Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Jan 2018 (study initiation to sacrifice of rearing animals)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
28 Jul 2011, incl. correction of 2 Oct 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection: The doses were selected based on signs of toxicity noted at dose levels of 100 and 300 mg/kg bw/d in a previously conducted dose range-finding reproduction / developmental toxicity study in Sprague-Dawley Rats which preceded this definitive extended one-generation reproduction toxicity study.
- Inclusion of extension of Cohort 1B
- Termination time for F2: PND 4 after culling or PND 21 after weaning
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B
- Inclusion of developmental immunotoxicity Cohort 3
- Route of administration: Orally by gavage.
- Choice of species: The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 03508136W0
- Purity: 99.9 g / 100 g
- Physical state / appearance: solid / white
- Expiry date: 13 Mar 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in 1% Sodium carboxymethyl cellulose suspension in drinking water over a period of 4 days at room temperature and over a period of 7 days in a refrigerator has been verified prior to the start of the study in a comparable batch. Before and during administration, the preparations will be kept homogeneous with a magnetic stirrer.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the test substance preparations, the specific amount of test substance will be weighed, topped up with 0.5% Sodium carboxymethyl cellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD
Details on species / strain selection:
The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH / Charles River Laboratories, Italy
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males about 6 weeks, females about 5 weeks
- Housing: 2 - 3 animals per cage in polysulfonate cages type 2000P; from delivery to randomization (F0 animals), during mating, gestation, lactation, females after weaning, for functional observational battery and motor activity measurements housing in polycarbonate cages type III, 1 animal per cage except during mating (1 male / 1 female per cage) and during rearing up to PND 21 / 22 (1 dam with her litter)
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse / rat "GLP"; ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70% (till 09.11.2017) and 45 - 65% (from 10.11.2017)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (6 am to 6 pm illumination)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% suspension in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% Sodium carboxymethyl cellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer. The test substance preparations were prepared at intervals which guaranteed that the test substance concentrations in the vehicle remained stable.
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: overnight until there is evidence of copulation or a maximum period of 14 days has elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory during the study period (at the beginning, towards the middle and towards the end) for verification of the concentrations. The samples, which were taken for the concentration control analyses at the beginning of the administration period, were also used to verify the homogeneity for the samples of the low and the high concentrations (20 and 180 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running. The samples of the middle of the study were not analyzed because no imprecision occurs during the analysis of the samples from the start and end of the study.
Duration of treatment / exposure:
After an acclimatization period of at least 5 days, the F0 animals, with the exception of the controls, received the test substance daily by gavage according to the time schedule (exception: no administration to animals being in labor) for approximately 10 weeks prior to breeding and continuing through breeding (up to two weeks), and for a maximum of 6 post-mating weeks (males) or gestation (three weeks) and lactation (three weeks) for females. Selected F1 offspring (cohorts 1A, 1B, 2A, 3) received the test substance daily by gavage from PND 21 until one day before sacrifice.
Frequency of treatment:
once daily
Details on study schedule:
F0 generation parental animals were mated to produce F1 generation pups. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 5 different cohorts which were subjected to specific postweaning examinations. Cohort 1B (= F1 generation parental animals) was selected to produce F2 pups.
On arrival and shortly before the beginning of the administration period, all F0 animals were examined for signs of illness. Only healthy animals were used for the study. F0 females were nulliparous and non-pregnant at the beginnng of the study.
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- F0 generation parental animals: 24
- F1 rearing animals, cohort 1A (Reproductive PND 90): 20
- F1 rearing animals, cohort 1B (= F1 generation parental animals): 24
- F1 rearing animals, cohort 2A (Neurotoxicity PND 75 - 90): 10
- F1 rearing animals, cohort 2B (Neurotoxicity PND 22): 10
- F1 rearing animals, cohort 3 (Immunotoxicity): 10
- F1 rearing animals, positive control animals: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on signs of toxicity noted at dose levels of 100 and 300 mg/kg bw/d in a previously conducted dose range-finding reproduction/developmental toxicity study in Sprague Dawley Rats which preceded this definitive extended one-generation reproduction toxicity study.
In the dose range-finding reproduction/developmental toxicity study, clinical signs of parental toxicity were observed in the high-dose F0 animals (300 mg/kg bw/d), such as mildly reduced food consumption and body weight gain during several sections of the study. Males were more affected than females. In addition, dilated cecum, enlarged and discolored kidneys, enlarged livers, reduced terminal body weights and distinctly increased absolute/relative kidney weights were noted in the 300 mg/kg bw/d F0 males at necropsy. Kidney weights were still significantly increased in F0 males at 100 mg/kg bw/d. Histopathology revealed dose-dependently increased incidences of findings in the cecum (thickening of wall, increased apoptosis), kidneys (tubular degeneration/regeneration, tubular dilation, medulla mineralization, lymphoid infiltration) and liver (lymphoid infiltration) in F0 males and females at 100 and 300 mg/kg bw/d.
F0 parental females (300 mg/kg bw/d) had a prolonged estrous cycle. All 8 pregnant females had a significantly lower average number of implants compared to the concurrent control (10.4 vs. 15.8). In addition, post-implantation loss was significantly increased (34.6 vs 3.6%) and 2 of the 8 pregnant females had complete intrauterine litter lasses. These effects resulted in a significantly lower live litter size (10.3 vs. 15.0). Newborn pups developed normally.
The selected high dose for the present study was expected to evoke moderate systemic toxic effects in the parental animals. The present dose selection, however, also considered the need of generating a sufficient number of F1 offspring to serve the purpose of an extended one-generation study which includes the full set of reproductive (extended to F2), neurotoxicity and immunotoxicty investigations along with additional learning/memory testing and histopathological investigations of the mammary gland. Thus, excessive impairment of reproductive performance in the F0/F1 parental animals was avoided. This procedure to select the high dose in a regulatory study meets the principles of guideline OECD 443 (adopted 2011), as well as ECHA practical guide 10 ("how to avoid unnecessary testing on animals"; chapter 4 "animal welfare"; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Directive 86/609/EEC on animal protection.

