Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
ongoing - see attached rational for prolonged study period - final report proposed for 22 May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals : 10 weeks
- Basis for dose level selection : see attached range finder study, Charles River Study 490131
- Inclusion/exclusion of extension of Cohort 1B : yes
- Inclusion of F2 : delayed sexual maturation in females pups of the F1 triggered F2 generation,
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : no, no signs of neurotoxicity from overall data
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : no, no signs of immunotoxicity from overall data
- Route of administration : feeding, as substance leads to irritation in stomach if given in bolus

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyldecan-1-amide
EC Number:
238-405-1
EC Name:
N,N-dimethyldecan-1-amide
Cas Number:
14433-76-2
Molecular formula:
C12H25NO
IUPAC Name:
N,N-dimethyldecanamide
Test material form:
liquid
Details on test material:
Identification: N, N-dimethyldecan-1-amide
Lot: 0020615289
BASF Compound Number: 10/0294-3
Production Date: 22 Mar 2019
Retest Date: 21 Mar 2021
Physical Description: Liquid
Correction Factors: 1.02
Purity: 97.9 area%
Storage Conditions: Ambient, room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat
Strain: Han Wistar CRL:WI (Han)
Condition: Purpose-bred, naïve
Source: Charles River UK Limited, Margate, Kent, UK
Sex:
male/female
Details on test animals or test system and environmental conditions:
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

HUSBANDRY
Housing
Animals will initially be socially housed 2 or 3 per cage by sex (unless reduced by mortality) in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid
tops and solid bottoms.
Bedding material will be sterilised white wood shavings which will be provided with a certificate of analysis for significant contaminants. An analytical certificate for each batch of
bedding used will be retained at the Test Facility. There are no known contaminants in the bedding that would interfere with the objectives of the study.
A few days prior to mating, F0 males (and Cohort 1B males will be transferred to individual cages with solid bottoms. F0 and Cohort 1B females will be transferred to these cages for
mating.
Mated females will be transferred to individual solid bottomed cages. White paper tissue will be supplied as nesting material from Gestation Day 20. Females with un-weaned litters will
be retained in this type of cage until weaning or termination. On a suitable day after completion of mating, the males will be re-housed with their original cage mates.
Cohort 1A and 1B will be socially housed 2 or 3 per cage by sex per cage in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid
bottoms.

Environmental Conditions
The targeted conditions for animal room environment will be as follows:
Temperature: 19 – 23°C
Humidity: 40 – 70%
Ventilation: A minimum of 10 air changes per hour
Light Cycle: 12 hours light and 12 hours dark (except when interrupted
by study procedures/activities).
There will be automatic control of temperature and humidity which will be continuously monitored and recorded. Information on actual temperature and humidity ranges versus
target ranges will be presented in the study report. There will be automatic control of light cycle.

Animal Enrichment
Animals will be socially housed when possible for psychological/environmental enrichment and will be provided with items such as a device for hiding in and an object for chewing,
except when interrupted by study procedures/activities. Objects for chewing and devices for hiding in will be provided with a certificate of analysis
for significant contaminants. An analytical certificate for each batch of objects for chewing and devices for hiding in used will be retained at the Test Facility.
Other items may be included to enrich the cage environment. Details will be given in the study report.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: SDS VRF-1 breeder diet
Details on exposure:
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

The test or control items will be administered ad libitum in the diet.

DIET PREPARATION
The control diet will be administered as received. Upon receipt, the control diet will be dispensed daily for administration to Group 1 control animals.
Test item dosing formulations will be prepared based on a method established at the Test Facility (Formulation Process Document 998849-19-098) at appropriate concentrations to meet dosage level requirements. The dosing formulations will be prepared at least weekly, stored at -20°C, dispensed as required, and allowed to thaw at ambient temperature.

