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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 September 2020 - 02 March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
no
Remarks:
The study described in this report was conducted in a facility which operates in accordance with Good Laboratory Practice principles, however no claim of GLP compliance was intended nor is made for this study.

Test material

Constituent 1
Chemical structure
Reference substance name:
Isononanoic acid, C16-18 (even numbered)-alkyl esters
EC Number:
601-141-6
Cas Number:
111937-03-2
Molecular formula:
Unspecified
IUPAC Name:
Isononanoic acid, C16-18 (even numbered)-alkyl esters
Test material form:
liquid
Details on test material:
CAS number: 111937-03-2
Appearance: Clear, colorless liquid
Storage conditions: Ambient (15-25°C) and desiccated (protected from humidity)
Supplier: Sponsor
Batch number: 0020643508
BASF compound number 12/0400-3
Purity: 98.2 area%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
RccHan™;WIST rat.Supplier Envigo RMS (UK).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RccHan™;WIST rat.Supplier Envigo RMS (UK).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males 76 to 82 days old. Females 69 to 75 days old.
- Weight at study initiation: Males 314 to 357 g. Females 183 to 213 g.
- Fasting period before study:
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5d
ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24°C
- Humidity (%):40-70%.
- Air changes (per hr):Filtered fresh air which was passed to atmosphere and not recirculated. Change rate not reported.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed.
Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.
Frequency of preparation: Twice-weekly.
Storage of formulation: Refrigerated temperature (2 to 8°C).
Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations during storage
were confirmed as part of another study, Covance Study
Number FR58MT.

VEHICLE
- Justification for use and choice of vehicle (if other than water): common vehicle
- Concentration in vehicle: approximately 50%
- Amount of vehicle (if gavage): Volume dosed total: 4 mL/kg body weight.
Details on mating procedure:
- M/F ratio per cage: one male and one female
- Length of cohabitation: Up to two weeks, till positive evidence of mating was detected.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal
smear as day 0
- Further matings after two unsuccessful attempts:no
- After successful mating each pregnant female was caged (how): separatley
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
no
Remarks:
Formulation Analysis Stability and homogeneity The homogeneity and stability of formulations during storage were confirmed as part of another study, Covance Study Number FR58MT. No formulation analysis was performed in this study.
Details on analytical verification of doses or concentrations:
see Covance Study Number FR58MT
Duration of treatment / exposure:
Duration of Treatment:
F0 animals: For a minimum of 15 days prior to pairing until scheduled termination,after weaning of the F1 (See Section 4).
F1 animals From Day 21 of age (weaning)@ to Day 28 of age.
@ Although direct exposure started at weaning on Day 21 of age, all offspring had potential indirect exposure in-utero and through the milk during lactation.

***The following deviations from study plan occurred:
Following the ninth daily dose of the two-week (15 day) pre-pairing treatment period,
Group 3 female No. 73 (replacement animal for Group 3 female No. 62 that died) was
paired with a male in error and showed evidence of mating that night. It was considered
that the pre-pairing treatment period for this animal was too short and the gestation and
lactation data for this animal would not be suitable for reporting. Subsequently, no data
for this animal is reported and the female group size at 300 mg/kg/day was seven.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on acute toxicity and structure

Examinations

Parental animals: Observations and examinations:
Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages were inspected daily for evidence of animal ill-health amongst the
occupant(s). Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
During the acclimatization period, observations of the animals and their cages were recorded
at least once per day.

Signs Associated with Dosing:
Detailed observations were performed to establish and confirm a pattern of signs in
association with dosing according to the following schedule:
F0 animals Week 1 - daily.
Weeks 2 to 4 - twice weekly
Week 5 onwards - once each week
Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 20 of
lactation for F0 females.

Clinical Signs:
A detailed physical examination was performed on each animal to monitor general health
according to the following schedule:
F0 animals Once each week for all F0 animals.
After mating of F0 females: Days 0, 5, 12, 18 and 20 after
mating and Days 1, 7, 14 and 21 of lactation.

Mortality:
A viability check was performed near the start and end of each working day. Animals were
killed for reasons of animal welfare where necessary.

Body Weight
The weight of animals was recorded as follows:
F0 males Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded as follows:
F0 animals Twice weekly until pairing (See Section 4).
For females after mating food consumption was performed to
match the body weight recording:
Days 0-3, 3-6, 6-10, 10-14, 14-17 and 17-20 after mating.
Days 1-4, 4-7, 7-11, 11-14, 14-18 and 18-21 of lactation.

Oestrous cyclicity (parental animals):
Estrous Cycle
Dry and wet smears were taken as follows:
Dry smears From beginning of treatment until animals were paired for
mating using cotton swabs.
Wet smears After pairing until evidence of mating confirmed, using pipette
lavage.