- Rationale for animal assignment: Male and female Sprague Dawley Rats were randomized according to their weight and allocated to the dose groups before the beginning of the administration period.

Positive control:
Developmental immunotoxicity examinations in cohort 3 animals:
10 male and 10 female surplus offspring derived from test group 00 (control group) were selected at weaning to become a positive control group in this study.

- Name of positve control substance: Cyclophosphamide monohydrate
- Batch number: MKBX1822V
- CAS No.: 6055-19-2
- Purity: 100%
- Identity: Confirmed
- Homogeneity: given
- Expiry date: 19 Dec 2018
- Physical state: solid, white
- Storage conditions: Refrigerator
- Oral administration of 10 mL/kg bw once daily by gavage for about 4 weeks; for immunization intraperitoneal treatment of 1mL per animal split into 2 portions of 0.5 ml

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily; during the administration period all animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and / or signs of overt toxicity

MORTALITY: Yes
- Time schedule: twice daily on working days; once daily on Saturdays, Sundays or public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the administration period on day 0 and subsequently once per week

BODY WEIGHT: Yes
- Time schedule for examinations: generally once a week at the same time of the day (in the morning)
- During pregnancy: weekly on GD 0, 7, 14, and 20
- During lactation: PND 1, 4, 7, 10, 14, 17, 19, and 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- During pregnancy: weekly for GD 0-7, 7-14, and 14-20
- During lactation: PND 1-4, 4-7, 7-10, 10-14, 14-17, 17-19, and 19-21

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: generally once a week

OTHER:
- Mortality: twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays
- Urinalysis
- Clinical pathology (Hematology, clinical chemistry, hormones, sperm parameters)
- T-cell dependent antibody response (all animals of cohort 3)
- Splenic lymphocyte subpopulation analysis
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluated preparing vaginal smears for all F0 and F1 female (= cohort 1B) parental animals during a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75.
Additionally, on the day of scheduled sacrifice, the estrous status was determined for each F0 and F1 female and cohort 1A female.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation and in all cohort 1A males:
testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology, spermatic head count in the testis
- sperm motility, sperm morphology, sperm head count (cauda epididymis, testis)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, body weight, anogenital distance (AGD), presence of nipples/areolae in male pups, puberty onset, sex ratio

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
- Auditory startle response habituation in cohort 2A animals
- Functional observational battery (FOB) in cohort 2A animals
- Motor activity measurement in cohort 2A animals
- Learning and memory test (Morris water maze) in cohort 2A animals

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
- Cyclophosphamide dependent immune system response in cohort 3 animals
Postmortem examinations (parental animals):
SACRIFICE
- All F0 parental animals will be sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues (see below) were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PND 77 (cohort 2A), PND 22 (cohort 2B) (no information about days of age of cohorts 1A and cohort 3)

GROSS NECROPSY
- Gross necropsy consisted of organ weights, length and width of brain

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues (see below) were prepared for microscopic examination and weighed, respectively. The brain, spleen and thymus were weighed in one surplus F2 weanling per sex per litter
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- DUNNETT-Test (two-sided)
- Fisher's exact test
- KRUSKAL-WALLIS and WILCOXON test (two-sided)
- WILCOXON test (one-sided)
Reproductive indices:
- mating index
- fertility index
- gestation index
- Live birth index
- Postimplantation loss
Offspring viability indices:
- Viability index (pups)
- Lactation index (pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent temporary salivation was assumed to be test substance-induced, however, was considered to be no sign of systemic toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female was sacrificed moribund on Day 63. Histopathological findings were consistent with a gavage error.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights were statistically significantly increased in mid-dose females during premating days 7 – 14, in mid-dose male animals during the weeks 3 - 4 after the premating period (study weeks 14 - 15), and in mid-dose females during premating days 0 - 7. Body weight change of high-dose males was statistically significantly below the concurrent control values during premating days 56 - 63 (about 26%). All findings were considered as spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 females was statistically significantly increased during major parts of the premating period and during GD 14 - 20 (up to 36% and 8%, respectively). Food consumption of the mid-dose F0 females was statistically significantly increased during premating days 0 - 7, 28 - 35 and during GD 14 - 20 (up to 9%, 14% and 6%, respectively).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly increase in males during major parts of the pre-mating period (up tp 22%) in 180 mg/kg bw/day and females of the high dose group (180 mg/kg bw/day) during major parts of the pre-mating and gestation period (up to 25% and 28%, respectively).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- Mean corpuscular hemoglobin (MCH) significantly increased in males at the end of administration calculated from measured hemoglobin and red blood cell counts. Both measured parameters were not statistically significantly altered. Therefore, the MCH change was regarded as incidental and not treatment-related.

