Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
with Reproduction/Developmental Toxicity Screening
Type of information:
experimental study
Adequacy of study:
key study
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)
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
Limit test:
no
Specific details on test material used for the study:
Solubility and stability of the test substance in the solvent/vehicle: test substance preparations (in corn oil) are stable over a period of 7 days at room temperature

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males (11-12 weeks old) and females (10 weeks old)
- Weight at study initiation: weight variation did not exceed 20% of the mean weight of each sex
- Housing:
Pretreatment: up to 5 animals per sex and cage,
Study period: housed individually, exept during overnight matings: male and female mating partners housed together, pregnant animals and their litters: housed together until PND 13
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
- Food: free of chemical and microbiological contaminants
- Drinking water: regularly assayed for chemical contaminants and presence of (pathogenic) microorganisms

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12


Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed, topped up with corn oil and intensely mixed until completely dissolved

VEHICLE
- Justification for use and choice of vehicle: test substance completely miscible with corn oil
- Concentration of test substance in vehicle: 0, 1.25g/100mL, 3.75g/100mL, 12.50g/100mL
- Amount of vehicle (if gavage): 4 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration control analysis: correctness of the preparations verified (90-110%)
Duration of treatment / exposure:
The duration of treatment covered a 2-weeks premating period and mating in both sexes , 7 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals (min. 34 days for males and min. 55 days for females).
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE
The high dose was selected based on signs of toxicity noted at a dose level of 1000 mg/kg bw/d in a previously conducted dose range-finding study in Wistar rats, which preceded this definitive OECD 422 study. At this dose, significantly increased water consumption (up to 30.5%), significantly reduced food consumption (up to 15.6%) and significantly reduced body weight gain (up to 43.5%) were noted in males, while females showed moderately increased water consumption (up to 11.8%), significantly reduced food consumption (up to 17.6%) and they significantly lost body weight (weight change -139.7%) during the course of the study. Also, a massive increase of liver weights was noted in both sexes (rel. 42%/48%) and kidney weights were moderately increased (rel. 14%/18%). In addition, both sexes frequently exhibited salivation, piloerection and unsteady gait. A dose level of 300 mg/kg bw/d produced similar effects on food consumption, body weight gain and liver weight, but less severe.

Positive control:
not applicable
Observations and examinations performed and frequency:
MORTALITY:
A check for moribund or dead animals was made twice daily or once daily on Saturdays, Sundays or public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS:
- Time schedule: at least daily, for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration.
- The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. For observation, the animals were removed from their cages by the investigator and placed in a standard arena.

Following parameters were assessed:
Abnormal behavior in “handling”
fur
skin
posture
salivation
respiration
activity/arousal level
tremors
convulsions
abnormal movements
gait abnormalities
lacrimation
palpebral closure
exophthalmos (protruding eyeball)
assessment of the feces excreted during the examination (appearance/consistency)
assessment of the urine excreted during the examination
pupil size

BODY WEIGHT:
Time schedule for examinations: weekly, with following exceptions:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly

FOOD CONSUMPTION:
weekly, with following exceptions:
- Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
- Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13. in dams gestation days 0-7, 7-14, 14-20 and lactation days 1-4, 4-7, 7-10 and 10-13

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery (FOB) was tested in the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The observations were performed at random.

Home cage observations
The animals were observed for a short period (about 10-30 seconds) in their cages with the lids closed in the rack, while disturbing influences (touching of the cage and loud noises) were avoided.
While other abnormalities were recorded, particular attention was paid to the following parameters:
Posture
tremors
convulsions
abnormal movements
gait
other findings

OPEN FIELD OBSERVATIONS.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena.Besides noting other abnormalities, the following
parameters were assessed:
Behavior on removal from cage
fur
skin
salivation
nasal discharge
lacrimation
eyes/pupil size
posture
palpebral closure
respiration
tremors
convulsions
abnormal movements/stereotypy
gait
activity/arousal level
feces (appearance/ consistency) within 2 minutes
urine (amount/color) within 2 minutes
rearing within 2 minutes
other findings

SENSORY MOTOR TESTS/REFLEXES
The animals were removed from the open field and were subjected to the sensory motor and reflex tests listed below:
Reaction to an object being moved towards the face (Approach response)
touch sensitivity (Touch response)
vision (Visual placing response)
pupillary reflex
pinna reflex
audition (Startle response)
coordination of movements (Righting response)
behavior during handling
vocalization
pain perception (Tail pinch)
other findings
grip strength of forelimbs
grip strength of hindlimbs
landing foot-splay test

MOTOR ACTIVITY MEASUREMENT
Measurement of motor activity (MA) took place at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.


