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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Jan 2021 - 28 Jan 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
- Premating exposure duration for parental (P0) animals: For ten weeks before pairing until termination after litters were weaned.

- Basic test design: Cohort 1A and 1B without extension. Cohorts 2A and 2B (developmental neurotoxicity) and Cohort 3 (developmental immunotoxicity) were excluded, since there was no particular concern for developmental neuro- or immunotoxicity.

- Route of administration: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

- Choice of species/strain: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan®:WIST) strain was used because of the historical control data available at this laboratory.

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:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan®:WIST) strain was used because of the historical control data available at this laboratory.
Supplier: Envigo RMS Limited
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS (F0 GENERATION)
- Sex: 120 males and 120 females were ordered. Spare animals were removed from study room after treatmnet commenced.
- Age of animals at start of treatment: 28 to 34 days
- Weight range of F0 animals at start of treatment: Males 58 to 94 g. Females 57 to 89 g. At commencement of the study body weight of animals did not exceed ±20% of the mean for each sex.
- Duration of acclimatization: Six days before commencement of treatment.

SELECTION OF OFFSPRING (F1 GENERATION)
- Selection: on day 18 to 20 of age
- The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals
- For F1 Cohort 1A and 1B where possible, two male and two females were selected from each selected litter.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 40-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated
- Lighting: Artificial lighting, 12 hours light: 12 hours dark
- Housing: Solid (polycarbonate) bottom cages with a stainless steel mesh lid were used throughout the study except during pairing. During pairing grid bottomed cages were used.
- Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week
- Number of animals per cage: During acclimatization, selected F1 maturation and females after weaning and males to termination were housed in groups up to four animals of one sex. During pairing one male and one female were placed in one cage. Females after mating and during littering were housed individually (+ litter).
- Diet: ad lib (diet was removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection)
- Water: ad lib (except overnight for blood sampling for hematology, blood chemistry, urinalysis and thyroid hormone investigations for F0 and F1 Cohort 1A animals)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
TEST ITEM PREPARATION:
- Formulations were prepared once each week and used within the 8-day period of stability
- Dose: 0, 20, 250, 800 mg/kg/day
- Formulated concentration: 0, 5, 62.5, 200 mg/mL
- Volume dose: 4 mL/kg
Details on mating procedure:
- F0 pairing commenced: After ten weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups (sibling pairing was not permitted).
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating (copulation plugs in cage tray and sperm in the vaginal smear)
- Day 0 of gestation: When positive evidence of mating was detected
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Remarks:
Labcorp Study Number: 8439630 Samples of each formulation prepared for administration in Week 1 (F0 and F1 generation), Week 6 (F0 generation) and the last week of treatment (F1 generation) were analyzed for achieved concentration of the test item.
Details on analytical verification of doses or concentrations:
The mean concentrations were within 10% of the nominal concentration, confirming the
accuracy of formulation, except for Week 6 (F0 generation) Group 2 which was +15.4% from
the nominal concentration. The difference from mean and coefficient of variation remained
within 4%, confirming precise analysis except for Week 1 (F0 generation) Group 2 and
Week 6 (F0 generation) Group 3 which were 10.24% and 13.74% respectively. Two out of
three procedural recoveries remained within the range established during the validation, as
per SOP, confirming the continued accuracy of the analytical procedure.
One control sample from Week 1 (F0 generation) and two control samples from Week 6
(F0 generation) had a response at the retention time of the test item peak, therefore these have
been reported as not quantifiable (NQ).
For the Last week (F1 generation), four results for each group have been reported, which
were two original sample and two contingency samples.
Duration of treatment / exposure:
F0 ANIMALS:
For ten weeks before pairing until termination after litters were weaned.