20 mg/kg bw/day
- In males relative basophil cell counts significantly lower compared to controls, but values were not dose-dependently changed. Thus, alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
60 mg/kg bw/day
- In females, cholesterol values significantly increased. However, change was not dose-dependent and, thus, was regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In males and females, urine pH values decreased (not statistically significant in females) which may be treatment-related but was regarded as non-adverse due to absence of other findings in the urine.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
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):
180 mg/kg bw/day
- in 21 out of 24 males, increased mineralization noted in the transition from medulla to cortex (minimal to severe)
- Nuclear crowding (accumulation of nuclei protruding into tubular lumen) in the kidneys in 22/24 male animals (minimal to moderate)
- Tubular dilation in the kidneys in 13/24 male animals (minimal to slight)

In males, the absolute and relative weights of the adrenal glands were significantly increased in test groups 02 and 03, which was regarded to be treatment-related. No further findings were noted macroscopically or histopathologically in the adrenal glands. This effect was regarded to be not adverse.
In male animals, the absolute and relative weights of the kidneys were significantly increased in test group 03. There were correlating macroscopic (enlarged) and histopathological findings in test group 03: increased mineralization, nuclear crowding and dilation of tubules in the outer zone of the outer medulla. Findings in the kidney in test group 03 were assessed as treatment-related and adverse.
The increased relative liver weight in test group 03 females were assumed to be treatmentrelated, but in the absence of any other findings (histopathology or clinical chemistry) regarded as non-adverse.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- No treatment-related alterations of T4 and TSH levels in males and females of all test groups

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Overall regular cycles in all females. However, mean estrous cycle duration was slightly but statistically significantly increased in the high dosed females (4.1 days vs 3.9 days of control).
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
- incidence of abnormal sperm in the cauda epididymidis: no treatment-related effects
- sperm head counts in the testis and in the cauda epididymidis: no treatment-related effects
- motility of the sperm: significantly lower in males of test groups 01, 02 and 03 (20, 60 and 180 mg/kg bw/d) compared to controls; however, the lowest value (84% motile sperm) exactly matched the control value in the F1 generation and the overall span of sperm motility in this study reflects a normal range of biological variation in rat (multi)generation studies; no dose-dependency
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Male mating index: 100%
- Male fertility index: between 91% and 100% (one control male, two males of 20 mg/kg bw/day and one male of 180 mg/kg bw/day did not generate F1 pups) reflecting a normal range of biological variation
- Female mating index: 100% (mean duration of copulaton varied between 2.0 and 2.3 days)
- Female fertility index: between 91% and 100% (one control female, two females of 20 mg/kg bw/day and one female of 180 mg/kg bw/day did not become pregnant) reflecting a normal range of biological variation
- Female gestation index: 100%

Details on results (P0)

Delivery data: effects observed, treatment-related
- Mean duration of gestation: 22.0 days in all test groups
- Implantation unaffected
- Mean number of resorptions (0.5 / 0.8 / 1.3* / 1.5**) and postimplantation loss (3.1% / 5.9% / 9.4%* / 10.5%**) statistically significantly above control values in 60 and 180 mg/kg bw/day groups (control/low/mid/high; *=p≤0.05; **=p≤0.01)
- F1 pups delivered per dam lower in mid-and high dose groups, although not statistically significant (14.9 / 14.0 / 13.5 and 12.7 pups/dam, respectively in control/low/mid/high dose group)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Organ:
kidney
Treatment related:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Transient salivation noted for several males and females in 180 mg/kg bw/day which was considered to be test substance-induced, however not considered a sign of systemic toxicity
- one female showed vaginal hemorrhage during study weeks 4-5 (not treatment-related)
- No substance-related findings in F1B females of all dose groups during gestation period for F2 litter
- one female showed vaginal hemorrhage on GD 23 (incidential)
- one female of 60 mg/kg bw/day and one female of 180 mg/kg bw/day group did not deliver F2 pups despite being sperm positive (not associated with treatment)
- no clinical findings during lactation period for F2 litter
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One female animal was found dead on pre-mating day 3. As the animal was partly cannibalized and no histopathological examination was conducted the cause of the death was not determined.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Body weights of high- and mid-dose females were statistically significantly increased during major parts of the premating period (up to 10% and 9%, respectively) and for mid-dose females additionally on PND 14 (about 6%).
- Body weight change of high-dose females was statistically significantly increased during premating days 0 - 21 and 0 – 70 (up to 18% and 10%, respectively) and for mid-dose females statistically significantly increaseds during premating days 0 - 21 (up to 20%).