CLINICAL PATHOLOGY (HAEMATOLOGY, CLINICAL CHEMISTRY)
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter:
Leukocyte count
erythrocyte count
hemoglobin
hematocrit
mean corpuscular volume
mean corpuscular hemoglobin
mean corpuscular hemoglobin concentration
platelet count
differential blood count
reticulocytes

Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. Clotting tests were carried out using a ball coagulometer.
Parameter assessed:
Prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY:
- Parameters examined:
Alanine aminotransferase
aspartate aminotransferase
alkaline phosphatase
γ-Glutamyltransferase
Sodium
potassium
chloride
inorganic phosphate
calcium
urea
creatinine
glucose
total bilirubin
total protein
albumin
globulins
triglycerides
cholesterol
bile acids

THYROID HORMONE(S)
Blood samples taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormone (T4); analyzed via ELISA.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Special attention given to the reproductive organs.

ORGAN WEIGHTS: Yes
Organs assessed in all animals:
Epididymides
ovaries
prostate
seminal vesicles with coagulating glands
testes
thyroid glands (fixed)
uterus (with cervix)

Organs assessed in 5 animals per sex/test group (females with litters only, same animals as used for clinical pathological examinations):
Adrenal glands
brain
heart
kidneys
liver
spleen
thymus

HISTOPATHOLOGY: Yes
The following organs or tissues of all parental animals were fixed in 4% neutral-buffered formaldehyde:
All gross lesions
adrenal glands
brain
cecum
cervix
coagulating glands
colon
duodenum
heart
ileum
jejunum
kidneys
liver
lungs
lymph nodes (axillary and mesenteric)
oviducts
prostate gland
Peyer's patches
rectum
sciatic nerve
seminal vesicles
skeletal muscle
spinal cord (cervical, thoracic, lumbar)
spleen
sternum with marrow
stomach (forestomach and glandular stomach)
thymus
thyroid glands
trachea
urinary bladder
uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964)
vagina

Following organs or tissues were initially fixed in modified Davidson's solution:
Epididymides
ovaries
testes

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings in all organs and tissues listed.
Special attention to stages of spermatogenesis in testes and histopathology of interstitial testicular cell structure

Other examinations:
See entry "key.BASF2018.85R0080/13R165.Combined repeated dose with reproduction/develpmental tox_rat oral" in the chapter "Toxicity to reproduction".
Statistics:
CLINICAL EXAMINATIONS
- DUNNETT: Food consumption (parental animals), body weight and body weight change
- KRUSKAL-WALLIS & WILCOXON: rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity

CLINICAL PATHOLOGY
- KRUSKAL-WALLIS & WILCOXON: Blood parameters

PATHOLOGY
- KRUSKAL-WALLIS & WILCOXON: Weight parameters
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
CLINICAL OBSERVATIONS

HIGH-DOSE (500 mg/kg bw/d):
- transient salivation: all male and female animals: noted during major parts of the treatment period
- "ploughed nose first into bedding"

MID-DOSE (150 mg/kg bw/d):
- transient salivation: all male and several female animals: during several parts of the treatment period
- "ploughed nose first into bedding"

LOW-DOSE (50 mg/kg bw/d):
- transient salivation: a few male animals and one single female animal during a few occations during the treatment period

This dose-dependent pattern of temporary salivation and animal “ploughed nose first into bedding” was considered to be test substance-induced. From the temporary, short appearance of both findings immediately after dosing it is likely, that they were induced by a bad taste of the test substance and/or local affection of the upper respiratory and/or digestive tract. It is,however, not considered to be a sign of systemic toxicity.