Parturition Observations and Gestation Length:
Duration of gestation Time elapsing between the detection of mating and
commencement of parturition.
Parturition observations From Day 20 after mating, females were inspected three times
daily for evidence of parturition. The progress and completion of
parturition was monitored, numbers of live and dead offspring
were recorded and any difficulties observed were noted.
Litter observations:
Records Made During Littering Phase
The records maintained were as follows:
Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and
then daily for evidence of ill-health or reaction to treatment.
Litter size Daily records were maintained of mortality and consequent
changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than ten offspring were
reduced to eight by random culling, leaving, whenever possible,
four male and four female offspring in each litter.
Sex ratio of each litter Recorded on Days 1, 4 (before and after culling) and on Day 21
of age.
Individual offspring body
weights
Recorded on Days 1, 4 (before culling), 7, 14, 17 and 21 of age.
Selection of offspring Offspring were weaned on Day 21 of age; this was when they
were separated from the dam. The allocation of offspring to
form the F1 generation was made before weaning on Day 21 of
age.
Postmortem examinations (parental animals):
Time of Necropsy:
F0 males After litters established, after nine weeks of treatment.
F0 females Day 21 of lactation.
Unselected offspring Culled - Day 4 of age.
Scheduled kill - Day 21 of age.
Selected spares after establishment of the F1 generation.
F1 selected animals Day 29 of age.

Method of Kill:
Animals 14 days and older Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring before Day 14 of age Intraperitoneal injection of sodium pentobarbitone.
Sequence To allow satisfactory inter-group comparison.

Necropsy
All adult F0 and selected F1 animals were subject to a complete macroscopic examination;
tissues retained as specified. Abnormal tissues were retained at the discretion of necropsy
staff.
After a review of the history of each animal, a full macroscopic examination of the tissues
was performed. All external features and orifices were examined visually. Any abnormality
in the appearance or size of any organ and tissue (external and cut surface) was recorded and
the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.

Females
The following were recorded:
Each uterine horn Number of implantation sites.
Postmortem examinations (offspring):
Offspring
Premature deaths (excluding culled offspring):
Where possible, a fresh macroscopic examination with an
assessment of stomach for milk content was performed. Abnormal tissues retained in an appropriate fixative.
Culled offspring on Day 4 of age:
Culled offspring with no clinical signs on Day 4 of age were killed and discarded without necropsy examination.
Unselected F1 offspring (including selected spares):
Subject to complete macroscopic examination; tissues retained and organs weighed as specified. Any abnormal tissues retained in appropriate fixative.
Statistics:
Summary statistics (e.g. means and standard deviations) presented in this report were
calculated from computer-stored individual raw data. The summary statistics and the
individual data were stored in the computer to a certain number of decimal places, different
for each parameter. For presentation purposes, however, they were usually rounded to fewer
places. It is, therefore, not generally possible to reproduce the presented means and standard
deviations exactly using the presented individual data.
Further information on statistics is given in any other section.
Reproductive indices:
Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately
for the following: Percentage mating (%), Conception rate (%), Fertility index (%).
Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day
on which offspring were first observed, with Day 1 = day of mating for calculation purposes.
Offspring viability indices:
Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and
live at Days 1, 4 (before and after culling), 7, 14, 17 and 21 of age. Group mean litter size and
SD were calculated from the individual litter values.
Survival Indices
The following were calculated for each litter as Post implantation survival index (%).Post-implantation survival index was expressed as 100% where the number of offspring
exceeded the number of implantation sites recorded. Further Live birth index (%), Viability index (%), Lactation index (%) were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs related to treatment in males during the nine week treatment
period, or in females during the two-week pre-pairing period, gestation and lactation.
One female receiving 300 mg/kg/day and one female receiving 1000 mg/kg/day had transient
piloerection on Day 1 of lactation; however, in the absence of any other signs in any other
animals at this or lower doses, they were attributed to the parturition process that both
animals had completed shortly before.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male (No. 29) receiving 1000 mg/kg/day was killed for welfare reasons on Study
Day 45. The animal was underactive and had labored breathing, piloerection, red discharge
(blood) from nose, partially closed eyelids, hunched posture and pallor. Subsequent
macroscopic examination revealed dark contents (blood) in the stomach, jejunum, duodenum
and cecum and both kidneys were pale. The reason for the death of this animal was not
known, but may have been due to trauma and, as all other animals at this dose remained in
good clinical condition throughout the study period, it was considered not to be related to
treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gain in males during the nine week treatment period and in females
during the two-week pre-pairing period, gestation and lactation was considered to be
unaffected by treatment at 100, 300 or 1000 mg/kg/day.
The high overall body weight gain (138% of Control) during lactation at 1000 mg/kg/day was
attributed to some high individual animal gains and not related to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Overall food consumption during the two-week pre-pairing period in males and females and
during gestation and lactation was considered to be unaffected by treatment at 100, 300 or
1000 mg/kg/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
It was considered that estrous cycles, pre-coital interval, mating performance, fertility and
gestation length and gestation index were unaffected by treatment at 100, 300 or
1000 mg/kg/day.
Three females receiving 100 or 300 mg/kg/day and two receiving 1000 mg/kg/day were
acyclic (at least ten days without estrus) and one female receiving 300 mg/kg/day showed an
irregular cycle (at least one cycle of two, three or six to ten days), during the two-week
pre-pairing period. However, as all females returned to estrus and mated within 4 days of
pairing with a male, the incidence of these findings was considered not to be toxicologically
significant.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
It was considered that estrous cycles, pre-coital interval, mating performance, fertility and
gestation length and gestation index were unaffected by treatment at 100, 300 or
1000 mg/kg/day.
All females were pregnant and all, with the exception of one female (No. 71) receiving
1000 mg/kg/day (with one implantation site and failed to litter) littered within 22-23 days of
mating. As this single incidence of poor fertility was seen following one of seven pairings it
was considered not to be toxicologically significant.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: unaffeffected parameters at 1000 mg/kg bw/d