- Body weights of the high- and mid-dose male F1B rats were statistically significantly decreased on premating day 14 (about 9% and 7%, respectively). This was a single event which is unlikely to be treatment-related.
- Decreased body weight changes of high-dose males during pre-mating days 7-14, 42-49 and 63-70 (up to 27%), mid-dose males during pre-mating days 7-14 (about 29%), and low-dose males during pre-mating days 7-14 (about 18%). Statistically significantly increased body weight change in the high-dose male animals during premating days 14 - 21 and in the mid-dose males during premating days 14 - 21, 35 - 42 and study weeks 3 - 4. - 21, 35 - 42. All of these changes were inconsistent in terms of timing and direction of the apparent effect. Thus, they are not considered to be test substance-related.
- Statistically significantly decreased body weight change in the high-dose female animals during GD 14 - 20, 0 - 20 and PND 14 - 17 as well as mid-dose females during PND 14-17 were considered as spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- High dose females showed statistically significantly increased food consumption during pre-mating days 28-49 and 0-70 (up to 22% and 13%, resp.)
- During pre-mating days 7-14, food consumption was statistically significantly increased in mid-dose females (about 13%)
- Statistically significantly decreased food consumption in the high-dose males during premating days 7 - 14 was considered as spontaneous in nature.
- Statistically significantly decreased food consumption in the high-dose females during PND 4 - 7 was considered as spontaneous in nature.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- High dose females showed increased water consumption during major parts of the pre-mating period (up to 26%)
- Mid dose females showed increased water consumption during pre-mating days 14-17 and 42-45 (about 14% and 15%, resp.)
- Statistically significantly increased water consumption in the mid-dose males during premating days 0 - 17 was considered as spontaneous in nature.
- Statistically significantly decreased water consumption in the high-dose females during PND 4 - 5 was considered as spontaneous in nature.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In males, absolute (not statistically significant) and relative increased adrenal gland weights (108% and 113%, resp.)
- In males, absolute and relative increase in kidney weights (126% and 132%, resp.)
- In males, abolute and relative decrease in liver weights (90% and 94%, resp.). Possibly treatment-related.
- In females, increased absolute kidney weight (107%).

60 mg/kg bw/day
- In males, absolute and relative increased adrenal gland weights (113% and 111%, resp.)
- In males, absolute and relative increased kidney weights (113% and 110%, resp.)
- In females, increased absolute terminal body weight (104%)

In females, the terminal body weight was statistically significantly increased in 60 mg/kg bw/d and without statistical significance in 180 mg/kg bw/d (106%). This possibly explains the increased absolute kidney weights in 180 mg/kg bw/d as no changes were seen in relative weights. Additionally, there were no histopathological findings in the kidneys of female animals of test group 180 mg/kg bw/d.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In males, enlarged kidneys found in 10/24 animals

The female animals, which were not pregnant as well as the male mating partners did not show relevant gross findings consistent with impaired fertility.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related effects on mammary gland and mammary fat pad were seen in cohort 1B male animals.
The female animals, which were not pregnant as well as the male mating partners did not show relevant histopathological findings consistent with impaired fertility.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- 3.9 days in control, 4.0 in the low- and mid-dose group and 4.1 days in the high-dose group (statistically comparable to control)
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Male mating index: 100%
- Male fertility index: between 96% and 100% (one mid-dose male and one high-dose male did not generate F2 pups). Normal range of biological variation
- Female mating index: 100%
- Mean duration until sperm was detected: 2.4 - 3.0 days without any relation to dose
- All females delivered pups except one mid-dose female and one high-dose female which did not become pregnant
- Female fertility index: between 96% and 100%. Normal range of biological variation
- Gestation index: 100%, 100%, 95%, 91% for control, low-, mid-, and high-dose groups, resp.

Details on results (P1)

Delivery data: effects observed, treatment-related
- Mean duration of gestation: 21.9 days and 22.0 days without any relation to dose
- mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (15.2 / 14.6 / 15.4 and 13.7 implants/dam)
- Number of resorptions and postimplantation loss were statistically significantly increased in the high-dose group (0.9 / 0.8 / 1.1 / 3.3** [*= p ≤ 0.05 / **= p ≤ 0.01] and 6.4% / 5.3% / 11.1% and 24.6%** in control/low/mid/high dose group, respectively). Two high-dose females had a complete litter loss.
- Mean number of F2 pups was statistically significantly decreased in the high-dose group (14.3 / 13.8 / 14.9 and 11.4** pups/dam, respectively for control/low/mid/high dose)

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (P1)

Critical effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Organ:
kidney
Treatment related:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
- Lower mean number of F1 pups (14.9 / 14.0 / 13.5 and 12.7 pups/dam, respectively in control/low/mid/high dose group, resp.) in mid-and high dose groups (subsequent to higher resorption rate), no statistical significance
- Number of liveborn pups statistically significantly below control in high dose group (340 / 289/ 322 / 285*, respectively in test groups 00 - 03).
- Rate of liveborn pups indicated by live birth indices of 99% / 98% / 99% and 97% in control/low/mid/high dose group showed no significant differences
- Number of stillborn pups statistically significantly above control in high dose group (2 / 5 / 3 / 8*, respectively in test groups 00 - 03). Eight, however, does not seem unusually compared to 6 stillborn pups in F2 control

REARING ANIMALS
Cohort 1A
- Transient salivation noted for several males and females in 180 mg/kg bw/day which was considered to be test substance-induced. However, no sign of systemic toxicity.

Cohort 2A
- Transient salivation was noted for a few high-dose (180 mg/kg bw/d) male and female animals during several parts of the treatment period. This dose-dependent temporary salivation was considered to be test substance-induced. However, no sign of systemic toxicity.

Cohort 3
- no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
PUPS/LITTER
- The number of cannibalized and dead F1 pups were evenly distributed about the groups.
- Viability index: 99%, 97%, 99%, and 99% (during early lactation, PND 0-4) for control, low, mid, and high dose, resp.
- The lactation index indicating pup survival on PND 4 - 21 was 100% in all test groups.

REARING ANIMALS
Cohort 1A
- one female animal showed abdominal position and gaspin gon study day 0 and was found dead thereafter on day 0. No association to test substance assumed.

