Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility information (OECD 408, with extended protocol and OECD 443 rangefinder)


NOAEL systemic = 100 mg/kg bw/day


NOAEL fertility, reproduction ≥ 1000 mg/kg bw/day

Link to relevant study records
Reference
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
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.
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.
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
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
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.
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: 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.
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
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.
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
Key result
Reproductive effects observed:
no
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.


Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Isononanoic acid, C16-18 alkyl esters (CAS 111937-03-2) is a substance in Cramer class I, indicating low concern with respect to systemic toxicity. In the 28-day repeated dose toxicity study, no toxicologically relevant effects were observed up to and including the highest dose level of 1000 mg/kg bw/day (Pitterman, 1993). In a developmental study with a study protocol similar to OECD 414, the test substance was administered to pregnant rats on gestation day 6-15 in doses up to 1000 mg/kg bw/day (Pitterman, 1997). No treatment-related effects on reproduction parameters or systemic toxicity were noted in the P-females, and no developmental effects were observed in the F1-pups up to and including the highest dose level of 1000 mg/kg bw/day (limit dose).


 


Futher in a GLP-compliant subchronic toxicity study in the rat via the oral route (OECD 408, GLP) (BASF SE 2018) an extended standard protocol with additional sperm motility parameters was performed and careful examination of reproductive organs/tissues to assess the potential effects of isononanoic acid isononanoate on fertility was performed. In summary there were no effects observed which influence the fertility up to and including the highest tested dosage of 1000 mg/kg bw/d.


 


Further within a similar to OECD 421 conducted study 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.
The following results are observed:
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.


Based on the results of this preliminary study, it is concluded that a high dose of
1000 mg/kg/day may be a NOAEL and can be selected as the high dose in the subsequent extended one-generation reproduction study.


 


EOGRT is ongoing and will be reported soon, see waiver.


 


A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.



Short description of key information:
Developmental toxicity study (OECD 414), rat, oral:
NOAEL systemic (P, F1, OECD 443 rangefinder) ≥ 1000 mg/kg bw/day
NOAEL fertility (P, OECD 443 rangefinder) ≥ 1000 mg/kg bw/day
NOAEL reproduction (P, OECD 443 rangefinder) ≥ 1000 mg/kg bw/day


Fertility information (OECD 408, with extended protocol and OECD 443 rangefinder)


NOAEL systemic = 100 mg/kg bw/day


NOAEL fertility ≥ 1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information
Prenatal developmental toxicity study (OECD 414), rat, oral:
NOAEL teratogenicity (F1) ≥ 1000 mg/kg bw/day
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jun - 21 Jul 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study with acceptable restrictions. Short treatment period (Day 6-15 of gestation), body weight was recorded on Day 0, 6, 16 and 20 only, food consumption was not recorded, the analytical purity of the test substance was not specified.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
exposure from Day 6 to Day 15 of gestation, body weight was recorded on Day 0, 6, 16 and 20, food consumption was not recorded
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley, CD
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 202.6 ± 20.4 g - 219 ± 25.6 g (range of group mean values)
- Housing: the animals were housed individually in Makrolon Type M3 cages (Ebeco, Castrop-Rauxel, Germany), on standard softwood bedding (ARWI-Center, Essen, Germany)
- Diet: pelleted Altromin Maintenance Diet 1324 (Fa. Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 48-82
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (lux units 50 - 550)

IN-LIFE DATES: From: 28 Jun 1994 To: 21 Jul 1994
Route of administration:
oral: gavage
Vehicle:
other: 0.5% sodium carboxymethylcellulose + 0.25% Cremophor in aqua dest.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared daily before administration, adjusted to the body weight measured on Day 6 of gestation.

VEHICLE
- Concentration in vehicle: 1, 3, 10% (10, 30, 100 mg/mL)
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mixture of the test item was analysed once to verify the actual concentration. The measured concentration was within the expected range. The nominal concentrations 1 % (100 mg/kg bw/day), 3 % (300 mg/kg bw/day) and 10 % (1000 mg/kg bw/day) were measured to be 1.1%, 3.0% and 10.5%, respectively.
Details on mating procedure:
- Any other deviations from standard protocol: Primiparous time-mated females were used. The females were mated at the supplier with an accurate day of mating and received at the testing facility on gestation day 0.
Duration of treatment / exposure:
Day 6-15 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
10 days; Day 6-15 of gestation
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal dose
No. of animals per sex per dose:
23 P females (100 and 300 mg/kg bw/day)
24 P females (control, 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0 (prior to administration), 6, 16 and 20 of gestation