VEHICLE
Control Item(s)
Identification: VRF-1 Diet
Supplier: Special Diet Services (SDS)
Batch (Lot) Number: To be recorded in the study data
Expiration Date: To be recorded in the study data
Physical Description: Solid (Ground)
Storage Conditions: Ambient
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

Dose formulation samples will be collected for analysis. All samples to be analysed will be transferred to the analytical laboratory at the Test Facility.
Analyses described below will be performed by UPLC with mass detection using a validated analytical procedure (AP No. 422966)
Frequency of treatment:
continous
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
4 000 ppm (nominal)
Dose / conc.:
12 500 ppm (nominal)
No. of animals per sex per dose:
F0 = 28
1A = 20
1B = 25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: see range finder study, Charles River Study 490131
- Fasting period before blood sampling for clinical biochemistry: not specified

Examinations

Parental animals: Observations and examinations:
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.
F0 Animals
The in-life procedures, observations and measurements listed below will be performed for all
F0 animals and their litters.
Mortality/Moribundity Checks
Frequency: Twice daily, once at the start and once towards the end of the working day throughout the study.
Procedure: Animals will be observed for general health/mortality and moribundity. Animals will not be removed from the cage
during observation, unless necessary for identification or confirmation of possible findings.
Clinical Observations
Detailed Clinical Observations
Frequency: Weekly, beginning Week -1.
Procedure: Animals will be removed from the cage for examination. The examinations will include, but are not limited to, changes in
skin, fur, eyes, mucous membranes, palpebral closure, vocalisation, rearing, arousal, stains and autonomic activity (lacrimation, salivation,
piloerection, unusual respiratory pattern). Changes in gait and posture, as well as the presence of clonic or tonic movements, stereotypy or
bizarre behaviour will be assessed. This reflects normal procedures and no separate recordings will be made for these specific signs.
Cage Side Observations
Frequency: Once daily, beginning Week -1
Procedure: Animals will not be removed from the cage during observation, unless necessary for identification or confirmation of possible
findings.
Body Weights
Frequency: Males: weekly beginning Week -1.
Females: weekly beginning Week -1 until pairing for mating and then on Gestation Days 0, 7, 14 and 20 and Lactation Days 1, 7, 14 and 21.
Pups will be weighed individually on Lactation Days 1, 4, 7, 14 and 21 according to Section 14.4 and 14.4.1.
Procedure: F0 animals will be weighed individually. Pups in litters will be weighed individually. A weight will also be recorded on the
day of scheduled necropsy.
Food Consumption
Frequency: Starting Week -1, weekly for both sexes until pairing for mating.
Mated females: over the periods Gestation Days 0-7, 7-14 and 14-20, and Lactation Days 1-7, 7-14 and 14-21.
Males weekly on a suitable day after mating and re-housing.
Procedure: Food consumption will be quantitatively measured.
Water Consumption
Frequency: Regular basis throughout the study.
Procedure: Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted,
consumption may be assessed by weight.
Oestrous cyclicity (parental animals):
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

Estrous Cycle Monitoring
Frequency: F0 females: From 2 weeks prior to pairing (Study Day 57) until day of detection of a copulatory plug in situ and/or of sperm in
the lavage. On the morning of necropsy to determine the stage of estrous cycle to allow correlation with histopathology of ovaries.
Procedure: Vaginal lavages will be taken early each morning and the stages of estrous observed will be recorded.
Sperm parameters (parental animals):
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

Sperm Evaluation – F0 and Cohort 1A Males

Computer Aided Sperm Assessment (CASA)
From all F0 and Cohort 1A males only, the right cauda epididymis will be placed in
0.3% BSA in Medium 199 (as per SOP/PAT/069) and the sperm will be allowed to “swim
out” into the medium. An appropriate dilution of the sperm suspension will be prepared and
examined using a Hamilton Thorne sperm motility analyser.

Sperm Count and Morphological Analysis
The cauda epididymis will be minced and suspended. Dilutions of this sperm suspension will
be counted using a haemocytometer to obtain a total sperm count which will be expressed per
cauda epididymis and per gram of cauda epididymis. Optionally, the cauda epididymis will
be frozen prior to assessment.
From all samples of the sperm suspension described in the preceding paragraph, a sperm
smear will be prepared and stained with eosin Y solution. At least two hundred sperm per
animal will be evaluated for morphological abnormalities using criteria described by
Wyrobek and Bruce (1975)

Spermatid Count
The right testes will be decapsulated and homogenized. The homogenate may be sonicated to
reduce tissue debris etc, if required. The number of homogenization resistant spermatids in
dilutions of this suspension will be counted using a haemocytometer to obtain a total
spermatid count which will be expressed per testis and per gram of testis. Optionally, the
testis will be frozen prior to assessment.
Litter observations:
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