Cohort 2A
- no adverse effects observed

Cohort 3
- one animal of the low-dose was found dead on study day 18. Not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
180 mg/kg bw/day
- mean body weights of male and female pups statistically significantly increased during PND 1-4 (up to 10%)
- mean body weight change of male pups statistically significantly increased during PND 1-4 (about 16%)

60 mg/kg bw/day
- mean body weights of male and female pups statistically significantly increased during PND 1-7 and on PND 21 (up to 12%, 11% and 6%, respectively).
- mean body weight change of male and female pups during PND 1-4 (about 19% and 16%, resp.)

20 mg/kg bw/day
- no effects observed on body weight
- mean body weight change of female pups during PND 14-21 (about 7%)

Per se, these effects do not constitute an adverse effect. Increased weights may reflect the advantageous nutritional condition of the pups in the smaller mid- and high-dose litters.

REARING ANIMALS
Cohort 1A
- Body weights of high and mid-dose females statistically significantly increased on study days 14 and 28 (up to 7%)
- Body weight change of high dose males statistically significantly decreased during study days 56-63 (up to 52%). As this had no impact on average body weights this is considered as an incidental finding.
- Body weight change of high and mid dose females statistically significantly increased during study days 7-14 and 0-7 (about 19% and 9%, respectively).
- The statistically significantly decreased body weight change in the mid-dose females during study days 56 - 63 was considered as spontaneous in nature.

Cohort 2A
- Comparable body weight and body weight changes of all treated groups to control

Cohort 3
- Comparable body weight and body weight changes of all treated groups to control
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
180 mg/kg bw/day
- Females showed increased food consumption during major parts of the study period (up to 21%)

REARING ANIMALS
Cohort 1A
- Females of the high-dose showed increased food consumption during major parts of the study period (up to 21%)

Cohort 2A
- no effects observed

Cohort 3
- no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
PUPS/LITTER
180 mg/kg bw/day
- Females showed increased water consumption during major parts of the study period (up to 25%)

60 mg/kg bw/day
- Females showed increased water consumption during days 7-17 (up to 20%)

REARING ANIMALS
Cohort 1A
- High-dose females showed statistically significant increase during major parts of the study period (up to 25%)
- Females of the mid-dose showed increased water consumption during days 7-17 (up to 20%)

Cohort 2A
- no effects observed

Cohort 3
- no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
180 mg/kg bw/day
- In males, prothrombin time (HQT, Hepatoquick's test) was prolonged at the end of adminstration period. Because this was the only measured plasma coagulation parameter besides platelet counts which can be affected by a dysregulated coagulation, this alteration has to be regarded as treatment-related and adverse.
- In males, hemoglobin values significantly higher. However, this was the only changed red blood cell parameter (i.e., hemoglobin, hematocrit and red blood cell (RBC) counts). Therefore, the hemoglobin change alone, was regarded as maybe treatment-related, but non-adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
- No alterations in absolute and relative lymphocyte subpopulation cell counts in spleen tissue (B-, T-lymphocytes, CD4-, CD8-T-lymphocytes and natural killer (NK) cells) in both sexes
180 mg/kg bw/day
- In females, total protein, albumin and calcium levels were significantly increased at the end of administration period which was regarded as treatment-related and adverse.

- Sodium levels in all treated groups were significantly increased. However, mean and median values of sodium in the test groups were not changed dose-dependently and sodium was the only altered electrolyte parameter among these individuals. In males of the low-dose group, calcium levels were significantly decreased, but again these values were not dose-dependently altered. Therefore, the mentioned alteration in this paragraph were regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
180 mg/kg bw/day
- In females, urine volume significantly higher and urine specific gravity significantly lower reflecting the physiological function of the kidneys towards higher fluid income and thus, without any other alterations in the urine, it was regarded as adaptive and non-adverse finding.
Sexual maturation:
no effects observed
Description (incidence and severity):
- Vaginal opening: Statistically significantly later puberty in low dose females was spontaneous in nature. Normal range of biological variation. At 20, 60 and 180 mg/kg bw/d test groups, 32.3, 33.2* (* = p≤0.05), 32.3 and 31.8 days, respectively.
- Preputial separation: No toxicologically relevant effect noted. At 20, 60 and 180 mg/kg bw/d test groups, 43.1, 43.0, 42.5 and 43.3 days, respectively.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
PUPS/LITTER
- no effects observed
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
PUPS/LITTER
- no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
180 mg/kg bw/day
- In males, increased absolute and relative kidney weights (112% and 117%, resp.)
- In males, decreased absolute liver, prostate, spleen, and thymus weights (86%, 90%, 83%, and 81%, resp.)
- In males, increased relative adrenal glands (113%)
- In males, decreased relative liver and spleen weights (91% and 87%, resp.)

60 mg/kg bw/day
- In males, decreased absolute prostate and absolute and relative spleen weight (91%, 91%, and 92%, resp.)

Absolute and relative spleen weights did not follow conspicious histopathological examination and no findings in clinical chemistry were found. Thus, this was regarded as equivocal and as not adverse. No findings in clinical chemistry were found regarding decreased absolute thymus weight in the highest dose group and thus, this was regarded to be an equivocal finding and as not adverse. The absolute weights of the prostate in test groups 60 and 180 mg/kg bw/day were decreased but no significant change was seen in relative weights, therefore the decrease in absolute weights was regarded as incidental. Decreased absolute and relative liver weights in 180 mg/kg bw/d group was regarded as possibly treatment-related.

