Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

EOGRT, OECD 443, diet, rat, GLP: NOAEL(fertilty) : ongoing

Link to relevant study records
Reference
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
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
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.
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
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
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.
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.
Dose descriptor:
other: Study ongoing
Remarks on result:
other: Study ongoing - final report pending
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.
Dose descriptor:
other: Study ongoing
Remarks on result:
other: Study ongoing - final report pending
Reproductive effects observed:
no

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.

Additional information

There is valid information available investigating the effects on fertily of N,N-dimethyldecanamide (CAS 14433 -76 -2).

EOGRT:

Within an OECD 443.......

Study currently ongoing, draft report not available at actual date, see attached justification of conducting laboratory.

In summary there are no effects on fertility up to the highest tested dosage of 12500 ppm.

Key study assignment:

There is one cruxial study available which is used as key study for this endpoint.


Effects on developmental toxicity

Description of key information

- oral, rat, gavage, 6-15 post coitum, mixture of N,N-dimethyl-decanamide and N,N-dimethyl-octanamide,

OECD 414: NOAEL (maternal) 50 mg/kg bw/d; NOAEL (fetal) 150 mg/kg bw/d; no teratogenic potential up to 450 mg/kg bw/d. (RCC 1991)

- oral, rabbit, gavage, 6-18 post coitum, mixture of N,N-dimethyl-decanamide and N,N-dimethyl-octanamide,

OECD 414: NOAEL (maternal) 300 mg/kg bw/d; NOAEL (fetal) 1000 mg/kg bw/d; no teratogenic potential (Bayer 1991)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug - Sep 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 12, 1981
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 903069
- Expiration date of the lot/batch: November 17, 1990

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability of the test substance in the solvent/vehicle: at least 2 hours at room temperature
Species:
rabbit
Strain:
Chinchilla
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dr. Karl Thomae GmbH / Birkendorferstrasse 65 / D-W-7950 Biberach / Riss
- Age at pairing: between 4 and 6 months
- Weight at study initiation: 2810 - 4825 grams (day 0 post coitum)
- Housing: individually
- Diet: ad libitum; Pelleted standard Kliba 341 rabbit maintenance diet ("Kliba" / Klingentalmuehle AG / CH 4303 Kaiseraugst / Switzerland)
- Water: Tap water was available ad libitum by an automatic system.
- Acclimation period: minimum of 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.
Route of administration:
oral: gavage
Vehicle:
other: Bi-distilled water with 0.5 % Cremophor (BASF)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The mixtures of the test article and vehicle were prepared daily before administration. The test article was weighed into a glass beaker on a tared precision balance (Mettler PE 360) and the vehicle added (w/w). The mixtures were prepared using a homogenizer. During the daily administration period,
homogeneity was maintained using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 25 mg/ml, 75 mg/ml, 250 mg/ml
- Amount of vehicle (if gavage): adjusted to a total application volumen 4ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Photometric analytical verification (UV/Vis)
Concentrations (25, 75, 250 mg/ml) and stability in bidestilled water with 0.5% Cremophor was verified.
Result: Recovery between 95.3 and 101.0%
Homogeneity varies from -5 to + 3%
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
until copulation was observed. The day of mating was designated as day 0 post coitum.
Duration of treatment / exposure:
- The test article was administered orally, by gavage, once daily in the morning from day 6 through to day 18 post coitum.
- Dose volume: 4 ml/kg body weight (daily adjustment of the individual volume to the actual body weight)
Frequency of treatment:
daily from day 6 through day 18 post coitum
Duration of test:
28 days (from successful mating to termination, excluding acclimatisation etc)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
16 mated female rabbits
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of the dose range-finding study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: mortalities, signs of reactions of treatment and/or symptoms of ill health

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 28 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: days 0-6, 6-11, 11-15, 15-19, 19-24 and 24-28 post coitum

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: yes
- Sacrifice on day 28 post coitum
- Post mortem examination, including gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions (embryonic resoption): Yes
- Number of late resorptions (fetal resorption): Yes
- Number of Pre-Implantation loss: yes
- Number of Post-Implantation loss: yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-one t-test), based on a pooled variance estimate, was applied for the comparison between the treated groups and the control group.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Indices:
see "Any other information on materials and methods incl. tables"
Historical control data:
Historical data of Chinchilla Rabbits (Hybrids, SPF Quality) from 1987, 1988 and 1989:
- Reproduction data of dams
- Spontaneous abnormal findings of fetuses (external, vsceral or skeletal examination)
- Skeletal examination of fetuses (stage of development) on fetus basis or on litter basis
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- group 4 (1000 mg/kg): in 1 female slight dyspnoea and ventral recumbency a few hours prior to death on day 12 post coitum and 1 female with dyspnoea on day 9 post coitum during 5 hours