Cohorts
The in-life procedures, observations, and measurements listed below will be performed for all applicable Cohort 1A and 1B animals, including Cohort 1B litters (i.e. F2 generation).
Mortality/Moribundity Checks
Frequency: Twice daily, once at the start and once towards the end of the working day throughout the study.
Procedure: Animals will be observed for general health/mortality and moribundity. Animals will not be removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical Observations
Detailed Clinical Observations
Frequency: Weekly from weaning on PND21, starting on a suitable day within one week of weaning of all litters (Nominal Week 4).
Procedure: Animals removed from the cage for examination.
Cage Side Observations
Frequency: Once daily, beginning Week -1
Procedure: Animals will not be removed from the cage during observation, unless necessary for identification or confirmation of possible
findings.
Body Weights
Frequency: Cohort 1A (both sexes) and Cohort 1B Males:Weekly from weaning, starting on a suitable day within one week of weaning of the
majority of litters (Nominal Week 4).
Cohort 1B Females: weekly from weaning until pairing.
Gestation Day 0, 7, 14 and 20.
Lactation Day 1, 7, 14 and 21.
F1 Pups will be weighed individually on Lactation Days 1, 4, 7, 14 and 21 according to Section 14.4 and 14.4.1.
F2 Pups will be weighed individually, by sex, on Lactation Days 1, 4, 7, 14 and 21.
Procedure: Cohort animals will be weighed individually. Pups in litters will be weighed individually. A weight will also be recorded
on the day of scheduled necropsy.
Food Consumption
Frequency
Cohorts 1A: Weekly, starting on a suitable day within one week of weaning of all litters (Nominal Week 4).
Cohort 1B: Starting from weaning, weekly for both sexes until pairing for mating. Mated females: over the periods Gestation Days 0-7,
7-14 and 14-20, and Lactation Days 1-7, 7-14 and 14-21.
Males weekly on a suitable day after mating and re-housing.
Procedure: Food consumption will be quantitatively measured.
Water Consumption
Frequency: Regular basis throughout the study.
Water Consumption
Frequency: Regular basis throughout the study.
Procedure: Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may
be assessed by weight.
Estrous Cycle Monitoring (Cohort 1A and 1B)
Frequency Cohort 1A: From the day after vaginal patency, continuing until the first confirmed estrous has been determined.
From PND75 (at least 14 consecutive days) Vaginal smears will also be taken on the morning of necropsy to determine the stage of estrous
cycle to allow correlation with histopathology of ovaries.
Cohort 1B: From 2 weeks prior to mating until detection of a copulatory plug in situ and/or of sperm in the lavage.
Procedure: Vaginal lavages will be taken early each morning and the stages of estrous observed will be recorded.
Postmortem examinations (parental animals):
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

Clinical Pathology
Sample Collection
Blood will be collected via orbital sinus under non-recoverable isoflurane anaesthesia or via venous collection either from the jugular vein or tail vein (without anaesthetic) from animals detailed in the table below. Effort should be made to avoid sampling bias by randomising blood collection across treatment groups. Animals will be fasted overnight prior to blood sampling.
Urine will be collected in ascending animal order over 6 hours with absence of food and presence of water.
After collection, samples will be transferred to the appropriate laboratory for processing.

Haematology Parameters

Red blood cell count
Haemoglobin concentration
Haematocrit
Mean corpuscular volume
Red Blood Cell Distribution Width
Mean corpuscular haemoglobin
concentration
Mean corpuscular haemoglobin
Reticulocyte count (absolute)
Platelet count
White blood cell count
Neutrophil count (absolute)
Lymphocyte count (absolute)
Monocyte count (absolute)
Eosinophil count (absolute)
Basophil count (absolute)
Large unstained cells (absolute)
A blood smear will be prepared from each haematology sample. Blood smears will be labelled, stained, and stored.