Cohort 2A
- No mean absolute and relative weights were significantly changed in any test group

Cohort 2B
- No mean absolute and relative weights were significantly changed in any test group

Cohort 3
180 mg/kg bw/day
- In males, significantly decreased relative thymus weight (81%). However, similar weight changes occurred in cohort 1A without histopathological correlations and clinical chemistry findings. Thus, the decrease of the relative thymus weight was regarded as equivocal and non-adverse.

- Relative spleen weights were decreased in all treated groups, however, without reaching statistical significance. There were no correlates in clinical chemistry and there was no statistical significance, therefore, this was regarded as incidental.
- The positive control (Cyclophosphamide mynohydrate) showed expected results.

SURPLUS F1 GENERATION (F1 WEANLINGS NOT SELECTED FOR COHORTS)
- In males at 60 mg/kg bw/d, increased absolute brain and thymus weight (105% and 117%, resp.)
- In males at 20 mg/kg bw/d, increased absolute and relative thymus weight (124% and 120%, resp.)
- In males at 20 mg/kg bw/d, increased relative spleen weight (119%)
- In females at 60 mg/kg bw/d, decreased relative brain weight (91%)

All observed significant weight changes were assumed to be secondary to the increased terminal body weights in test groups 20 and 60 mg/kg bw/d (not significant) in both males and females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
PUPS/LITTER
- A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored eye(s), empty stomach and hemorrhagic testis. Without any relation to dosing, these findings were considered to be not treatment-related.

REARING ANIMALS
Cohort 1A
- no effects observed

Cohort 2A
- no effects observed

Cohort 2B
- no effects observed

Cohort 3
- no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
REARING ANIMALS
Cohort 1A
180 mg/kg bw/day
- Increased mineralization in the kidneys in the transition from medulla to cortex in 7/20 male animals (minimal to moderate)
- Nuclear crowding in the kidneys in 6/20 male animals (minimal to slight)
- Tubular dilation in the kidneys in 7/20 male animals (minimal to slight)
- Increased incidence of atrophy in the mammary gland (7/20) and mammary fat pad (7/20)

Cohort 2A
- No treatment-related findings were seen

Cohort 2B
- No treatment-related findings were seen

Cohort 3
- no histopathology performed
Other effects:
no effects observed
Description (incidence and severity):
PUPS/LITTER
- Sex ratio: no substantial differences between control and treated groups; slight differences regarded to be spontaneous in nature

REARING ANIMALS
Cohort 1A
- Estrous cycle regular (4.1 days in all test groups)
- No treatment-related alterations of T4 and TSH levels in all test groups. Significant decrease of the TSH values in female PND4 pups of 20 and 180 mg/kg bw/d. However, these changes were not dose-dependent and therefore, they were regarded as incidental and not treatment-related.
- Spermanalysis: no effects observed
- No differences observed in differential ovarian follicle count

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 2A
- No influence of the test substance on auditory startle habituation (maximum amplitude and latency) observed
- FOB: Home cage observations without findings, open-field observations did not reveal any test-substance related findings, sensorimotor tests/reflexes without findings, quantitative parameter revealed no impaired parameters
- Motor activity measurement: not influenced by the test compound. There were statistically significant increases in activity during interval 11 in low-, mid- and highdose males. This isolated finding was not related to the dose and did neither influence the total session beam interruptions significantly, nor did it impair habituation. Thus, it was not considered to be related to the test substance.
- Learning and memory test: No influence in the aibility of rats to acquire position of a hidden platform. Memory was also considered to be unaffected.
- All length and width measurements were without any findings
- All morphometric brain measurements were without any findings. Only some single parameters in male (Nucleus caudatus width left, Corpus callosum width) or female (Nucleus caudatus width left) animals of test group 180 mg/kg bw/d showed a statistically significant increase or decrease. As no other values were changed, this minimal width in-/decrease is assumed as incidental and not related to treatment.

Cohort 2B
- All length and width measurements were without any findings

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Cohort 3
- No changes in SRBC IgM titer found in males and females. Positive control (Cyclophosphamide) vaild.

Details on results (F1)

ANIMALS OF THE POSITIVE CONTROL
A significant decrease in absolute and relative weights of the spleen and a not statistically significant decrease of these weight parameters in the thymus occurred in the positive control male and female animals. The decreased weights of spleen and thymus were the expected result.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Generation:
F1
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1 (cohort 1B)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased post-implantation loss in F1 progeny
Dose descriptor:
NOAEL
Remarks:
developmental neurotoxicity
Generation:
other: Cohort 2A and 2B
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1 (cohort 3)
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Organ:
kidney
mammary gland
Treatment related:
yes

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Mean number of F2 pups delivered per dam (average litter size) was statistically significantly decreased in the high-dose group (14.3 / 13.8 / 14.9 and 11.4** pups/dam, respectively in control/low/mid/high dose group, resp.).
- Rate of liveborn pups not affected (live birth indices: 98%, 99%, 99%, and 98% for control, low, mid, and high-dose, resp.)
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- The number of cannibalized and dead F2 pups were evenly distributed about the groups.
- Viability index: 98%, 100%, 98%, 99% for control, low, mid, and high-dose, resp.
- Lactation index (indicating pup survival): 100%, 100%, 100%, and 99% for control, low, mid, and high-dose, resp.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of the high-dose female pups were statistically significantly above the concurrent control values on PND 1 (about 7%). However, this isolated finding was considered to be incidental.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few F2 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, empty stomach, dilated renal pelvis and small testis. These findings occurred without any relation to dosing. Thus, all these findings were not considered to be associated to the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Sex ratio: no substantial differences; slight differences were regarded to be spontaneous in nature.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased post-implantation loss in F1 progeny