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross macroscopic examination of all reproductive and gender-specific organs, with emphasis on the uterus, uterine contents, position of the 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: Yes
- Number of late resorptions: Yes
- Other: weight of fetuses
Fetal examinations:
- External examinations: Yes, all fetuses
- Soft tissue examinations: Yes, half per litter (146 fetuses of group 1, 133 fetuses in group 2, 169 fetuses in group 3 and 162 fetuses in group 4)
- Skeletal examinations: Yes, half per litter (159 fetuses of group 1, 144 fetuses in group 2, 178 fetuses in group 3 and 172 fetuses in group 4)
- Head examinations: No

Group 1: control
Group 2: 100 mg/kg bw/day
Group 3: 300 mg/kg bw/day
Group 4: 1000 mg/kg bw/day
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on pooled variance, was applied for comparion between groups. The Steel-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 (Bonferroni-Holm-corrected).
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There was no mortality during the study period. No signs of systemic toxicity were observed. The mean body weight of the mid-dose group was statistically significantly increased on Day 16 and 20 of the study period (see Table 1). This considered to be an incidental observation, as the body weight gain over the study period was comparable between the control and treatment groups (58.9, 67.8, 57.4 and 59.6 g for the control, 100, 300 and 1000 mg/kg bw/day group, respectively). One female in each of the control group and the low-dose group was not pregnant, while all the pregnant females had viable fetuses (see Table 2).

No substance-related effects on the reproductive parameters (number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss, embryonic deaths, embryonic resorptions, fetal resorptions, live fetuses, dead fetuses) were observed (see Table 3). The necropsy and macroscopic examination did not show any treatment-related effects. One female in the high-dose group had blood in the uterine horn, but this is not considered to be treatment-related as no other effects were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The external examination of the foetuses did not reveal any treatment-related macroscopical effects. The skeletal examination showed a statistically significant increase in the number of foetuses with 6 ossified sternebrae in the high-dose group (see Table 5). As there were no increases in ossification anywhere else and no overall increase in abnormal findings for this group, the result is considered to be incidental. The results for the remaining offspring parameters (body weight, placental weight, sex ratio) were comparable between the control and treatment groups.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Body weights

Dose group

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Gestation day 0

212.3

202.6

219.7

209.0

Gestation day 6

263.1

255.5

273.5

259.4

Gestation day 16

340.7

338.6

361.7*

341.5

Gestation day 20

403.3

400.7

428.0*

407.9

*Dunnett-Test based on pooled variance, p < 0.05

 

Table 2: Summary of mating performance of the females

Dose group

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

No. of mated females

24

23

23

24

No. of pregnant females*

23

22

23

24

No. of premature litters**

0

0

0

0

No. of mortalities

0

0

0

0

*Included in the statistical analysis

**Premature litter is an event in the cage immediately before the caesarean section

 

Table 3: Reproduction parameters for dams with live foetuses

Dose group

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Number of dams

23

22

23

24

Corpora lutea (total)

376

343

403

390

Corpora lutea (mean±SD)

16.3±1.5

15.6±1.2

17.5±1.6

16.2±2.3

Implantation sites (total)

316

301

367

346

Implantation sitesaas:

-% of corp. lutea

- mean±SD

 

84.0

13.7±3.7

 

87.8

13.7±2.5

 

91.1

16.0±1.9

 

88.7

14.4±3.1

Pre-implantation loss (total)b

60

42

36*

44

Pre-Implantation lossas

% of corpora lutea

16.0

12.2

8.9

11.3

Post-implantation lossb

11

24

20

12

Post-implantation loss as

% of implantation sites

3.5

8.0

5.4

3.5

Embryonic deaths totalb

11

24

20

12

Embryonic resorptions (total)a

9

18

20

10

Embryonic resorptions as

% of implantation sites (mean±SD)

2.8±0.4

6.0±0.8

5.4±0.9

2.9±0.4

Foetal resorptions (total)a

2

6

0

2

Foetal resorptionsas

% of implantation sites(mean±SD)

0.6±0.1

2.0±0.3

0

0.6±0.1

Fetuses per dam (mean±SD)