These isolated findings were considered to be incidental and not test article related effects.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- group 2 (100 mg/kg): 3 females died during the dosing period; 1 on day 7 post coitum (2nd day of dosing) and 1 on day 15 post coitum (10th day of dosing) to intubation errors, 1 on day 12 post coitum (7th day of dosing) spontaneously without any signs of toxic effects
- group 3 (300 mg/kg): 1 female was found dead in the morning of day 28 post coitum (day of Caesarean Section, ten days after the last dosing) without any previous signs of toxic effects.
- group 4 (1000 mg/kg): 1 female died on day 12 post coitum (7th day of dosing) without any previous signs of toxic effects.

These Isolated cases of spontaneous deaths In any dose groups were considered to be incidental because no relation to the number of administrations or to the dosages was evident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- group 4 (1000 mg/kg): slightly reduced mean body weight gain during the dosing period (in particular between days 6 and 11 post coitum (1st and 6th day of dosing))
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- group 4 (1000 mg/kg): reduction in food consumption (-21.2% compared to vehicle control) during the dosing period (days 6 to 19 post coitum), statistically significant between days 11-15 post coitum
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- the total post-implantation loss in two females of group 3 (300 mg/kg) was considered to be incidental, because no female with total post-implantation loss was evident in group 4 (1000 mg/kg)
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- groups 1 (vehicle control) and 3 (300 mg/kg): 2 runts (body weight <19.0 g)
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - groups 1 (vehicle control) and 3 (300 mg/kg): 2 runts (body weight <19.0 g)
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
no dead fetuses
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
1/158 fetuses in 1/16 litters in group 1 (vehicle control);
2/145 fetuses in 2/14 litters in group 2 (100 mg/kg);
3/120 fetuses in 3/12 litters in group 3 (300 mg/kg);
1/147 fetus in 1/15 litters in group 4 (1000 mg/kg).

The findings noted were: thoracic vertebral bodies and/or arches (hemicentric, missing or fused), sternebrae abnormally ossified and/or fused, rib(s) bifurcated or fused and caudal vertebrae hemicentric or bipartite.

The types and the incidences of the abnormal findings noted did not indicate test article specific effects.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- group 2 (100 mg/kg): 1/145 fetuses with dilated aorta and missing arch of aorta
- group 3 (300 mg/kg): 1/120 fetuses with hemidiaphragm and 1 fetus with oval foramen in the diaphragm
- group 4 (1000 mg/kg): 1/147 fetuses with hydronephrosis (both kidneys)

These isolated abnormal findings noted did not indicate an association with administration of the test article.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The fetal parameters were not affected up to and including the highest dose level of 1000 mg/kg body weight/day.
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The fetal parameters were not affected up to and including the highest dose level of 1000 mg/kg body weight/day.
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
The test substance did not reveal any teratogenic potential up to and including the highest dose level of 1000 mg/kg body weight/day.
The no-observed adverse effect level for the maternal organism was considered to be 300 mg/kg and for the fetal organism 1000 mg/kg body weight/day.
Executive summary:

The purpose of this study was to assess the effects of the test substance on embryonic and fetal development in pregnant Chinchilla rabbits.

Each group consisted of 16 mated female rabbits. The test substance was administered orally by gavage once daily from day 6 through to day 18 post coitum, at dose levels of:

Group 1:       0 mg/kg body weight/day (vehicle control)

Group 2:       100 mg/kg body weight/day

Group 3:       300 mg/kg body weight/day

Group 4:       1000 mg/kg body weight/day

The dosages were based on the results of a dose range-finding study. A standard dose volume of 4 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bi-distilled water with 0.5 % Cremophor).

Females were sacrificed on day 28 post coitum and the fetuses were removed by Caesarean section. The examination of the dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

MATERNAL DATA

GENERAL TOLERABILITY

- There were no deaths, clinical signs or necropsy findings in the females of any dose group which were considered to be related to administration of the test substance.

- Test article-related reduced food consumption (statistically significant between days 11-15 post coitum) was evident during the dosing period at 1000 mg/kg. During this period, the body weight gain was slightly reduced (without statistical significance).

No effects on food consumption and body weight development were noted at 100 mg/kg or at 300 mg/kg.

REPRODUCTION PARAMETERS

- None of the differences between the vehicle control group and any dose group noted were considered to be an effect of administration of the test substance. The differences evident were considered to be incidental because of missing dose-relation.

FETAL DATA

- During external and fresh visceral examination as well as at examination of fetal heads by Wilson technique and skeletal examination of fetuses, no abnormal findings were evident which indicated test article-related effects.

The isolated common abnormal findings (with the lowest incidence at 1000 mg/kg) were within the normal range of spontaneously occurring findings in this rabbit strain and were considered to be incidental.

- The other fetal parameters recorded - sex ratios, body weights and stage of skeletal development - resulted in similar values in all groups.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - September 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 12, 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 903069
- Expiration date of the lot/batch: November 17, 1990

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability of the test substance in the solvent/vehicle: at least 2 hours
Species:
rat
Strain:
Wistar
Remarks:
SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. / Wolferstrasse 4 / CH 4414 Fullinsdorf / Switzerland
- Age at study initiation (pairing): 11 week minimum
- Weight at study initiation (day 0 post coitum): 179-226 g
- Housing: single
- Diet (e.g. ad libitum): ad libitum; Pelleted standard Kliba 343 rat/mouse maintenance diet ("Kliba", Klingentalmuehle AG, CH 4303 Kaiseraugst/Switzerland)
- Water (e.g. ad libitum): Tap water was available ad libitum
- Acclimation period: 10 days under test conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light):12 hours artificial fluorescent light/12 hours dark with background music (Schweizerischer Telefonrundspruch) played at a centrally defined low volume for at least 8 hours during the light period

IN-LIFE DATES: From: July, 27 To: August 31, 1990
Route of administration:
oral: gavage
Vehicle:
other: Bi-distilled water with 0.5% Cremophor (BASF)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The mixtures of the test article and vehicle were prepared daily before administration. The test substance was weighed into a glass beaker on a tared precision balance (Mettler PE 360) and the vehicle added (w/w). The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer.

The test article was administered orally, by gavage, once daily in the morning from day 6 through to day 15 post coitum. All groups received a dose volume of 10 ml/kg body weight, with a daily adjustment of the individual volume to the actual body weight. Control animals were similarly dosed with the vehicle alone
VEHICLE
- Justification for use and choice of vehicle (if other than water): common vehicle
- Concentration in vehicle: 5 mg/ml, 15 mg/ml, 45 mg/ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Photometric analytical verification (UV/Vis)
Concentrations (5, 15, 150 mg/ml) and stability in bidestilled water with 0.5% Cremophor was verified.
Result: Recovery between 99.4 and 103.8%
Homogeneity varies from -4 to + 5%
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no information
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post coitum
Duration of treatment / exposure:
from day 6 through to day 15 post coitum
Frequency of treatment:
once daily in the morning
Duration of test:
21 days (from successful mating to termination, excluding acclimatisation etc)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of a dose range-finding study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: Signs of mortalities, reaction to treatment and of ill health were observed.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until 21 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: days 0-6, 6-11, 11-16 and 16-21 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: post mortem examination, including gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea
- Uteri (and contents) of all females with live fetuses were weighed at necropsy
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions (embryonic resoption): Yes
- Number of late resorptions (fetal resorption): Yes
- Number of Pre-Implantation loss: yes
- Number of Post-Implantation loss: yes
Fetal examinations:
- External examinations: Yes; all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes; half per litter
- Head examinations: not specified
Statistics:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-one t-test), based on a pooled variance estimate, was applied for the comparison between the treated groups and the control group.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Indices:
see "Any other information on materials and methods incl. tables"
Historical control data:
- historical reproductive data of Wistar rats (SPF quality); 1987/ 1988/ 1989/ 1990
- spontaneous abnormal findings of Wistar rats (SPF quality); 1987/ 1988/ 1989/ 1990
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- The dams at 450 mg/kg body weight/day group showed severe clinical signs of reaction to treatment (in the main ruffled fur, ventral recumbency, dyspnoea and apath), in particular from days 8 to 14 post coitum (3th to 9th day of dosing period, respectively).
- In 4 animals, respectively, on day 10 and/or 11 post coitum and in 1 animal on day 12 post coitum, comatose state was noted during a few hours.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- group 4 (450 mg/kg) dams: stagnation of the body weight gain between days 6 and 9 post coitum; during the rest of the treatment period slightly retarded body weight gain; slightly reduced corrected body weight gain (corrected for uterus weight) (4.9% vs. 7.8 % in the vehicle control)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- group 4 (450 mg/kg) dams: reduction in food consumption (-24.1 and -18% between days 6-11 or 11-16 post coitum compared to vehicle control)
- group 3 (150 mg/kg) dams: reduction in food consumption (-6.1% compared to vehicle control)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- one female of group 1 (vehicle control) and 2 females of group 3 (150 mg/kg): blood in the uterus
- one female of group 4 (450 mg/kg): hairloss on the abdomen
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
- group 4 (450 mg/kg bw/d) dams: marginally increased post-implantation loss (9.4% vs. 5.6% in the vehicle control)
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- 1 in group 3 (150 mg/kg) and 1 in group 4 (450 mg/kg): runts

Isolated occurrence of runts are not unusual in rats of this strain. Therefore, these findings were considered to be incidental and not related to treatment with the test article.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - group 4 (450 mg/kg): the mean fetal body weight was significantly reduced by 8.5% in comparison with the vehicle control
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
no dead fetuses
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- 1 fetus of group 2 (50 mg/kg) and 1 fetus of group 3 (150 mg/kg): caudal malposition of the right or of both hind legs

Isolated occurrence of caudally malpositioned hindlegs are not unusual in rats of this strain. Therefore, these findings were considered to be incidental and not related to treatment with the test article.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
5/150 control fetuses (in 4 litters),
4/150 fetuses at 50 mg/kg (in 4 litters),
3/137 fetuses at 150 mg/kg (in 3 litters) and
12/147 fetuses at 450 mg/kg (in 9 litters)

- mainly wavy ribs and dumbbell shaped thoracic vertebral bodies (common findings in rats of this strain)
- statistical significant differernces in group 4 (450 mg/kg) from vehicle control: retardation in skeletal development (non-ossified cervical vertebrae or phalangeal nuclei or incompletely ossified sternebrae)
- slightly increased incidence of abnormal findings in group 4 (450 mg/kg) in comparison with the concurrent vehicle control group was related to the reduced mean fetal body weight and was not considered to be a specific teratogenic effect of the test article
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- 1/137 fetuses of the vehicle control and 1/134 fetuses of group 4 (450 mg/kg): pelvic dilation of the right kidney

These findings were considered to be incidental.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
External and visceral examination of the fetuses, did not reveal any treatment related effects up to and including the dose level of 450 mg/kg body weight/day.
The retardation in skeletal development noted for the fetuses at 450 mg/kg correlated with the reduced mean fetal body weight noted in this group. The abnormal skeletal findings in the 450 mg/kg group were not considered to be a specific teratogenic effect of the test article.
Developmental effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Table 1: Differences in food comsumption of dams, mean (g/animal/day)

Group         Days post coitum

0 - 6                     6 - 11                       11 - 16              6 - 16**            16 - 21

(mg/kg)    g (%)*                   g (%)*                       g (%)*               

1                 17.5                       19.5                            23.3                     21.4                      22.5

(0)

2                 17.7                      18.8                             23.1                     21.0                      22.6

(50)          (+1.1)                 (-3.6)                        (-0.9)               (-1.9)                  (+0.4)

3                 17.8                      18.2                             21.9                     20.1                     22.4

(150)          (+1.7)                    (-6.7)                       (-6.0)              (-6.1)                  (-0.4)

4                  18.1                      14.8                            19.1                     17.0                     21.8

(450)         (+3.4)                 (-24.1)                    (-18.0)            (-20.6)                 (-3.1)

* = Percentages refer to the values of group 1.

** = The calculations of food consumption and body weight gain during the treatment period started on day 6 post coitum (immediately prior to the first administration)and ended on day 16 post coitum(approximately 24 hours after the last administration).

Conclusions:
Based on these results, the no-observed adverse effect level for the maternal organism was considered to be 50 mg/kg body weight/day and for the fetal organism 150 mg/kg body weight/day.
Under the conditions described in this study, the test substance did not reveal any teratogenic potential up to and including the highest dose level of 450 mg/kg body weight/day.
Executive summary:

The purpose of this study was to assess the effects of the test substance on embryonic and fetal development in pregnant Wistar rats. Each group consisted of 25 mated female rats. The test substance was administered orally by gavage once daily from day 6 through to day 15 post coitum, at dose levels of:

Group 1: 0 mg/kg body weight/day (vehicle control)

Group 2: 50 mg/kg body weight/day

Group 3: 150 mg/kg body weight/day

Group 4: 450 mg/kg body weight/day

The dosages were based on the results of a dose range-finding study. A standard dose volume of 10 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bi-distilled water with 0.5% Cremophor). Females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. The examination of the dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

MATERNAL DATA

GENERAL TOLERABILITY

- Administration of the test substance caused severe clinical signs of reaction to treatment at 450 mg/kg during the dosing period. Ruffled fur, ventral recumbency, dyspnoea, apathy and in 4 dams comatose state were observed. No clinical signs of reaction to treatment were noted at 50 or 150 mg/kg.

- Dose related reduced food consumption was noted at 150 and 450 mg/kg during the dosing period. This finding was considered to be test article related. No effects of test article on the food consumption of the dams were noted at 50 mg/kg.

- Stagnation of the body weight gain from the first to the fourth day of the dosing period and retardation of the body weight gain during the rest of the treatment period were noted for the dams at 450 mg/kg. No test article related effects on the body weight of the dams were evident at 50 or 150 mg/kg.

- At terminal necropsy of the dams, no test article related abnormal findings were noted.

REPRODUCTION PARAMETERS

- At 450 mg/kg, the marginally increased post-implantation loss was considered to be an effect of treatment with the test article. The maternal reproduction parameters of the dams at 50 or 150 mg/kg were not affected by treatment with the test substance.

FETAL DATA

- Sex ratios of the fetuses were not affected by treatment with the test article.

- At 450 mg/kg, test article related reduction in mean fetal body weight was noted.

No effects on the mean body weight of the fetuses were evident at 50 or 150 mg/kg.

- None of the few abnormal findings noted at external or visceral examination by Wilson technique of the fetuses indicated a test article related effect.

- At skeletal examination of the fetuses, the slightly increased incidence of fetuses with abnormal findings and the retardation in skeletal development noted for the fetuses at 450 mg/kg correlated with the reduced mean fetal body weight noted in this group. The abnormal skeletal findings in the 450 mg/kg group were not considered to be a specific teratogenic effect of the test article.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached RA justification
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The fetal parameters were not affected up to and including the highest dose level of 1000 mg/kg body weight/day.
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
The fetal parameters were not affected up to and including the highest dose level of 1000 mg/kg body weight/day.
Developmental effects observed:
no
Conclusions:
The test substance did not reveal any teratogenic potential up to and including the highest dose level of 1000 mg/kg body weight/day.
The no-observed adverse effect level for the maternal organism was considered to be 300 mg/kg and for the fetal organism 1000 mg/kg body weight/day.
Executive summary:

The purpose of this study was to assess the effects of the "analog test substance" on embryonic and fetal development in pregnant Chinchilla rabbits.

Each group consisted of 16 mated female rabbits. The test substance was administered orally by gavage once daily from day 6 through to day 18 post coitum, at dose levels of:

Group 1:       0 mg/kg body weight/day (vehicle control)

Group 2:       100 mg/kg body weight/day

Group 3:       300 mg/kg body weight/day

Group 4:       1000 mg/kg body weight/day

The dosages were based on the results of a dose range-finding study. A standard dose volume of 4 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bi-distilled water with 0.5 % Cremophor).

Females were sacrificed on day 28 post coitum and the fetuses were removed by Caesarean section. The examination of the dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

MATERNAL DATA

GENERAL TOLERABILITY

- There were no deaths, clinical signs or necropsy findings in the females of any dose group which were considered to be related to administration of the test substance.

- Test article-related reduced food consumption (statistically significant between days 11-15 post coitum) was evident during the dosing period at 1000 mg/kg. During this period, the body weight gain was slightly reduced (without statistical significance).

No effects on food consumption and body weight development were noted at 100 mg/kg or at 300 mg/kg.

REPRODUCTION PARAMETERS

- None of the differences between the vehicle control group and any dose group noted were considered to be an effect of administration of the test substance. The differences evident were considered to be incidental because of missing dose-relation.

FETAL DATA

- During external and fresh visceral examination as well as at examination of fetal heads by Wilson technique and skeletal examination of fetuses, no abnormal findings were evident which indicated test article-related effects.

The isolated common abnormal findings (with the lowest incidence at 1000 mg/kg) were within the normal range of spontaneously occurring findings in this rabbit strain and were considered to be incidental.

- The other fetal parameters recorded - sex ratios, body weights and stage of skeletal development - resulted in similar values in all groups.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached RA justification
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Abnormalities:
no effects observed
Dose descriptor:
NOAEL
Remarks:
fetotoxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
External and visceral examination of the fetuses did not reveal any treatment related effects up to and including the dose level of 450 mg/kg body weight/day.
The retardation in skeletal development noted for the fetuses at 450 mg/kg correlated with the reduced mean fetal body weight noted in this group. The abnormal skeletal findings in the 450 mg/kg group were not considered to be a specific teratogenic effect of the test article.
Developmental effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Conclusions:
Based on these results, the no-observed adverse effect level for the maternal organism was considered to be 50 mg/kg body weight/day and for the fetal organism 150 mg/kg body weight/day.
Under the conditions described in this study, the test substance did not reveal any teratogenic potential up to and including the highest dose level of 450 mg/kg body weight/day.
Executive summary:

The purpose of this study was to assess the effects of the "analog test substance" on embryonic and fetal development in pregnant Wistar rats. Each group consisted of 25 mated female rats. The test substance was administered orally by gavage once daily from day 6 through to day 15 post coitum, at dose levels of:

Group 1: 0 mg/kg body weight/day (vehicle control)

Group 2: 50 mg/kg body weight/day

Group 3: 150 mg/kg body weight/day

Group 4: 450 mg/kg body weight/day

The dosages were based on the results of a dose range-finding study. A standard dose volume of 10 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bi-distilled water with 0.5% Cremophor). Females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. The examination of the dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

MATERNAL DATA

GENERAL TOLERABILITY

- Administration of the test substance caused severe clinical signs of reaction to treatment at 450 mg/kg during the dosing period. Ruffled fur, ventral recumbency, dyspnoea, apathy and in 4 dams comatose state were observed. No clinical signs of reaction to treatment were noted at 50 or 150 mg/kg.

- Dose related reduced food consumption was noted at 150 and 450 mg/kg during the dosing period. This finding was considered to be test article related. No effects of test article on the food consumption of the dams were noted at 50 mg/kg.

- Stagnation of the body weight gain from the first to the fourth day of the dosing period and retardation of the body weight gain during the rest of the treatment period were noted for the dams at 450 mg/kg. No test article related effects on the body weight of the dams were evident at 50 or 150 mg/kg.

- At terminal necropsy of the dams, no test article related abnormal findings were noted.

REPRODUCTION PARAMETERS

- At 450 mg/kg, the marginally increased post-implantation loss was considered to be an effect of treatment with the test article. The maternal reproduction parameters of the dams at 50 or 150 mg/kg were not affected by treatment with the test substance.

FETAL DATA

- Sex ratios of the fetuses were not affected by treatment with the test article.

- At 450 mg/kg, test article related reduction in mean fetal body weight was noted.

No effects on the mean body weight of the fetuses were evident at 50 or 150 mg/kg.

- None of the few abnormal findings noted at external or visceral examination by Wilson technique of the fetuses indicated a test article related effect.

- At skeletal examination of the fetuses, the slightly increased incidence of fetuses with abnormal findings and the retardation in skeletal development noted for the fetuses at 450 mg/kg correlated with the reduced mean fetal body weight noted in this group. The abnormal skeletal findings in the 450 mg/kg group were not considered to be a specific teratogenic effect of the test article.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Additional information

There are no valid teratogenicity in vivo studies for pure N,N-dimethyldecanamide. Nevertheless teratogenicity was investigated with a mixture of N,N-dimethyldecanamide and N,N-dimethyloctanamide (with traces of N,N-dimethyl-dodecanamide and N,N-dimethyl-hexanamide). Due to the fact that a high amount in the mixture was N,N-dimethyl-decanamide and the rest of the mixture are homologues with a lower and higher molecular weight which can be assumed to have a similar toxicological behaviour it is concluded that the mixture has an nearly similar toxicological behaviour like pure N,N-dimethyldecanamide, further details can be found in the attached RA justification in chapter 13.

Valid data are available to assess the teratogenicity in rats and rabbits.

Teratogenicity study with a mixture of N,N-dimethyldecanamide and N,N-dimethyloctanamide in rats:

In a study according to OECD 414 (RCC 1991) the mixture was administered orally by gavage, once daily, to mated female Wistar rats at dosages of 50, 150 or 450 mg/kg body weight/day from day 6 through to day 15 post coitum in order to assess the effects on embryonic and fetal development. In the dams at 450 mg/kg body weight/day, severe clinical signs of reaction to treatment, reduced food consumption and reduced body weight gain were observed. Additionally, at this dose level slightly increased post-implantation loss, reduced mean fetal body weights, an increased incidence of fetuses with common abnormal skeletal findings and retardations in skeletal development were noted. At 150 mg/kg body weight/day, there was a slight reduction in food consumption during the dosing period. At this dose level no effects on the maternal reproduction parameters or on the fetal parameters were noted. External and visceral examination of the fetuses, did not reveal any treatment related effects up to and including the dose level of 450 mg/kg body weight/day. Based on these results the author concluded that, the no-observed adverse effect level (NOAEL) for the maternal organism was considered to be 50 mg/kg body weight/day and for the fetal organism 150 mg/kg body weight/day and that under the conditions described in this study,the test substancedid not reveal any teratogenic potential up to and including the highest dose level of 450 mg/kg body weight/day.

Overall the study shows toxic effect to maternal and fetal organism, whereas the maternal organism reacts earlier and the fetus is only affected in higher dosages but the study did not show any teratogenic effect up to and including the highest dosage tested. The NOAELs derived were only based on weight reduction/slight reduced food comsumption by the author and considered by the applicant to be less relevant in the view of all available information.

Teratogenicity study with a mixture of N,N-dimethyldecanamide and N,N-dimethyloctanamide in rabbits:

A teratogenicity study according to OECD 414 was conducted to assess the effects of the test substance on embryonic and fetal development in pregnant Chinchilla rabbits (Bayer 1991). Therefore groups of 16 mated female rabbit received the test substance orally by gavage once daily from day 6 through to day 18 post coitum, at dose levels of:   0 mg/kg body weight/day (vehicle control), 100 mg/kg body weight/day, 300 mg/kg body and 1000 mg/kg body weight/day. The dosages were based on the results of a dose range-finding study. A standard dose volume of 4 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bi-distilled water with 0.5 % Cremophor).

Females were sacrificed on day 28 post coitum and the fetuses were removed by Caesarean section. The examination of the dams and fetuses was performed in accordance with international recommendations.

The following results were obtained:

Concerning maternal general tolerabiltiy there were no deaths, clinical signs or necropsy findings in the females of any dose group which were considered to be related to administration of the test substance. Further test article-related reduced food consumption (statistically significant between days 11-15 post coitum) was evident during the dosing period at 1000 mg/kg. During this period, the body weight gain was slightly reduced (without statistical significance) but no effects on food consumption and body weight development were noted at 100 mg/kg or at 300 mg/kg.

Concerning reproductions parameters none of the differences between the vehicle control group and any dose group noted were considered to be an effect of administration of the test substance. The differences evident were considered to be incidental because of missing dose-relation.

Concerning fetal data, during external and fresh visceral examination as well as at examination of fetal heads by Wilson technique and skeletal examination of fetuses, no abnormal findings were evident which indicated test article-related effects. The isolated common abnormal findings (with the lowest incidence at 1000 mg/kg) were within the normal range of spontaneously occurring findings in this rabbit strain and were considered to be incidental. Further the other fetal parameters recorded - sex ratios, body weights and stage of skeletal development - resulted in similar values in all groups.

Taken together no signs of teratogenicity are observed within OECD 414 studies conducted in rats and rabbits. Concluding there is no concerning teratogenicity for N,N-Dimethyldecanamid.

Key study assignment:

As there is only one reliable and relevant study per species available, this study is used as key study.

Justification for classification or non-classification

Due to citeria of legislation GHS (Regulation (EU) 1272/2008) for reproductive toxicans ("Suspected human reproductive toxicant Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information......") the substance is not be classified as reproductive toxican as there is no evidence from the current available data. Also according to DSD (67/548/EEC) the available test results led not to a classification as reproductiv toxican.

Labelling for toxicity to reproduction:

GHS: no classification

DSD: no classification

Additional information