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
180 mg/kg bw/day (actual dose received)
Treatment related:
no

Any other information on results incl. tables

Table 1: Summary of absolute organ weights of F0 parental animals

F0

Male animals

Test group (mg/kg bw/d)

01

(20)

02

(60)

03

(180)

Adrenal glands

104%

109%*

112%*

Kidneys

96%

104%

116%**

Thymus

113%

113%*

93%

* : p <= 0.05, **: p <= 0.01

Table 2: Summary of relative organ weights of F0 parental animals

F0

Male animals

Female animals

Test group (mg/kg bw/d)

01

(20)

02

(60)

03

(180)

01

(20)

02

(60)

03

(180)

Adrenal glands

106%

109%*

116%**

 

 

 

Kidneys

97%

104%

120%**

 

 

 

Liver

 

 

 

100%

102%

107%**

Thymus

116%

112%*

96%

 

 

 

* : p <= 0.05, **: p <= 0.01

Table 3: Summary of gross pathology of F0 parental animals

F0

Male animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals

24

24

24

24

Cecum

 

 

 

 

·       Enlarged

0

0

0

3

Kidneys

 

 

 

 

·       Enlarged

0

0

0

6

Table 4: Summary of histopathology of F0 parental animals

F0

Male animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals

24

24

24

24

Mineralization, medulla, (m)f

0

0

1

21

·       Grade1

 

 

1

11

·       Grade2

 

 

 

7

·       Grade3

 

 

 

1

·       Grade4

 

 

 

2

Nuclear crowding

0

0

0

22

·       Grade1

 

 

 

11

·       Grade2

 

 

 

8

·       Grade3

 

 

 

3

Dilation, tubular

0

0

0

13

·       Grade1

 

 

 

7

·       Grade2

 

 

 

6

Table 5: Summary of female reproductive data of F0 parental animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals (N)

23

21

24

23

Postimplantation loss

 

 

 

 

·       Total

11

16

32

35

·       Mean

0.5

0.8

1.3*

1.5**

·       SD

0.67

0.89

0.92

1.62

% Postimplantation loss

 

 

 

 

·       Mean

3.1

5.9

9.4*

10.5**

·       SD

4.31

7.70

7.23

10.86

·       N

23

21

24

23

* : p <= 0.05, **: p <= 0.01

Table 6: Summary of absolute organ weights of F1A animals

F1A

Male animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

Kidneys

97%

103%

112%**

Liver

102%

99%

86%**

Prostate

96%

91%*

90%**

Spleen

93%

91%*

83%**

Thymus

96%

100%

81%*

* : p <= 0.05, **: p <= 0.01

Table 7: Summary of relative organ weights of F1A animals

F1A

Male animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

Adrenal glands

98%

98%

113%**

Kidneys

98%

103%

117%**

Liver

104%

100%

91%**

Spleen

94%

92%*

87%**

* : p <= 0.05, **: p <= 0.01

Table 8: Summary of histopathology of F1A animals

 

F1A

Male animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals

20

20

20

20

Mineralization, medulla, (m)f

0

0

1

7

·       Grade1

 

 

1

2

·       Grade2

 

 

 

2

·       Grade3

 

 

 

3

Nuclear crowding

1

1

0

6

·       Grade1

1

1

 

5

·       Grade2

 

 

 

1

Dilation, tubular

0

0

2

7

·       Grade1

 

 

2

4

·       Grade2

 

 

 

3

 

Mammary gland

 

F1A

Male animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals

20

18

20

20

Atrophy, (multi)focal

1

0

2

7

·       Present

1

0

2

7

 

Mammary gland fat pad

 

F1A

Male animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals

10

10

10

10

Atrophy,(multi)focal

1

0

2

7

·       Present

1

0

2

7

Table 9: Summary of absolute organ weights of F1B animals

F1B

Male animals

Female animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

11

(20)

12

(60)

13

(180)

Terminal body weight

 

 

 

98%

104%*

106%

Adrenal glands

105%

113%**

108%

 

 

 

Kidneys

102%

113%**

126%**

98%

103%

107%*

Liver

104%

99%

90%*

 

 

 

* : p <= 0.05, **: p <= 0.01

Table 10: Summary of relative organ weights of F1B animals

F1B

Male animals

Test group (mg/kg bw/d)

11

(20)

12

(60)

13

(180)

Adrenal glands

105%

111%*

113%**

Kidneys

103%

110%**

132%**

Liver

105%

97%

94%*

* : p <= 0.05, **: p <= 0.01

Table 11: Summary of gross pathology of F1B animals

F1B

Male animals

Test group (mg/kg bw/d)

00

(0)

01

(20)

02

(60)

03

(180)

No. of animals

24

24

24

24

Kidneys

 

 

 

 

·       Enlargedd

0

0

1

10

Table 12: Summary of female reproductive data of F1B animals

Test group (mg/kg bw/d)

10

(0)

11

(20)

12

(60)

13

(180)

No. of animals (N)