No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups during the study.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and body weight change of all male and female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study.
There were no test item related changes in terminal body-weight.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of all male and female F0 animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.

HIGH-DOSE (500 mg/kg bw/d):
- no changes were observed
MID-DOSE (150 mg/kg bw/d):
- females: mean corpuscular hemoglobin concentration (MCHC) significantly increased
- females: hemoglobin values were significantly higher compared to controls
LOW-DOSE (50 mg/kg bw/d):
- in females: hemoglobin values were significantly higher compared to controls.

Both parameters were not dose-dependently changed and hemoglobin values were within the historical control range. Therefore, these changes were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.

HIGH-DOSE (500 mg/kg bw/d):
- no changes observed
MID-DOSE (150 mg/kg bw/d):
- male and female animals: cholesterol values significantly increased
LOW-DOSE (50 mg/kg bw/d):
- female animals: cholesterol values significantly increased

However, values were not dose-dependently changed --> changes incidental, not treatment-related

- other clinical chemistry parameters: no changes observed

THYROID HORMONES
no treatment-related alterations of T4 levels were observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery
- Home cage observations:
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations:
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes:
There were no test substance-related findings in male and female animals of all test groups.
- Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups

Motor activity measurement
No treatment-related changes of motor activity (summation of all intervals) were observed in animals of all dose groups in comparison to the concurrent control group.

The statistically significantly lower value in the high-dose males during interval 2 was assessed as incidental, as the summation of all intervals was not statistically significantly decreased, the values during interval 2 were within the historical control range and no such effect was seen in female animals which were treated for a considerably longer period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
LIVER:
Minimal increased mean absolute and relative weights in males and females treated with 500 mg/kg bw/d. The absolute and relative mean weights in both male and female animals were above the range of the historical control values. In the 500 mg/kg bw/d dose group, minimal centrilobular hepatocellular hypertrophy was observed, microscopically. Most probably, this hepatocellular hypertrophy represents the morphologic hallmark of a treatment-related
enzyme induction. Although a marginal and non-adverse change, the hypertrophy is believed to have resulted in minimal increased mean liver weights.
Males:
mean absolute weights (% of ctrl): 95%, 100% and 116% in 50, 150 and 500 mg/kg bw/d dose group, respectively
mean relative weights (% of ctrl): 98%, 102% and 118%* in 50, 150 and 500 mg/kg bw/d dose group, respectively
Females:
mean absolute weights (% of ctrl): 105%, 113%, 128%** in 50, 150 and 500 mg/kg bw/d dose group, respectively
mean relative weights (% of ctrl): 110%, 118% and 128%** in 50, 150 and 500 mg/kg bw/d dose group, respectively
*p<= 0.05; **p<= 0.01;

ADRENAL GLANDS
Females: Slightly, but statistically significantly increase in absolute and relative weights within the range of the historical control values for females.
mean absolute weights (% of ctrl): 112%, 118% and 131%* in 50, 150 and 500 mg/kg bw/d dose group, respectively
mean relative weights (% of ctrl): 118%, 122% and 131*% in 50, 150 and 500 mg/kg bw/d dose group, respectively
*p<= 0.05

THYMUS
Slightly, but not significantly decreased mean absolute and relative thymic weights in males and females treated with 50, 150 and 500 mg/kg bw/d. The mean values did not show a dose-relationship.

THYROID GLANDS
Slightly, but not significantly increased mean absolute and relativ weights in males and females treated with 500 mg/kg bw/d.