13.3±3.6

12.6±3.1

15.1±2.4

13.9±2.7

Live foetusesa

305

277

347

334

Dead foetusesa

0

0

0

0

Malformed foetuses

0

0

0

0

Uterus weightc(mean±SD)

81.3±24.1

77.4±18.0

97.0±15.7*

88.8±18.3

aSteel Test

bFishers Exact Test (Bonferroni-Holm-Corrected)

cDunnett-Test based on pooled variance

*p < 0.05

 

Table 4: Developmental parameters for offspring

Dose group

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Number of live foetuses (m/f)

305 (154/151)

277 (141/136)

347 (162/185)

334 (165/169)

Sex ratio (m/f)

0.51/0.49

0.51/0.49

0.47/0.53

0.49/0.51

Weights of live foetuses

(mean±SD)

4.1±0.8

4.1±0.6

4.4±0.8

4.2±0.6

No. of runts

1

2

1

2

Table 5: results of skeletal examination of offspring

Dose group

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

No abnormal findings, number (% of total)

10 (6.3%)

12 (8.3%)

32 (18.0%)**

10 (5.8%)

6 ossified sternebrae, number (% of total)

124 (78.0%)

120 (83.3%)

151 (84.8%)

153 (89%)**

** Fishers Exact Test (two-sided), p < 0.01

 

Conclusions:
The test substance had no effect on intrauterine development
CLP: not classified
DSD: not classified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfill the standard information requirements set out in Annex VIII-IX, 8.7 of Regulation (EC) 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction – (pre-natal) development

The potential of isononanoic acid, C16-18 alkyl esters (CAS 111937-03-2) to cause developmental toxicity was assessed in a study performed using a study protocol similar to OECD guideline 414 (Pitterman, 1997). 23-24 pregnant female Sprague-Dawley rats/dose level were administered 0, 100, 300 and 1000 mg/kg bw/day of the test substance by gavage on gestation Day 6 to 15.

No signs of systemic toxicity were observed in the P-females and no treatment-related effects were observed. One female in the high-dose group had blood in the uterine horn, but this is not considered to be treatment-related as no related effects were observed. One female in each of the control group and the low-dose group did not become pregnant, while all the pregnant females had viable foetuses. No treatment-related effects on the reproductive parameters (number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss, embryonic deaths, embryonic resorptions, foetal resorptions, live foetuses, dead foetuses) were observed.

The external examination of the F1-foetuses did not reveal any treatment-related macroscopic findings. The skeletal examination showed a statistically significant increase in the number of foetuses with 6 ossified sternebrae in the mid-dose group (300 mg/kg bw/day). As there were no increases in ossification anywhere else and no overall increase in abnormal findings for this group, the result is considered to be incidental. The results for the remaining offspring parameters (body weight, placental weight, sex ratio) were comparable between the control and treatment groups. 

The NOAEL developmental is considered to be ≥ 1000 mg/kg bw/day.

The most recent version of OECD guideline 414, adopted in January 2001, stipulates that the dams should be dosed from implantation (gestation Day 0) to the day prior to scheduled caesarean section (around gestation day 19 for rats). The study was performed according to the previous version of OECD guideline 414, adopted in May 1981, which recommended exposure during organogenesis; gestation Day 6-15 for rats. The available data on repeated dose toxicity and developmental toxicity is of high quality and does not show treatment-related adverse effects at dose levels up to and including the highest dose of 1000 mg/kg bw/day, indicating that adverse effects are unlikely to occur in the period prior to Day 6 of gestation (Pitterman, 1993, 1997). Based on the available it is not considered necessary to perform an additional, dedicated, developmental toxicity study.

Conclusions for developmental toxicity

A developmental toxicity study performed with isononanoic acid, C16-18 alkyl esters (CAS 111937-03-2), using a protocol similar to OECD guideline 414 is available (Pitterman, 1997). Pregnant rats were administered the test substance from gestation Day 6-15. No effects on development (survival, body weight, sex ratio, external appearance, skeletal development) in the P1-pups were observed up to and including the highest dose level of 1000 mg/kg bw/day.

Thus, isononanoic acid, C16-18 alkyl esters is not considered to cause adverse effects on intrauterine development.

A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and within CSR.


Justification for selection of Effect on developmental toxicity: via oral route:
There is only one study available.

Justification for classification or non-classification

The available data on the toxicity to reproduction of the substance do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.

Additional information