Coagulation Parameters

Activated partial thromboplastin time
Fibrinogen
Prothrombin time
Sample Quality

Clinical Chemistry Parameters

Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
Gamma-glutamyltransferase
Creatine Kinase
Total bilirubina
Urea
Creatinine
Calcium
Phosphate
Bile Acids
Total protein
Albumin
Globulin
Albumin/globulin ratio
Glucose
Cholesterol
Triglycerides
Sodium
Potassium
Chloride
Sample Quality

Urinalysis Parameters

Colour
Appearance/Clarity
Specific gravity
pH
Volume
Protein
Glucose
Bilirubin
Ketones


Bone Marrow Smear Evaluation

Bone marrow smears will be collected and prepared as described in the Tissue Collection. Evaluation of stained smears may be added by amendment at the discretion of the Study Director in consultation with the pathologist and the Sponsor.

Thyroid Stimulating Hormone (TSH) and Thyroxine (T4)
Blood Collection
Samples for TSH and T4 Evaluation will be collected according to the following table.
Postmortem examinations (offspring):
Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and may change once final report is available.

Blood Collection – F0 and Cohort 1A Animals
Blood (1 mL) will be collected from the jugular vein or from the tail vein from adult animals
within a short timeframe nominally between 8 am and 10 am on the morning of the day of
necropsy. Additional blood samples may be obtained (e.g. due to clotting of non-serum
samples) if permissible sampling frequency, timing and blood volume are not exceeded.
Effort should be made to avoid sampling bias by randomising blood collection across
treatment groups. Animals will be fasted prior to sampling.

Blood Collection – F1 Unselected Pups (PND 22-24)
Blood (1 mL) will be collected from unselected pups from the orbital sinus under terminal
anaesthesia (without recovery). Samples will be taken from a short timeframe (nominally
between 8am and 10am). Animals will be fasted prior to sampling.

Blood Collection – Culled PND4 Pups
Blood samples will be collected from available culled pups on PND4 via cardiac puncture
under non-recoverable isoflurane into uniquely labelled tubes, without anticoagulant. At
least 0.5 mL of blood per litter (from up to 4 pups per sex, per litter, as necessary) will be
collected. Samples will be collected from all litters at each dose level with sufficient
numbers of culled pups. Samples will be taken within a short timeframe on the morning of
necropsy, nominally between 8 am and 10 am.

Analysis (Thyroid Hormones)
For logistical reasons, samples may be stored prior to assessment. The validated stability
period is 84 days for TSH (at the Test Site) and 14 days for T4 (at the Test Facility) at -20ºC.
Samples will be analysed for T4 at the Test Facility using validated analytical procedures.
T4 will be measured on Advia Centaur CP Immunoassay System by using solid phase,
competitive chemiluminescent enzyme immunoassays.

F1 and F2 will be proceed to necropsy and tissue collection, organ weights will be also be determined.

Litter/Pup Examinations
Offspring Found Dead or Euthanised Prematurely Before PND14
Where practicable, these animals will be sexed and then checked for the presence of milk in
the stomach and for the presence of any externally visible abnormalities. Any externally
abnormal pups will be fixed in 10% formalin for optional further examination. Externally
normal pups will be discarded.
Offspring Found Dead or Euthanised Prematurely On or After PND14
These animals will be subject to a gross necropsy. An external examination will be followed
by an inspection of the cranial, thoracic and abdominal contents. Internal sex will also be
confirmed. Representative samples of any abnormal tissues will be taken and fixed in neutral
buffered 10% formalin. These carcasses will then be discarded.
Scheduled Necropsy of Non-Selected Weanlings PND4
Non-selected pups on PND4 will be necropsied. This will consist of external examination
followed by macroscopic examination of the tissues and organs of the cranial, thoracic and
abdominal cavities in situ. Samples of any grossly abnormal tissues will be preserved in 10%
formalin or other appropriate fixative. The carcasses will be discarded.
Scheduled Necropsy of F2 Pups PND22
From each litter, 1 male and 1 female pup (where they are available) from 10 litters per group
will be necropsied. This will consist of external examination followed by macroscopic
examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ.
Samples of all listed tissues (see Section 16.6.4) and any grossly abnormal tissues will be
preserved in 10% formalin or other appropriate fixative.
Where 1 pup of either sex are not available for necropsy, or 1 pup of each sex are not
available for organ weight collection, additional pups of the opposite sex will be
necropsied/weighed such that 2 animals are necropsied, and 2 animals are weighed as far as
possible.
The remaining pups in each litter will be checked for externally visible abnormalities at the
time of killing. Any found to have such an abnormality will have a gross necropsy performed
and any abnormalities will be will be preserved in 10% formalin or other appropriate fixative.
The remaining carcasses will be discarded.

Necropsy
Adults, Unselected and Surplus pups
F0, Cohort 1A and 1B animals, as well as unselected pups on PND22-24 (following blood
collection) and surplus pups on PND22-24 (submitted for necropsy with their respective F0
dam), will be subjected to a complete necropsy examination, which will include evaluation of
the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and
external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their
associated organs and tissues.
The reproductive tracts of all F0 (and Cohort 1B females will be examined for signs of
implantation, the number of any implantation sites being recorded. The total number of
corpora lutea graviditatis will be recorded for each female. The uteri of all non-pregnant
females will be fixed in buffered formalin and stained using 10% aq (v/v) ammonium
omogeni solution and examined for implantation sites.
Necropsy procedures will be performed by qualified personnel with appropriate training and
experience in animal anatomy and gross pathology. A veterinary pathologist, or other
suitably qualified person, will be available.
At the discretion of the necropsy supervising pathologist, images may be generated for
illustration of for consultation on gross observations. Generation of such images will be
documented and communicated to the Study Director. Images and associated documentation
will be retained and archived.

Organ Weights
The organs identified for weighing in the Tissues Collection and Preservation table will be
weighed at necropsy for all scheduled euthanasia animals. Organ weights will not be
recorded for animals found dead or euthanised in poor condition or in extremis. Paired
organs will be weighed together. In the event of gross abnormalities, in addition to the
combined weight, the weight of each organ of a pair may be taken and entered as a tissue
comment. Terminal body weights will be used for organ weight analysis.

Tissue Collection and Preservation
Representative samples of the tissues identified in the Tissue Collection and Preservation
table will be collected from all animals and preserved in 10% neutral buffered formalin,
unless otherwise indicated. Additional tissue samples may be collected to elucidate abnormal
findings.
Statistics:
STATISTICAL ANALYSIS
All statistical analyses will be performed within the respective study phase, unless otherwise
noted. Numerical data collected on scheduled occasions will be summarised and statistically
analysed as indicated below according to sex and occasion or by litter.
Constructed Variables
Body Weight Gain = Calculated between appropriate scheduled
intervals
Food Consumption = Calculated between appropriate scheduled
intervals
Organ Weight relative to Body Weight Calculated between appropriate scheduled
intervals
Organ Weight relative to Brain Weight Calculated between appropriate scheduled
intervals
Additional or alternative body weight or food consumption intervals may be evaluated to
elucidate study results at the discretion of the Study Director.
Descriptive Statistical Analysis
Means, standard deviations, percentages, numbers, and/or incidences will be reported, as
appropriate by dataset.
Inferential Statistical Methods
All statistical tests will be conducted at the 5% significance level. All pairwise comparisons
will be conducted using two sided tests and will be reported at the 1% and 5% levels, unless
otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Analyses will be performed for
Body Weight
Body Weight Gains
Food Consumption
Haematology Variables
Coagulation Variables
Clinical Chemistry Variables
Urinalysis Variables
Organ Weights
Organ Weight relative to Body Weight
Organ Weight relative to Brain Weight
Ovarian Scoring (total number of oocytes per animal)
Parametric/Non-parametric
Levene’s test will be used to assess the homogeneity of group variances.
The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not
significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-
Wallis test is found to be significant, then pairwise comparisons will be conducted using
Dunnett’s or Dunn’s test.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and preliminary information which can change once final report is available.

Effect levels (P0)

Dose descriptor:
other: Study ongoing
Remarks on result:
other: Study ongoing - final report pending

Results: F1 generation

Details on results (F1)

Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and preliminary information which can change once final report is available.

Effect levels (F1)

Dose descriptor:
other: Study ongoing
Remarks on result:
other: Study ongoing - final report pending

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Study ongoing, draft report was not available at dossier update, see attached laboratory justification, entered details are based on study plan and preliminary information which can change once final report is available.

Applicant's summary and conclusion