UTERUS
Compared to the control group, the mean absolute and relative weights of treated animals were decreased, but within the historical control range values. The control group weights were above the historical control range values. One control group female showed a marked uterine dilation, hemometra and endometrial hyperplasia, consistent with a marked increase of the absolute uterine weight. Next to this, two other control females had moderate estrous cycle-related dilated uterine horns, all together responsible for the high mean uterine weight in this group.
mean absolute weights (% of ctrl): 35%, 32% and 37% in 50, 150 and 500 mg/kg bw/d dose group, respectively
mean relative weights (% of ctrl): 40%, 35% and 41% in 50, 150 and 500 mg/kg bw/d dose group, respectively

KIDNEYS
Females (150 mg/kg bw/d): A significantly increased mean relative weight of kidneys.
mean absolute weights (% of ctrl): 98%, 111% and 114% in 50, 150 and 500 mg/kg bw/d dose group, respectively
mean relative weights (% of ctrl): 103%, 115%** and 113% in 50, 150 and 500 mg/kg bw/d dose group, respectively
*p<= 0.01


Because of the absence of relevant histopathologic changes in the thymus, thyroid glands and adrenal glands, all these organ weight changes are considered to be of negligible toxicologic relevance. Furthermore, the weight changes for the thymus did not show a clear dose-relationship. The increase in mean relative kidney weight in females of the 150 mg/kg bw/d group was judged as incidental: the mean female kidney weights did not show a dose-relationship, and the kidneys in the high-dose group animals did not show relevant microscopic observations.

All other absolute and relative organ weight changes were not statistically significantly deviating from controls and were attributed to the normal biological variation in animals of this strain and age.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test-item related gross findings. All macroscopic findings are considered spontaneous and consistent with the usual pattern of findings in animals of this strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
LIVER
500 mg/kg bw/d:
- minimal centrilobular hepatocellular hypertrophy in 5/10 males and 6/10 females; considered an adaptive non-adverse response.
No test-item related histopathologic findings observed in other organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse test substance related effects observed
Critical effects observed:
no

Analyses confirmed the overall accuracy of the prepared concentrations in corn oil.

The stability of these preparations was also demonstrated over a period of 7 days under ambient conditions.

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 500 mg/kg bw/d, the highest dose tested.
Executive summary:

Dihydrorosan was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 50, 150 and 500 mg/kg bw/d in corn oil. The duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 7 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear.

A detailed clinical observation, food consumption and body weights of the F0 parents was determined. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed, their viability was recorded and at necropsy on PND 13, all pups were examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13.

Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).

The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature and correct concentrations of the test substance preparations were verified.

No test substance-related, adverse findings were observed concerning clinical examinations, clincal-/pathology and reproductive performance in the F0 parental animals and concerning clinical examinations and gross findings in the F1 pups at any dose level tested.

In the clinical examinations of the F0 parental animals, signs of a bad taste of the test substance and/or local affection of the upper respiratory and/or digestive tract such as temporary salivation and animal “ploughed nose first into bedding” were noted in a dose-dependent fashion. As they were transient and appeared only shortly after dosing they were not considered as signs of systemic toxicity.

Observed organ weight changes comprised of decreased mean thymus weights in males and females of all treated groups, increased mean liver and thyroid gland weights in males and females treated with 500 mg/kg bw/d, and increased mean adrenal gland weights in females of the 500 mg/kg bw/d treated group. Because the weight changes of the thymus, thyroid glands and adrenal glands were not supported by relevant histopathologic changes they were considered to be of negligible toxicologic relevance. The increase in mean liver weights in males and females of the 500 mg/kg bw/d treated group was considered to be related to minimal centrilobular hepatocellular hypertrophy, encountered microscopically. This hepatocellular hypertrophy is believed to represent the morphologic hallmark of a low level of enzyme induction and is as such considered an adaptive non-adverse response.

Accordingly, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 500 mg/kg bw/d, the highest dose tested.

For further information, see entry "key.BASF2018.85R0080/13R165.Combined repeated dose with reproduction/develpmental tox_rat oral" in the chapter "Toxicity to reproduction".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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)
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-2-isobutyl-4-methyl-2H-pyran
EC Number:
236-770-1
EC Name:
Tetrahydro-2-isobutyl-4-methyl-2H-pyran
Cas Number:
13477-62-8
Molecular formula:
C10H20O
IUPAC Name:
4-methyl-2-(2-methylpropyl)oxane
Test material form:
liquid
Details on test material:
Name of test substance as used in the study: Dihydrorosan; Tetrahydro-2-isobutyl-4-methyl-2H-pyran
Batch identification: 00000477L0
Purity: 98.2 area-% (DB Wax), 99.1 area-% (DB 1) as sum of two isomers (GC)
Physical state/Appearance: Liquid/ yellowish
Storage conditions: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males (11-12 weeks old) and females (10 weeks old)
- Weight at study initiation: weight variation did not exceed 20% of the mean weight of each sex
- Housing:
Pretreatment: up to 5 animals per sex and cage,
Study period: housed individually, exept during overnight matings: male and female mating partners housed together, pregnant animals and their litters: housed together until PND 13
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 3 weeks