24

24

22

23

Postimplantation loss

 

 

 

 

·       Total

22

18

25

76

·       Mean

0.9

0.8

1.1

3.3**

·       SD

0.97

0.79

1.28

3.94

% Postimplantation loss

 

 

 

 

·       Mean

6.4

5.3

11.1

24.6**

·       SD

6.43

5.71

21.25

30.10

·       N

24

24

22

23

* : p <= 0.05, **: p <= 0.01

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present modified extended 1-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 60 mg/kg bw/d for the F0 and F1 parental as well as adolescent animals, based on evidence for kidney toxicity, as well as corresponding effects on clinical-pathological parameters, which were observed at the LOAEL (Lowest Observed Adverse Effect Level) of 180 mg/kg bw/d. The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 180 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental toxicity in the F1 and F2 progeny is 20 mg/kg bw/d, based on increased postimplantation loss in the F1 progeny, which was observed at the LOAEL (Lowest Observed Adverse Effect Level) of 60 mg/kg bw/d. The NOAEL for developmental neurotoxicity for the F1 progeny is 180 mg/kg bw/d, the highest dose tested.
The NOAEL for developmental immunotoxicity for the F1 progeny is 180 mg/kg bw/d, the highest dose tested. Lower mean and median anti-SRBC IgM antibody titers of the positive control group (4.5 mg/kg bw/d cyclophosphamide, oral) demonstrated that the test system worked properly.
Executive summary:

The test substance was administered to groups of 24 male and 24 female healthy young Sprague-Dawley rats for test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 5 different cohorts which were subjected to specific postweaning examinations. Cohort 1B (=F1 generation parental animals) were selected to produce F2 pups. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 24 males and 24 females, selected from F1 pups to become F1 parental generation, were offered an aqueous preparation by stomach tube at different dosages (0, 20, 60 and 180 mg/kg bw/d) of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 parental animals. Control animals were dosed daily with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water).

RESULTS

The following test substance-related adverse effects/findings were noted:

180 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during major parts of the premating and gestation period (up to 25% and 28% above control, respectively)

• Increased food consumption in females during major parts of the premating period and during GD 14 - 20 (up to 36% and 8% above control, respectively)

• Increased postimplantation loss (10.5% vs. 3.1% in control)

• Increased absolute (+16%) and relative (+20%) weights of the kidneys in males

• Macroscopically enlarged kidneys in 6/24 males

• Increased mineralization in the kidneys in the transition from medulla to cortex in 21/24 male animals (minimal to severe)

• Nuclear crowding in the kidneys in 22/24 male animals (minimal to moderate)

• Tubular dilation in the kidneys in 13/24 male animals (minimal to slight)

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• Decreased number of liveborn pups subsequent to higher postimplantation loss

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during major parts of the study period (up to 25% above control)

• Increased food consumption in females during major parts of the study period (up to 21% above control)

• Increased body weights in females on study days 14 and 28 (up to 7% above control, respectively)

• Increased body weight change in females during study days 7 - 14 (about 19% above control, respectively)

• Prolonged prothrombin time in males

• Increased total protein, albumin and calcium values in females

• Increased absolute (+12%) and relative (+17%) weights of the kidneys in males

• Increased mineralization in the kidneys in the transition from medulla to cortex in 7/20 male animals (minimal to moderate)

• Nuclear crowding in the kidneys in 6/20 male animals (minimal to slight)

• Tubular dilation in the kidneys in 7/20 male animals (minimal to slight)

• Increased incidence of atrophy in the mammary gland and mammary fat pad

F1 PARENTAL ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during major parts of the premating period (up to 26% above control)

• Increased food consumption in females during premating days 28 - 49 and 0 - 70 (up to 22% and 13% above control, respectively)

• Increased body weights in females during major parts of the premating period (up to 10% above control)

• Increased body weight change in females during premating days 0 - 21 and 0 - 70 (up to 18% and 10% above control, respectively)

• Increased postimplantation loss (24.6% vs. 6.4% in control)

• Increased absolute (+26%) and relative (+32%) weights of the kidneys in males.

• Macroscopically enlarged kidneys in 10/24 males

F2 PUPS

CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS

• Decreased mean pups delivered subsequent to higher postimplantation loss

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

60 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during GD 17 - 20 (up to 22% above control)

• Increased food consumption in females during premating days 0 - 7, 28 - 35 and during GD 14 - 20 (up to 9%, 14% and 6% above control, respectively)

• Increased postimplantation loss (9.4% vs. 3.1% in control)

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during study days 7 - 17 (up to 20% above control)

• Increased body weights in females on study days 14 and 28 (up to 7% above control, respectively)

• Increased body weight change in females during study days 0 - 7 (about 9% above control, respectively)

F1 PARENTAL ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• Increased water consumption in females during premating days 14 - 17 and 42 - 45 (about 14% and 15% above control, respectively)

• Increased food consumption in females during premating days 7 - 14 (about 13% above control)

• Increased body weights in females during major parts of the premating period and on PND 14 (up to 9% and 6% above control, respectively)

• Increased body weight change in females during premating days 0 – 21 (up to 20% above control)

• Increased absolute (+13%) and relative (+10%) weights of the kidneys in males

F2 PUPS

CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

20 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 PARENTAL ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F2 PUPS

CLINICAL EXAMINATIONS/ PUP ORGAN WEIGHTS/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 2A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 3

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings