in females at 1000 mg/kg/day (1.12x of control). This change was considered treatment-related. Any other statistically significant changes in hematological parameters were considered unrelated to administration of the test item due to the minimal magnitude of the change.
Coagulation parameters of treated rats were considered not affected by treatment with the test item in females up to 1000 mg/kg bw/day. Increased platelet counts in males at 300 and 1000 mg/kg bw/day (up to 1.21x of control) were observed, which were considered test item-related. Mean values remained within the historical control range. Decreased platelet counts in females at 100 mg/kg bw/day (0.76x of control) were recorded. As no dose-relation was apparent, this change was considered not toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):

Increased alanine aminotransferase (ALAT) activity was seen in males at 1000 mg/kg/day (1.95x of control). Mean value was outside the historical control range. This change was considered treatment-related. Increased cholesterol levels was seen in males (1.21x of control) and females (1.18x of control, not statistically significant) at 1000 mg/kg bw/day. Mean values were outside the historical control range for males only. These changes were considered treatment-related. Increased bile acid concentrations was seen in males at 1000 mg/kg bw/day. Bile acids were increased as well in males at 300 mg/kg bw/day and in females starting at 100 mg/kg bw/day (not statistically significant). These changes were considered the result of slightly low control values and not related to treatment. Decreased aspartate aminotransferase (ASAT) activity was seen in males at 100 mg/kg bw/day and was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional tests were inadvertently not performed in females.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined male animals up to 1000 mg/kg bw/day. Grip strength of the fore and hind legs were generally lower in males of all treated groups compared to the concurrent controls. It should be noted that the mean control values were at the higher end of the historical control range. As mean values of treated groups remained within historical control range2 and in the absence of a dose-related trend these decreases were considered unrelated to treatment with the test item. In males, a trend towards lower values of total movements and ambulations was observed at 1000 mg/kg bw/day (0.77x and 0.76x of control, respectively, not statistically significant), which was considered possibly test item-related. 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
Description (incidence and severity):
There was a test item related increase in liver weight of males (relative to body weight) and females (absolute and relative to body weight) treated at 1000 mg/kg bw/day as indicated in Table 1. Increased mean liver weights were also noted in males and females at 300 mg/kg bw/day, mainly caused by two individuals for each sex. At this dose, changes compared to the concurrent controls were only slight (≤10%), reaching no statistical significance, and remained within normal ranges of biological variation. There was no microscopic correlate for this weight increase. Statistically significant increased heart weight in males at 300 mg/kg bw/day (absolute and relative to body weight) was considered not test item-related as no dose-related trend was apparent.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations. All the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related microscopic observations in the jejunum of males and females treated at 1000 mg/kg bw/day, as summarized in Table 2. Vacuolation in the lamina propria of the jejunum was observed in 3/5 males (up to slight degree) and 4/5 females (up to moderate degree). Based on the absence of any additional degenerative or inflammatory changes the finding was considered non-adverse. All remaining 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
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No toxicologically relevant effects observed
Key result
Critical effects observed:
no

Table 1: Mean Percent Liver Weight Differences from Control Groups

 

Males

Females

Dose level (mg/kg bw/day):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

               Absolute

0

9

11

2

8

14*

               Relative to body weight

3

10

15**

4

7

12**

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

 

Table 2: Summary Test Item-Related Jejunum Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg bw/day):

0

100

300

1000

0

100

300

1000

JEJUNUM a

5

5

5

5

5

5

5

5

    Vacuolation, lamina propria

 

 

 

 

 

 

 

 

       minimal

-

-

-

1

-

-

-

2

       slight

-

-

-

2

-

-

-

1

       moderate

-

-

-

-

-

-

-

1

a  =  Number of tissues examined from each group.

Data source

Reference
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
please refer to "Principles of methods if other than guideline" for details
Principles of method if other than guideline:
Deviations to OECD 422 (2016): no functional observations and motor acitivity measurements were performed at the end of treatment for female animals, for one male, the testis and epididymides were directly placed in formalin instead of first modified Davidson's fixative for a minimum of 24 h; no food consumption was recorded for one female during LD 1-4, no bw and nipple retention were determined on PND13 for pups in two of the litters of the control group, it could not be retrieved from the raw data if one of the females dosed at 300 mg/kg bw/day was dosed post coitum Day 5; the fasting period exceeded 24 h in 4 males (3-64 min exceedance) of the 100 mg/kg bw/day group
GLP compliance:
yes (incl. QA statement)
Remarks:
Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
Limit test:
no
Justification for study design:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-9-Octadecen-1-ol ethoxylated
EC Number:
500-016-2
EC Name:
(Z)-9-Octadecen-1-ol ethoxylated
Cas Number:
9004-98-2
Molecular formula:
n.a.
IUPAC Name:
(Z)-9-Octadecen-1-ol ethoxylated
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
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 or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: Males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: Males 261-355g, females 203-246g
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. 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
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-22
- Humidity (%): 39-57
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
From 12 FEB 2020 to 16 APR 2020



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Trial preparations were performed to select the vehicle (corn oil) and to establish a suitable formulation procedure.
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Test item dosing formulations were kept at room temperature until 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: Trial preparations were performed at the Test Facility to select corn oil as the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. 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. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analyses were performed using a validated analytical procedure (UPLC-MS based on ammonium adduct).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study (Test Facility Study No. 20218716).
Duration of treatment / exposure:
Males were treated for 29 days, up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days.
Females which failed to deliver were treated for 40-43 days.
Frequency of treatment:
Daily, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finding Study (Test Facility Reference No. 20218717) with oral gavage administration of (Z)-9-Octadecen-1-ol ethoxylated (1.2 EO) in rats, and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

BODY WEIGHT: 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.

FOOD CONSUMPTION: 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.

CLINICAL CHEMISTRY AND HAEMATOLOGY: Blood of all F0-animals was collected on the day of scheduled necropsy. Samples were collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthasia (isoflurane). F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

SERUM HORMONES: Measurement of total T4 was conducted for F0-males.

NEUROBEHAVIOURAL EXAMINATION: 5 males during Week 4 of treatment and 5 females during the last week of lactation (i.e. PND 6-13) were assessed. Tests were performed after dosing, after completion of clinical observations. All dose groups were assessed. The battery of functions tested were: sensory activity / grip strength / motor 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 estrous.
Sperm parameters (parental animals):
Parameters examined in F0 males: Testis weight, epididymis weight, seminal vesicle weight was recorded. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed.
Litter observations:
STANDARDISATION OF LITTERS
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

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, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

GROSS EXAMINATION OF DEAD PUPS: Pups found dead were examined for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Blood was collected from two or three pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals were sacrificed after the last litter of each generation was weaned.

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

HISTOPATHOLOGY: Histopathology was conducted for the following tissues: Aorta, nasopharynx, bone marrow, femur, sternum, brain, cervix, epididymides, esophagus, eye, adrenal, coagulation gland, harderian, lacrimal, mammary, parathyroidc, pituitary, prostate, salivary, seminal vesicle, thyroid,
gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea, urinary bladder, uterus, vagina.
Postmortem examinations (offspring):
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). 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.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two
surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the thyroid from two pups per litter was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:
For each group, the following calculations were performed. Group mean values of precoital time were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): Number of females mated/Number of females paired x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females/Number of females mated x 100

Gestation index (%): Number of females with living pups on Day 1 x 100
Offspring viability indices:
For each group, the following calculations were performed. Group mean values of duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100

For each group, the following calculations were performed:

Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during daily detailed clinical observations.No findings were noted during the weekly arena observations in this study. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups starting in Week 4 or Week 2 of treatment, respectively, was considered not toxicologically relevant, taking into account the nature and in general limited severity of the effect and its time of occurrence (i.e. after dosing). This observation was considered to be a physiological response 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 low incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no mortalities that could be attributed to treatment with the test item.
One female of the control group was euthanized on lactation Day 2, as she had a total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered not affected by treatment. In males at 1000 mg/kg bw/day, body weight gain was decreased with statistical significance on Day 8 of pre-mating. In females at 1000 mg/kg bw/day, body weight gain was increased with statistical significance on Day 7 post-coitum and Day 7 of lactation. These and any remaining statistically significant changes were considered not toxicologically relevant as no trend was apparent regarding dose and duration of treatment and mean values were within normal ranges of biological variation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. In females at 1000 mg/kg bw/day, absolute and relative food consumption on Days 1-8 of pre-mating were slightly lower (0.86x of control for absolute food consumption, not statistically significant). This change was considered treatment-related. However, as complete recovery of food consumption was recorded during Days 8-15 of treatment, no toxicological relevance was attributed to this finding.
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):
Hematology parameters were considered not affected by treatment with the test item in males up to 1000 mg/kg bw/day.
Decreased white blood cell (WBC) counts was seen in females at 1000 mg/kg bw/day (0.69x of control), caused by a slight decrease in neutrophils, lymphocytes and eosinophils. This change was considered treatment-related. Increased red blood cell distribution width (RDW) was seen in females at 1000 mg/kg/day (1.12x of control). This change was considered treatment-related. Any other statistically significant changes in hematological parameters were considered unrelated to administration of the test item due to the minimal magnitude of the change.
Coagulation parameters of treated rats were considered not affected by treatment with the test item in females up to 1000 mg/kg bw/day. Increased platelet counts in males at 300 and 1000 mg/kg bw/day (up to 1.21x of control) were observed, which were considered test item-related. Mean values remained within the historical control range. Decreased platelet counts in females at 100 mg/kg bw/day (0.76x of control) were recorded. As no dose-relation was apparent, this change was considered not toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increased alanine aminotransferase (ALAT) activity was seen in males at 1000 mg/kg/day (1.95x of control). Mean value was outside the historical control range. This change was considered treatment-related. Increased cholesterol levels was seen in males (1.21x of control) and females (1.18x of control, not statistically significant) at 1000 mg/kg bw/day. Mean values were outside the historical control range for males only. These changes were considered treatment-related. Increased bile acid concentrations was seen in males at 1000 mg/kg bw/day. Bile acids were increased as well in males at 300 mg/kg bw/day and in females starting at 100 mg/kg bw/day (not statistically significant). These changes were considered the result of slightly low control values and not related to treatment. Decreased aspartate aminotransferase (ASAT) activity was seen in males at 100 mg/kg bw/day and was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined male animals up to 1000 mg/kg bw/day. Grip strength of the fore and hind legs were generally lower in males of all treated groups compared to the concurrent controls. It should be noted that the mean control values were at the higher end of the historical control range. As mean values of treated groups remained within historical control range2 and in the absence of a dose-related trend these decreases were considered unrelated to treatment with the test item. In males, a trend towards lower values of total movements and ambulations was observed at 1000 mg/kg bw/day (0.77x and 0.76x of control, respectively, not statistically significant), which was considered possibly test item-related. 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:
no effects observed
Description (incidence and severity):
There were test item-related microscopic observations in the jejunum of males and females treated at 1000 mg/kg bw/day, as summarized in Table 2. Vacuolation in the lamina propria of the jejunum was observed in 3/5 males (up to slight degree) and 4/5 females (up to moderate degree). Based on the absence of any additional degenerative or inflammatory changes the finding was considered non-adverse. All remaining 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
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not affected by treatment. All females had regular cycles of 4 days during pretest and premating. Extended di-estrous occurred in Female Nos. 42, 43 (Group 1), 63, 67, 68 (Group 3) and 77 (Group 4) during the mating period (all with normal litters). Given the absence of a dose-related incidence, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were 1/10 males of the control and 300 mg/kg bw/day group each that did not mate, 1/10 females of the control group with total litter loss, and 3/10 couples treated at 300 mg/kg bw/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this. Mating index was not affected by treatment. All females showed evidence of mating. Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within 4 days. One control female and one female at 300 mg/kg bw/day mated on the first day of the second mating period. Extended di-estrous during the mating period was considered the cause of absence of mating during the first mating period. Number of implantation sites was considered not to be affected by treatment. The mean number of implantation sites was 11.7, 11.8, 12.6 and 12.9 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. All individual values remained within the normal range of biological variation. Fertility index was considered not to be affected by treatment. The fertility indices were 100, 100, 70 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. A total of three females at 300 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment. Gestation index and duration of gestation were considered not to be affected by treatment. 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.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No toxicologically relevant effects observed

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 with the test item. These were: scab on ear and abscess on head for one pup at 1000 mg/kg bw/day and blue spot on flank for a pup in the same litter. Alopecia for a number of pups from 2 litters at 100 mg/kg bw/day. The nature and incidence of clinical signs remained within the range considered normal for pups of this age.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. The live birth indices were 94, 99, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Six pups of the control group and one pup at 100 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since 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 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. Viability indices were 98, 99, 97 and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 100% for the control, 100, 300 mg/kg bw/day groups, respectively, and 99% for the 1000 mg/kg bw/day group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test item.
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 considered not to be affected by treatment with the test item.
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 considered not to be affected by treatment with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 1000 mg/kg bw/day/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:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings (other than those reported as clinical signs) were noted among pups and these were considered not to be related to treatment with the test item. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No toxicologically relevant effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Mean Percent Liver Weight Differences from Control Groups

 

Males

Females

Dose level (mg/kg bw/day):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

               Absolute

0

9

11

2

8

14*

               Relative to body weight

3

10

15**

4

7

12**

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

 

Table 2: Summary Test Item-Related Jejunum Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg bw/day):

0

100

300

1000

0

100

300

1000

JEJUNUM a

5

5

5

5

5

5

5

5

    Vacuolation, lamina propria

 

 

 

 

 

 

 

 

       minimal

-

-

-

1

-

-

-

2

       slight

-

-

-

2

-

-

-

1

       moderate

-

-

-

-

-

-

-

1

a  =  Number of tissues examined fro

Applicant's summary and conclusion