DETAILS OF FOOD AND WATER QUALITY:
- Food: free of chemical and microbiological contaminants
- Drinking water: regularly assayed for chemical contaminants and presence of (pathogenic) microorganisms

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed, topped up with corn oil and intensely mixed until completely dissolved
VEHICLE
- Justification for use and choice of vehicle: test substance completely miscible with corn oil
- Concentration of test substance in vehicle: 0, 1.25g/100mL, 3.75g/100mL, 12.50g/100mL
- Amount of vehicle (if gavage): 4 mL/kg bw/d
Details on mating procedure:
Each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration control analysis: correctness of the preparations verified (90-110%)
Duration of treatment / exposure:
The duration of treatment covered a 2-weeks premating period and mating in both sexes , 7 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals (min. 34 days for males and min. 55 days for females).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE
The high dose was selected based on signs of toxicity noted at a dose level of 1000 mg/kg bw/d in a previously conducted dose range-finding study in Wistar rats, which preceded this definitive OECD 422 study. At this dose, significantly increased water consumption (up to 30.5%), significantly reduced food consumption (up to 15.6%) and significantly reduced body weight gain (up to 43.5%) were noted in males, while females showed moderately increased water consumption (up to 11.8%), significantly reduced food consumption (up to 17.6%) and they significantly lost body weight (weight change -139.7%) during the course of the study. Also, a massive increase of liver weights was noted in both sexes (rel. 42%/48%) and kidney weights were moderately increased (rel. 14%/18%). In addition, both sexes frequently exhibited salivation, piloerection and unsteady gait. A dose level of 300 mg/kg bw/d produced similar effects on food consumption, body weight gain and liver weight, but less severe.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
MORTALITY:
A check for moribund or dead animals was made twice daily or once daily on Saturdays, Sundays or public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS:
- Time schedule: at least daily, for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration.
- The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. For observation, the animals were removed from their cages by the investigator and placed in a standard arena.

Following parameters were assessed:
Abnormal behavior in “handling”
fur
skin
posture
salivation
respiration
activity/arousal level
tremors
convulsions
abnormal movements
gait abnormalities
lacrimation
palpebral closure
exophthalmos (protruding eyeball)
assessment of the feces excreted during the examination (appearance/consistency)
assessment of the urine excreted during the examination
pupil size

BODY WEIGHT:
Time schedule for examinations: weekly, with following exceptions:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly

FOOD CONSUMPTION:
weekly, with following exceptions:
- Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
- Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13. in dams gestation days 0-7, 7-14, 14-20 and lactation days 1-4, 4-7, 7-10 and 10-13

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery (FOB) was tested in the first 5 male and the first 5 female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The observations were performed at random.

Home cage observations
The animals were observed for a short period (about 10-30 seconds) in their cages with the lids closed in the rack, while disturbing influences (touching of the cage and loud noises) were avoided.
While other abnormalities were recorded, particular attention was paid to the following parameters:
Posture
tremors
convulsions
abnormal movements
gait
other findings

OPEN FIELD OBSERVATIONS.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena.Besides noting other abnormalities, the following
parameters were assessed:
Behavior on removal from cage
fur
skin
salivation
nasal discharge
lacrimation
eyes/pupil size
posture
palpebral closure
respiration
tremors
convulsions
abnormal movements/stereotypy
gait
activity/arousal level
feces (appearance/ consistency) within 2 minutes
urine (amount/color) within 2 minutes
rearing within 2 minutes
other findings

SENSORY MOTOR TESTS/REFLEXES
The animals were removed from the open field and were subjected to the sensory motor and reflex tests listed below:
Reaction to an object being moved towards the face (Approach response)
touch sensitivity (Touch response)
vision (Visual placing response)
pupillary reflex
pinna reflex
audition (Startle response)
coordination of movements (Righting response)
behavior during handling
vocalization
pain perception (Tail pinch)
other findings
grip strength of forelimbs
grip strength of hindlimbs
landing foot-splay test

MOTOR ACTIVITY MEASUREMENT
Measurement of motor activity (MA) took place at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.