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Offspring Clinical Signs
All offspring remained in good clinical condition and the incidence of clinical observations
(dark skin colour (bruising), decreased activity, shallow breathing, thin build, little/no milk in
stomach and umbilical cord attached) showed no relationship with parental treatment at 100,
300 or 1000 mg/kg/day.
There were no clinical signs related to treatment or signs associated with dosing during the
eight-day period of direct treatment (Days 21-28 of age) at 100, 300 or 1000 mg/kg/day.
One female at 100 mg/kg/day showed vocalisation on Day 25 of age only.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Litter Size, Sex Ratio and Survival Indices
Litter size, post implantation survival index (%), live birth index (%), viability index on
Day 4 of age (%) and lactation index on Day 21 of age (%) and the ratio of male and female
offspring were unaffected by treatment at 100, 300 or 1000 mg/kg/day.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring Body Weight
Absolute body weight on Day 1 of age at 1000 mg/kg/day was marginally but statistically
significantly low (89% of Control) and was attributed to the marginally larger litter size at
this dose (110% of Control); subsequent body weight gain and absolute body weight on Day
21 of age was unaffected by treatment.
Absolute body weight on Day 1 and overall body weight gain to Day 21 of age was
unaffected by treatment at 100 or 300 mg/kg/day.
Overall body weight gain was unaffected during the eight-day period of direct treatment
(Days 21-28 of age) at 100, 300 or 1000 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Overall food consumption was unaffected during the eight-day period of direct treatment
(Days 21-28 of age) at 100, 300 or 1000 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic finding on Day 29 of age at 100, 300 or 1000 mg/kg/day.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parameters unaffected up to 1000 mg/kg bw/d

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this preliminary study, it is concluded that a high dose of
1000 mg/kg/day may be selected as the high dose in the subsequent extended one-generation
Executive summary:

The purpose of this study was to assess the influence of Isononanoic acid, C16-18-alkyl esters on reproductive performance in the Han-Wistar rat to assist in dose level selection for a main extended one-generation reproductive toxicity (EOGRTS) according to OECD443.
For the F0 generation, three groups of eight male and eight female rats received Isononanoic acid, C16-18-alkyl esters at 100, 300 or 1000 mg/kg/day by oral gavage administration at a volume dose of 4 mL/kg. Males were treated daily for 15 days before pairing and until termination. Females were treated daily for 15 days before pairing, throughout pairing, gestation and lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as the treated groups. The F1 generation that comprised of 10 male
and 10 female progeny from each group, were treated from weaning (Day 21 of age) at the same doses and volume dose as the respective F0 generation until Day 28 of age and were terminated on Day 29 of age.
Clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, parturition observations gestation length and macroscopic pathology investigations were undertaken on the F0 generation and the clinical condition, litter size and survival, sex ratio and body weight for all F1 offspring were assessed. After weaning, the F1 generation was assessed for clinical signs, body weight, food consumption and macroscopic pathology investigations.
Results
F0 generation and F1 offspring to weaning: there were no signs attributed to dosing or effect on clinical condition, body weight gain or food intake at 100, 300 or 1000 mg/kg/day and there were no treatment related macroscopic findings.
One male receiving 1000 mg/kg/day was killed for welfare reasons on Study Day 45. The death of this animal was considered not to be attributed to treatment.
Estrous cycles, pre-coital interval, mating performance, fertility, parturition and gestation length and gestation index were unaffected.
There was no adverse effect on post implantation survival, litter size, sex ratio, survival and clinical condition of the offspring to Day 21 of age.
Absolute body weight on Day 1 of age was marginally lower than Control (89%) and was attributed to the larger litter size (110% of Control). Subsequent growth was unaffected. There were no treatment related macroscopic findings in decedent or culled offspring (Days 4 or 21 of age).
F1 selected animals: there were no clinical signs or signs associated with dosing, or effect on body weight gain or food intake, during the eight-day period of direct treatment (Days 21-28 of age) and all animals were macroscopically normal on Day 29 of age.