CLINICAL PATHOLOGY (HAEMATOLOGY, CLINICAL CHEMISTRY)
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

HAEMATOLOGY:
The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter:
Leukocyte count
erythrocyte count
hemoglobin
hematocrit
mean corpuscular volume
mean corpuscular hemoglobin
mean corpuscular hemoglobin concentration
platelet count
differential blood count
reticulocytes

Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. Clotting tests were carried out using a ball coagulometer.
Parameter assessed:
Prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY:
- Parameters examined:
Alanine aminotransferase
aspartate aminotransferase
alkaline phosphatase
γ-Glutamyltransferase
Sodium
potassium
chloride
inorganic phosphate
calcium
urea
creatinine
glucose
total bilirubin
total protein
albumin
globulins
triglycerides
cholesterol
bile acids

THYROID HORMONE(S)
Blood samples taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormone (T4); analyzed via ELISA.
Oestrous cyclicity (parental animals):
For a minimum of 2 weeks prior to mating, estrous cycle normality was evaluated by daily analysis of vaginal smears for each F0 female parental rat. The evaluation continued throughout the pairing period until the female showed evidence of copulation. At necropsy, one additional vaginal smear was examined to determine the estrous cycle stage for each F0 female with scheduled sacrifice.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 4, the individual litters were standardized, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups were sacrificed. Standardization of litters was not performed in litters with ≤ 8 pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups .

PUP VIABILITY
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.

PUP CLINICAL OBSERVATIONS
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

PUP BODY WEIGHT
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 10 and 13.
The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

ANOGENITAL DISTANCE
Anogenital distance is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

NIPPLE/AREOLA ANLAGEN
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Special attention given to the reproductive organs.

ORGAN WEIGHTS: Yes
Organs assessed in all animals:
Epididymides
ovaries
prostate
seminal vesicles with coagulating glands
testes
thyroid glands (fixed)
uterus (with cervix)

Organs assessed in 5 animals per sex/test group (females with litters only, same animals as used for clinical pathological examinations):
Adrenal glands
brain
heart
kidneys
liver
spleen
thymus

HISTOPATHOLOGY: Yes
The following organs or tissues of all parental animals were fixed in 4% neutral-buffered formaldehyde:
All gross lesions
adrenal glands
brain
cecum
cervix
coagulating glands
colon
duodenum
heart
ileum
jejunum
kidneys
liver
lungs
lymph nodes (axillary and mesenteric)
oviducts
prostate gland
Peyer's patches
rectum
sciatic nerve
seminal vesicles
skeletal muscle
spinal cord (cervical, thoracic, lumbar)
spleen
sternum with marrow
stomach (forestomach and glandular stomach)
thymus
thyroid glands
trachea
urinary bladder
uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964)
vagina

Following organs or tissues were initially fixed in modified Davidson's solution:
Epididymides
ovaries
testes

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings in all organs and tissues listed.
Special attention to stages of spermatogenesis in testes and histopathology of interstitial testicular cell structure


Postmortem examinations (offspring):
PUP NECROPSY OBSERVATIONS
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations (T4). The remaining pups were sacrificed under isoflurane by decapitation. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:
DUNNETT: Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index
FISHER'S EXACT: Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups
WILCOXON & BONFERRONI-HOLM: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index
WILCOXON: % live male day x, %live female day x
KRUSKAL-WALLIS & WILCOXON: Number of cycles and Cycle Length, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, Blood parameters, Weight parameters
Reproductive indices:
MALE REPRODUCTION DATA
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero
Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) x 100
* defined by a female with implants in utero

FEMALE REPRODUCTION AND DELIVERY DATA
Female mating index (%) = (number of females mated*/ number of females placed with males)x 100
* defined as the number of females with vaginal sperm or with implants in utero
Female fertility index (%) =(number of females pregnant*/ number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero
Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100

Postimplantation loss (%) = ((number of implantations – number of pups delivered)/number of implantations) x 100

Sex ratio = (number of live male or female pups on day 0 and 13 / number of live male and female pups on day 0 and 13) x 100

Anogenital index = (anogenital distance [mm] / cubic root of pup weight [g])
Offspring viability indices:
Viability index (%) = (number of live pups on day 4* after birth / number of live pups on the day of birth) x 100
* before standardization of litters (i.e. before culling)

Suvival index (%) = (number of live pups on day 13 after birth / number of live pups on day 4* after birth) x 100
* before standardization of litters (i.e. before culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For details, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
For details, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
For details, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
For details, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For details, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For details, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
For details, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
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):
Minimal centrilobular hepatocellular hypertrophy in livers of 5/10 males and 6/10 females were found in the high dose group (500 mg/kg bw/d), and were considered an adaptive non-adverse response.
No treatment-related morphological findings in the reproductive organs of either sex were found. The spermatogenic staging profiles were normal for all males examined.
In the control group, 1 female had a marked spontaneous hemometra and uterine dilation, and a diffuse endometrial hyperplasia and 2 other females had moderate estrous cycle-related dilated uterine horns.
Other microscopic findings reported in individual animals are considered spontaneous or incidental and consistent with the usual pattern of findings in animals of this strain and age.

For further details on histopathological findings, see key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats) in Chapter 7.5.1.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups. The mean estrous cycle duration was similar in control and test groups, i.e. 3.93 - 4.17 days.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Male reproduction data
- For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Copulation was not confirmed for one control male. Thus, the male mating index was 90% in the control and 100% in test groups.
- Fertility was proven for the F0 parental males within the scheduled mating interval for F1 litter with the following exception: one control male did not generate pregnancy. Thus, the male fertility index ranged between 80% and 100% without showing any relation to dose levels. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data
- The female mating index calculated after the mating period for F1 litter was 90% in the control and 100% in test groups.
- The mean duration until sperm was detected (GD 0) varied between 2.7 and 3.0 days without showing any dose-relationship.
- All female rats delivered pups or had implants in the utero except for 2 control females, which did not become pregnant. The fertility index varied between 88.9% in the control and 100% in test groups.
- The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.6 days).
- The gestation index was 100% in control, low and mid dose test groups and 90% in the high dose test group, indicating no treatment-related changes.
- Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (9.8 / 12.3 / 12.3 and 13.0 implants/dam in control, low, mid and high dose groups, respectively).
- There were no indications for test substance-induced intrauterine embryofetal mortality since the post-implantation loss did not show any significant differences between the groups (8.0 / 15.9 / 10.1 and 14.5 mean% of implants/dam in control, low, mid and high dose groups, respectively),.
- The mean number of F1 pups delivered per dam remained unaffected (9.0 / 10.5 / 11.2 and 13.7 pups/dam in control, low, mid and high dose groups, respectively)
- The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97.2% / 100% / 98.2% and 100% in control, low, mid and high dose groups, respectively.
- The number of stillborn pups was not significantly different between the test groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse test substance related effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, cannibalized and dead F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study, as they are in the historical control range.
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 98.9%/ 98.0% /99.1% and 99.1% in control, low, mid and high dose groups, respectively.
The survival index indicating pup survival during advanced lactation (PND 4 - 13) was 100% in all test groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
500 mg/kg bw/d:
Decrease of mean body weights throughout lactation until PND 13 with statistically significant difference for female pups on PND 1 and both sexes combined on PND 1 and 4.
Pup body weights
Day 1 males (Deviation vs ctrl.): -9.1%
Day 4 males (Deviation vs ctrl.): -12.7%
Day 7 males (Deviation vs ctrl.): -8.7%
Day 13 males (Deviation vs ctrl.): -4.5%

Day 1 females (Deviation vs ctrl.): -11.8%*
Day 4 females (Deviation vs ctrl.): -13.5%
Day 7 females (Deviation vs ctrl.): -11.5%
Day 13 females (Deviation vs ctrl.): -7.9%
*p<=0.05

This decrease was rather small and showed a tendency to recover during the course of lactation until PND 13.
In addition, no test substance-related influence on body weight change values of F1 pups were noted in all test groups, and there were no corroborative effects on pup well-being or survival noted. Thus, these minor pup weight changes were not considered as adverse findings by themselves.

150 and 50 mg/kg bw/d:
no test compound-related influence on body weights

Runts:
high dose group (500 mg/kg bw/d): 2 males/3 females
mid dos group (150 mg/kg bw/d): 2 females
low dose group (50 mg/kg bw/d): 1 female
control group: no runts
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):
In male and female pups at PND13 (50, 150 and 500 mg/kg bw/d), no treatment-related alterations of T4 levels were observed.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous finings at gross necropsy, such as diaphragm hernia, dilated renal pelvis, post mortem autolysis and partly cannibalized. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences.
Thus, all these findings were not considered to be associated to the test substance.
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)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse test substance related effects observed

Overall reproductive toxicity

Reproductive effects observed:
no
Treatment related:
no

Any other information on results incl. tables

Analyses confirmed the overall accuracy of the prepared concentrations in corn oil.

The stability of these preparations was also demonstrated over a period of 7 days under ambient conditions.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 500 mg/kg bw/d, the highest dose tested.
Executive summary:

Dihydrorosan was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 50, 150 and 500 mg/kg bw/d in corn oil. The duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 7 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals. After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear.

A detailed clinical observation, food consumption and body weights of the F0 parents was determined. Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed, their viability was recorded and at necropsy on PND 13, all pups were examined macroscopically for external and visceral findings. Anogenital distance measurements were conducted on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13.

Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4).

The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature and correct concentrations of the test substance preparations were verified.

No test substance-related, adverse findings were observed concerning clinical examinations, clincal-/pathology and reproductive performance in the F0 parental animals and concerning clinical examinations and gross findings in the F1 pups at any dose level tested.

In the clinical examinations of the F0 parental animals, signs of a bad taste of the test substance and/or local affection of the upper respiratory and/or digestive tract such as temporary salivation and animal “ploughed nose first into bedding” were noted in a dose-dependent fashion. As they were transient and appeared only shortly after dosing they were not considered as signs of systemic toxicity.

Observed organ weight changes comprised of decreased mean thymus weights in males and females of all treated groups, increased mean liver and thyroid gland weights in males and females treated with 500 mg/kg bw/d, and increased mean adrenal gland weights in females of the 500 mg/kg bw/d treated group. Because the weight changes of the thymus, thyroid glands and adrenal glands were not supported by relevant histopathologic changes they were considered to be of negligible toxicologic relevance. The increase in mean liver weights in males and females of the 500 mg/kg bw/d treated group was considered to be related to minimal centrilobular hepatocellular hypertrophy, encountered microscopically. This hepatocellular hypertrophy is believed to represent the morphologic hallmark of a low level of enzyme induction and is as such considered an adaptive non-adverse response.

Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups during the entire study. F0 parental animals across all dose groups proved to be fertile and those control individuals failing to generate offspring did not show any specific gross or histopathological findings. Mating behavior, conception, implantation and parturition were not influenced.

A small decrease in pup body weights of the high-dose group was observed, but not noteworthy enough to be considered adverse, as it showed a tendency to recover during the course of lactation until PND 13. Neither a test substance-related influence on body weight change nor any corroborative effects on pup well-being or survival were noted. The determination of anogenital distance/index and the count of nipple/areola anlagen revealed no treatment-related changes up to and including a dose of the test item of 500 mg/kg bw/d. In male and female pups at PND13, no treatment-related alterations of T4 levels were observed.

Accordingly, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 500 mg/kg bw/d, the highest dose tested.

For further information, see entry "key.BASF2018.85R0080/13R165.Combined Repeated Dose (oral, Rats)” in the chapter "Repeated dose toxicity, oral".