F1 ANIMALS:
From weaning until termination of respective cohort.
- Unselected F1 offspring: Retention of organ weights. No direct treatment, killed on Day 22 of age.
- Cohort 1A : Primary assessment of effects upon reproductive systems and
of general toxicity. Treated from weaning to approximately 13 weeks of age (Day 90).
- Cohort 1B : Spare cohort. Treated from weaning to approximately 14 weeks of age.
Frequency of treatment:
Once daily at approximately the same time each day. Individual dose volume was calculated from the most recently recorded scheduled body weight. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Doses / concentrationsopen allclose all
Dose / conc.:
800 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
No. of animals per sex per dose:
F0 GENERATION:
28 animals/sex/group

F1 GENERATION:
- 1A: 20 animals/sex/group
-1B: 25 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
RATIONALE FOR DOSE LEVEL SELECTION:
The doses for this study (0, 20, 250 or 800 mg/kg/day) were selected based on the findings of the preliminary Reproductive/Development Toxicity Screening Study, in which there were no adverse compound-related effects at doses up to and including 1000 mg/kg/day and a 90 Day study. In the latter study, the test item was administered at dose levels of 0, 100, 300 or 1000 mg/kg bw/day. Findings observed at 1000 mg/kg bw/day consisted of prolonged prothrombin time in females, decreased total protein, albumin and globulin values in females, decreased calcium levels in females, increased urea and inorganic phosphate levels in females, and higher incidences of blood in the urine of male animals assumed to be related to alpha-2u-nephropathy in all male animals. Pathologically, kidney weight increase in male animals accompanied by alpha-2u-nephropathy were present in all male animals (not relevant in humans). Fatty change in kidneys of all females (minimal to slight), light brown discoloration in livers of 6 out of 10 female animals and slight to severe periportal fatty change in livers of all female animals. Less severe changes were observed at 300 mg/kg bw/day. Alpha-2u-nephropathy was present at 100 mg/kg body weight in males. No adverse effects occurred in females at this dose level.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily

DETAILED PHYSICAL EXAMINATION:
- Time schedule: once each week and F0 females on day 0, 5, 12 18 , 20 after mating and days 1, 7, 14 and 21 of lactation.

BODY WEIGHT:
- F0 males: Day that treatment commenced, each week and before necropsy.
- F0 females: Day that treatment commenced, each week until mating detected, every other day (0 - 20) after mating, on days 1, 4, 7, 14, 21 and 28 post-partum and before necropsy.

FOOD CONSUMPTION:
Weigt of food supplied to each cage, remaining and an estimate of any spilled was recorded weekly from the day that treatment commenced (not recorded during mating period).
For females after mating, food consumption was performed to match the body weight recording: Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating and days 1-4, 4-7, 7-14 and 14-21 of lactation.
Mean weekly or daily consumption per animal (g/animal/week or g/animal/day)was calculated for each relevant phase.

PARTURITION AND GESTATION:
- Duration of gestation
- Parturition observations: From day 20 after mating animals were checked 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.

MORTALITY:
Viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary or total litter loss.
A complete necropsy was performed in all cases.

OTHER: Particular attention was paid tp possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.
Oestrous cyclicity (parental animals):
DRY AND WET SMEARS:
Dry smears: For 15 days before pairing, using cotton swabs.
Wet smears: After pairing until evidence of mating confirmed. Four days before scheduled termination (days 25 to 28 post partum).

Litter observations:
CLINICAL OBSERVATIONS:
- Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.
- On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.

STANDARDISATION OF LITTERS
Performed on day 4 postpartum: Litters containing more than eight offspring were reduced to eight by random culling, leaving, whenever possible, four male and four female offspring in each litter.

SEX RATIO:
Number and sex of pups was recorded on days 1, 4 and on day 21 of age

BODY WEIGHTS:
Recorded on days 1, 4, 7, 14 and 21 of age.
Selected F1 offspring: Days 23, 25, 27 and 29.
Unselected F1 offspring: Day 22 of age.

ANO-GENITAL DISTANCE:
Day 1 of age - all offspring.

NIPPLE/AREOLAE COUNT:
Day 13 of age - male offspring.

SEXUAL MATURATION:
- Males: Daily examination from Day 38 of age until balano-preputial separation occurred.
-Females: Daily examination from Day 25 of age until vaginal opening occurred.

ESTROUS CYCLE MONITORING:
- F1 Cohort 1A: Dry and wet smears were taken as follows:
Wet smears (using pipette lavage): Following onset of vaginal patency until first cornified (estrus) smear was recorded. For at least three days prior to the start of the necropsy phase and on the day of termination.
Dry smears (using cotton swabs): For two weeks from approximately Day 75 of age.
- F1 Cohort 1B: Wet smears were taken as follows:
Wet smears (using pipette lavage): For at least three days prior to the start of the necropsy phase
and on the day of termination

SPERM ANALYSIS:
Immediately after scheduled sacrifice of each F1 Cohort 1A male and collection of blood, the
left vas deferens, epididymis and testis were removed and the epididymis and testis were
weighed. Sperm motility, morphology and count were analyzed.

HEMATOLOGY, BLOOD CHEMISTRY AND URINALYSIS:
F1 Cohort 1A: Ten male and ten female inimals per group at termination.

THYROID HORMONE ANALYSIS - TSH AND T4:
- F1 Offspring: Day 4 of age: Ten litters per group - pooled litter sample (T4). Day 22 of age: Ten male and ten female animals per group.
- F1 adults (cohort 1A): Ten male and ten female animals per group


Postmortem examinations (parental animals):
TIME OF NECROPSY:
- F0 males. After weaning of the F1 animals, after confirmation that no further mating required.
- F0 females. Day 28 post partum.
- F0 females failing to produce a viable litter and those with total litter loss.Terminated with first cohort of females with live litters.
- Unselected offspring F1 litters: Culled on Day 4, and Day 22 of age.
- Cohort 1A animals. At approximately 13 weeks of age.
- Cohort 1B animals. At approximately 14 weeks of age.

HISTOPATHOLOGY / ORGAN WEIGHTS
All animals were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. The following tissues and regions were examined: Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femurs (femorotibial joint and including bone marrow), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Optic nerves, Ovaries, Pancreas, Pituitary, Prostate (dorsolateral and ventral combined), Rectum, Sciatic nerves, Seminal vesicles (with coagulating gland), Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum - bone marrow, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix and oviducts, Vagina, Vas Deferens
Postmortem examinations (offspring):
HISTOPATHOLOGY / ORGAN WEIGHTS
All animals were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents offspring at 21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained.
For females of Cohort 1A, counts were performed for the number of ovarian follicles and
corpora lutea.
For males of Cohort 1A, sperm motility, morphology and count were analyzed.
The following tissues and regions were examined:
- Cohort 1A: Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides (only one examined), Esophagus, Eyes, Femurs (femorotibial joint and including bone marrow), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (mesenteric, left axillary), Optic nerves, Ovaries, Pancreas, Pituitary, Prostate (dorsolateral and ventral combined), Rectum, Sciatic nerves, Seminal vesicles (with coagulating gland), Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen (half of the spleen preserved for histopathological examination; half used for splenic lymphocyte subpopulation analysis), Sternum - bone marrow, Stomach, Testes (only one examined), Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix and oviducts, Vagina, Vas Deferens (only one examined)
- Cohort 1B: Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femurs (femorotibial joint and including bone marrow), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (mesenteric - left axillary), Optic nerves, Ovaries, Pancreas, Pituitary, Prostate (dorsolateral and ventral combined), Rectum, Sciatic nerves, Seminal vesicles (with coagulating glands), Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum - bone marrow, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix and oviducts , Vagina, Vas Deferens
-Unselected F1 offspring: Abnormalities, Brain (cerebellum, cerebrum, midbrain), Epididymides, Ovaries, Pituitary, Prostate (dorsolateral and ventral combined, Seminal vesicles (with coagulation gland), Skin with mammary glands (inguinal area), Spleen, Testes, Thymus, Uterus with cervix and oviducts, Vagina
Statistics:
See below.
Reproductive indices:
Individual data was tabulated. Group values were calculated for males and females separately
for the following:
PERCENTAGE MATING (%) = Number of animals mating / animals paired x 100.
CONCEPTION RATE (%) = Number of animals achieving pregnancy / Animals mated x 100.
FERTILITY INDEX (%) = Number of animals achieving pregnancy / Animals paired x 100.
GESTATION INDEX (%) = Number of live litters born / Number pregnant x 100.
Offspring viability indices:
POST IMPLANTATION SURVIVAL INDEX (%) = Total number of offspring born/ Total number of uterine implantation sites x 100 (Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded)

LIVE BIRTH INDEX (%) = Number of offspring on Day 1 after littering / Total number of offspring born x 100.
VIABILITY INDEX (%) = Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering x 100.
LACTATION INDEX (%) = Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling) x 100.
Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The litter from one female (No. 309) receiving 800 mg/kg/day were killed for welfare reasons
following parturition and before LD 1. There were no macroscopic findings or histopathological changes in the dam, to account for the loss of the litter and it was therefore considered not to be related to treatment.
One female (No. 265) receiving 250 mg/kg/day that was paired with a male for mating in Week 10 remained in pairing for 14 nights, but showed no evidence of mating. The animals were separated and during the following two days (Days 86 and 87) the female showed bleeding from the vagina and was killed for welfare reasons on Day 87. There were no macroscopic findings and the uterus contained 12 implantation sites. The death of this animal was not attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall body weight gain during lactation at 800 mg/kg/day was statistically significantly high (138% of Control); however, as absolute bodyweight on LD 28 was similar to Control it was considered not to be toxicologically important.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake in males receiving 800 mg/kg/day was marginally higher than Control
throughout the study period (Weeks 1-17), but the overall difference from Control did not
attain statistical significance. Overall food intake in females receiving 800 mg/kg/day was marginally, but statistically
significantly high (108% of Control) during the pre-pairing period (Weeks 1-10).
Overall food consumption in treated males and in females at 20 or 250 mg/kg/day was
unaffected by treatment.Food conversion efficiency during the pre-pairing period (Weeks 1-10) was unaffected by
treatment at 20, 250 or 800 mg/kg/day.
Overall food consumption during gestation (GD 0-20) was marginally, but statistically
significantly high at 800 mg/kg/day (113% of Control). Food intake at 20 or 250 mg/kg/day
was unaffected by treatment.
Food conversion efficiency during gestation (GD 0-20) was unaffected by treatment at 20,
250 or 800 mg/kg/day.
Overall food consumption during lactation (LD 7-21) was marginally, but statistically
significantly high at 800 mg/kg/day (108% of Control). Food intake at 20 or 250 mg/kg/day
was unaffected by treatment.
Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion efficiency during the pre-pairing period (Weeks 1-10) was unaffected by
treatment at 20, 250 or 800 mg/kg/day.
Food conversion efficiency during gestation (GD 0-20) was unaffected by treatment at 20,
250 or 800 mg/kg/day
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no clear toxicologically significant differences in the cellular composition of the blood, following treatment at 20, 250 or 800 mg/kg/day to males for 17 weeks and females on LD 28. All differences that attained statistical significance from Control were evident in males only and considered minor, or failed to show relationship to dose.
At 800 mg/kg/day, neutrophil count and subsequent overall white cell count were statistically significantly high (167% and 132% of Control, respectively). Mean cell volume was marginally, but statistically significantly low (97%) and red cell distribution width was marginally, but statistically significantly high (104%). When compared with Control, prothrombin time at 20, 250 or 800 mg/kg/day was marginally, but statistically significantly short (89%, 90% or 86%) and failed to show clear relationship with dose. There were no statistically or toxicologically significant differences from Control in females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males only at 250 or 800 mg/kg/day, urea concentrations were slightly high (113% or 123% of Control, respectively); attaining statistical significance at 800 mg/kg/day. There were no other toxicologically significant differences in the constituents of the blood, following treatment at 20, 250 or 800 mg/kg/day to males for 17 weeks and females on LD 28.
In males only at 800 mg/kg/day, cholesterol and calcium concentrations were slightly, but
statistically significantly high (136% and 103% of Control, respectively). At 20, 250 or
800 mg/kg/day, chloride concentrations were marginally, but statistically significantly low
(all 98% of Control). Total protein concentration at 250 or 800 mg/kg/day were marginally,
but statistically significantly high (103% or 106% of Control, respectively) and
albumin/globulin ratio at 800 mg/kg/day was statistically significantly low (90% of Control).
In females only, alanine amino-transferase activity was statistically significantly high at 20,
250 or 800 mg/kg/day (185%, 192% or 169% of Control, respectively), but failed to show
relationship to dose. Bilirubin concentration was statistically significantly high at
800 mg/kg/day and potassium concentration was statistically significantly high without
relationship to dose at 20, 250 or 800 mg/kg/day (108%, 113% or 107% of Control,
respectively).
Calcium concentration was marginally, but statistically significantly high (103% of Control)
in females only at 20 mg/kg/day.

All differences that attained statistical significance from Control were evident in only one sex and considered minor, or failed to show relationship to dose.
Endocrine findings:
no effects observed
Description (incidence and severity):
Serum concentrations of T4 were statistically significantly high in F0 generation males
treated at 250 or 800 mg/kg/day and were higher than the Historical Control Data (HCD)
range. However, there was no clear relationship to dose and T4 concentrations were
unaffected by parental treatment in the F1 offspring (PND 22) or in F1 (Cohort 1A) adults at
approximately 13 weeks of age was therefore judged non-adverse.
Serum concentrations of TSH were unaffected by treatment in the F0 or F1 generations at 20,
250 or 800 mg/kg/day.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Total urinary protein was statistically significantly high in males only at 800 mg/kg/day (177% of Control).
There were no other toxicologically significant differences in the composition of the urine, following treatment at 20, 250 or 800 mg/kg/day to males for 17 weeks and females on LD 28.
In males only at 800 mg/kg/day, urinary volume and total protein were statistically
significantly high (143% or 177% of Control, respectively) and total sodium concentration
was statistically significantly high (152% of Control). In females at 20, 250 or
800 mg/kg/day, total sodium and potassium concentrations were statistically significantly. high, without relationship to dose (174%, 174% or 124% and 155%, 152% or 122% of
Control, respectively). Chloride concentration was statistically significantly high at 20 or
250 mg/kg/day (172% or 173% of Control, respectively). All differences that attained statistical significance from Control were considered minor, or failed to show relationship to dose.
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to administration of Isononanoic acid, C16-18-alkyl esters were present in the kidneys of males and females and the liver and pancreas of females.
In males given 250 and 800 mg/kg/day, hyaline droplet accumulation was present in the kidney tubules with associated tubular basophilia and granular cast formation. These changes are consistent with alpha-2u-nephropathy, a common finding in untreated male rats (Khan KNM et al., 2002), clearance or accumulation of which can be altered when it is bound to a xenobiotic. Accumulation of this complex can lead to tubular damage and this is described as alpha-2µ-globulin nephropathy. Alpha-2µ-globulin is an androgen regulated by the liver of male rats only and therefore it’s accumulation and subsequent tubular damage is both sex and species specific (Alden et al 1984; Frazier et al 2012; Montgomery and Seely 1990; Short et al 1989; Swenberg et al 1989). In the context of this study, the hyaline droplet accumulation and subsequent associated pathology were considered to be adverse in the animals affected as degenerate changes were seen, but, due to the reasons specified above, are considered irrelevant in man.
In females given 800 mg/kg/day, vacuolation was present in the periportal region of the liver and the cortical tubules in the kidneys. Vacuolation of the cortical tubules of the kidney was also present in females given 250 mg/kg/day.
Secretory depletion in the acinar cells of the pancreas was present at a low incidence in females given 800 mg/kg/day. The occurrence in one female given 250 mg/kg/day is considered incidental.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycles during the two-week period before pairing and during LD 25-28 were unaffected by treatment at 20, 250 or 800 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance, Fertility index (%), Gestation length and gestation Index were unaffected by parental treatment at 20, 250 or 800 mg/kg/day.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 800 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No effects on fertility and reproduction at highest tested dosage

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Live birth index and the ratio of male to female offspring were unaffected by parental treatment at 20, 250 or 800 mg/kg/day.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were five decedents in the F1 generation, none were related to treatment.
One Control male (No. 420) was found dead on Day 11; macroscopic examination revealed the esophagus was perforated and the thoracic cavity contained clear fluid and adhesions involving multiple organs. The death of this animal was attributed to mis-dosing and not related to treatment.
One Control female (No. 803) was killed for welfare reasons on Day 26 due to biting and swallowing part of the dosing cannula. Macroscopic examination confirmed that the cannula
was retained in the stomach. The death of this animal was accidental and therefore not related to treatment.
One male (No. 424) receiving 20 mg/kg/day had a distended abdomen during Days 24-27 and was abnormally warm to touch on Day 27 and was killed for welfare reasons on Day 27. Macroscopic examination revealed adhesion of abdominal organ (not determined) and yellow
abdominal contents. Microscopic examination revealed that the cause of death was peritonitis
which may have been related to notable pelvic dilation in the kidneys and therefore the death of this animal was considered not to be related to treatment.
One male (No. 456) receiving 250 mg/kg/day was found dead on Day 10, with no previous clinical history. Macroscopic examination revealed the esophagus was perforated and the thoracic cavity contained clear fluid and pale caseous material. The death of this animal was attributed to mis-dosing and not related to treatment.
One female (No. 952) receiving 250 mg/kg/day had decreased activity, vocalisation, irregular
breathing, piloerection, dull eyes and whole body pallor and was killed for welfare reasons on Day 72. Macroscopic examination revealed the esophagus was perforated and the thoracic cavity contained clear fluid and pale caseous material, an edematous thymus and depressions in the glandular mucosa region of the stomach. The death of this animal was attributed to mis-dosing and not related to treatment.
One male (No. 437) was removed from the study on the third day of the Formal F1 generation, as macroscopic examination showed that the cause of death was mis-dose. The animal was killed for welfare reasons shortly after dosing at 20 mg/kg/day. The animalshowed decreased activity, rapid respiration, piloerection, and pale eyes and macroscopic examination revealed thin, red fluid in the thoracic cavity. A selected spare male (No. 583) was allocated to the study to replace this animal.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Offspring Body Weight
Absolute bodyweight on PND 1 was unaffected by parental treatment at 20, 250 or
800 mg/kg/day.
Overall body weight gain (PND 1-21) was marginally, but statistically significantly high in
males and females at 800 mg/kg/day (107% and 108% of Control, respectively) and in
females at 250 mg/kg/day (106%), however, the differences from Control were minor and
therefore not toxicologically significant.
Overall body weight gain was unaffected by parental treatment in males and females at
20 mg/kg/day or in males at 250 mg/kg/day.

F1 Generation Bodyweight:
Absolute body weight of selected males and females on PND 21 was marginally, but statistically significantly high at 800 mg/kg/day (104% and 107% of Control, respectively). Body weight gain following weaning (PND 21) to PND 25 was marginally but statistically significantly low in males at 250 or 800 mg/kg/day (both 94% of Control) and was similarly low in females at 800 mg/kg/day (93% of Control). However as absolute body weight of males and females at commencement of the Formal F1 generation (Week 4 of age) at all doses were similar to Control, a relationship to treatment is not inferred.
Overall body weight gain to termination was unaffected by treatment at 20, 250 or 800 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall food consumption to termination was statistically significantly high in males and females (106% and 108% of Control, respectively) at 800 mg/kg/day; however, the difference from Control was minor and therefore not toxicologically significant.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant differences in the cellular composition of the blood following 10 weeks of treatment (approximately Week 13 of age) to males and females at 20,
250 or 800 mg/kg/day. All differences that attained statistical significance from Control were
evident in only one sex and considered minor.
At 800 mg/kg/day, hemoglobin concentration was marginally, but statistically significantly
low (97% of Control) in males and lymphocyte count was marginally, but statistically
significantly low (78% of Control) in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Following 10 weeks of treatment (approximately Week 13 of age) to males and females at 800 mg/kg/day, urea concentration was marginally, but statistically significantly high in males and females, when compared with Control (122% and 113%, respectively).
There were no other toxicologically significant differences in the constituents of the blood plasma at 20, 250 or 800 mg/kg/day. All differences that attained statistical significance from Control were evident in only one sex and were considered minor, or failed to show relationship to dose.
In males only, alkaline phosphatase activity was statistically significantly high at 250 or 800 mg/kg/day (139% or 113% of Control, respectively) and aspartate amino-transferase activity was statistically significantly high at 800 mg/kg/day (134% of Control). Sodium concentration was marginally, but statistically significantly high at 800 mg/kg/day (101% of Control). In females, glucose concentration was marginally, but statistically significantly low without relationship to dose at 250 or 800 mg/kg/day (82% or 86% of Control, respectively) and phosphorous concentration was marginally, but statistically significantly high at 250 or 800 mg/kg/day (124% or 117% of Control, respectively).
When compared with Control, potassium concentration was marginally, but statistically significantly high in males at 250 or 800 mg/kg/day (108% or 107%, respectively) and in females at 20, 250 or 800 mg/kg/day (109%, 116% or 111%, respectively); however the differences failed to show relationship to dose.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant differences in the composition of the urine following 10 weeks of treatment (approximately Week 13 of age) to males and females at 20, 250 or 800 mg/kg/day. The only difference that attained statistical significance from Control was the low total chloride concentration in males only at 800 mg/kg/day (69%).
Sexual maturation:
no effects observed
Description (incidence and severity):
Vaginal Opening - Cohort 1A
The interval between vaginal opening and first estrus was unaffected by treatment at 20,
250 or 800 mg/kg/day.
Estrous Cycles - Cohort 1A
Estrous cycles from Day 75 of age and the stage of the cycle at termination were unaffected
by treatment at 20, 250 or 800 mg/kg/day.
Estrous Cycles - Cohort 1B
Estrous cycles during the 4-day period before termination at approximately 14 weeks of age
were unaffected by treatment at 20, 250 or 800 mg/kg/day.
Females: Ovarian follicle count was unaffected by treatment at 800 mg/lg/day.
Corpora lutea count at 800 mg/kg/day was statistically significantly higher than Control (120%). Individual animal values showed wide variation in both the Control and treated group (Control: 9-28 (Animal No. 808 considered atypically low, considered introduced by histological processing and was excluded); 800 mg/kg/day: 11-37). The significance of the higher corpora lutea count, in the absence of an effect on ovarian follicle count and test item related microscopic changes in the ovaries, was considered due to chance and therefore not toxicologically significant.

Males: Sperm motility, morphology or testicular or cauda epididymis sperm counts were unaffected by treatment in the F1 generation.
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: There was a statistically significantly higher kidney weight, both absolute and body weight relative, in males given 250 (109 and 108%) and 800 mg/kg/day (109% both). There was a statistically significantly higher liver weight in females, both absolute and body weight relative in those given 800 mg/kg/day (121 and 120%) and body weight relative only in those given 250 mg/kg/day (113%).
A statistically significantly lower absolute and body weight relative pituitary weight was observed in males given 250 mg/kg/day (89 and 92%) and 800 mg/kg/day (89 and 92%) and a higher epididymides weight which reached statistical significance relative to body weight in those given 800 mg/kg/day (108%). There was no histological correlate in the pituitary or epididymides and thus, these variations were not considered to be toxicologically significant.
Cohort 1B: There was a small, but statistically significantly higher absolute weight of the epididymides of males and the uterus/cervix of females given 800 mg/kg/day. After adjustment for body weight these changes were no longer of statistical significance. All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or relative to body weight but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Changes related to administration of Isononanoic acid, C16-18-alkyl esters were present in the kidneys of males and females and the liver and pancreas of females.In males given 250 and 800 mg/kg/day, hyaline droplet accumulation was present in the kidney tubules with associated tubular basophilia and granular cast formation. In females given 250 and 800 mg/kg/day, vacuolation was present in the periportal region of the liver and the cortical tubules in the kidneys.
Secretory depletion was present in the pancreas of females given 250 and 800 mg/kg/day. All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this animal strain. Therefore, they were
considered not test article related.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 800 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: No effects on fertility and reproduction at highest tested dosage

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity relevant to human health, in the F0 and F1 generations and for reproductive performance of the F0 generation and survival and growth of the F1 offspring was 800 mg/kg/day.
The NOAEL for kidney changes in male rats, consistent with alpha-2u-nephropathy and considered to be adverse in the animals affected was 20 mg/kg/day.
Executive summary:

The purpose of this study was to assess the influence of Isononanoic acid, C16-18-alkyl esters on reproductive performance when administered by oral gavage to Han Wistar rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrus cycles.


In the F0 generation, three groups of 28 male and 28 female rats received Isononanoic acid, C16-18-alkyl esters at dose levels of 20, 250 or 800 mg/kg/day by oral gavage administration at a volume dose of 4 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In the F1 generation, 45 males and 45 females were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. A similarly constituted Control group in each generation received the vehicle, corn oil, at the same volume dose as the treated groups and throughout the same relevant period.



For the F0 generation data were recorded on clinical observations, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and
parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid-related hormones, sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.


For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age. Serum samples that were collected from selected offspring on Day 22 of age were analyzed for thyroid-related hormones.


The F1 generation comprised of two cohorts:


For F1 Cohort 1A, data were recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid-related hormones, sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and spleen immunophenotyping investigations were performed.


For F1 Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Organ weight and macroscopic pathology
investigations were performed.



Results
There were no treatment related deaths, no signs associated with dosing and no treatment related clinical signs in the F0 generation. The clinical condition of the F1 generation offspring to weaning (PND 21) was unaffected by treatment and there were no treatment related deaths, no signs associated with dosing and no treatment related clinical signs in the F1 generation. There were no treatment related macroscopic findings in the F0 adults or in the F1 offspring at any life-stage.


Overall body weight gain in males and in unmated and pregnant females in the F0 generation and in the Formal F1 generation were unaffected by treatment.
Body weight gain during lactation in the F0 generation at 20, 250 or 800 mg/kg/day was marginally high and overall body weight gain of the F1 offspring to PND 21 was marginally high at in males and females at 800 mg/kg/day and in females at 250 mg/kg/day; however, these differences from Control were considered not to be toxicologically significant.

Food consumption in F0 and F1 generation males and females was unaffected by treatment. Food consumption during gestation and lactation in F0 females was marginally high; however, overall food conversion efficiency during all life stages of the F0 generation and in the F1 generation was unaffected by treatment.


Estrous cycles in the F0 and F1 generations and the interval between vaginal opening and first estrus in the F1 generation were unaffected by treatment.


Pre-coital interval, mating performance and fertility, gestation length and gestation index and the number of implantations were unaffected by treatment in the F0 generation.


Higher numbers of litters at 250 and 800 mg/kg/day had death of one or more offspring following parturition to PND 1 in the F0 generation (9 and 5 litters v 2 litters in the Control, respectively); however there was no relationship to dose and mean litter sizes and subsequent survival of the offspring was unaffected; therefore a relationship to treatment was not inferred.

The ratio of male to female offspring and ano-genital distance was unaffected by treatment to the F0 generation and no male offspring had nipples.


Maturation of the F1 generation was unaffected by treatment.


Hematology in the F0 and f1 generations was unaffected by treatment.

Serum concentrations of T4 and TSH in the F0 and F1 generation adults and serum concentrations of T4 and TSH in the F1 offspring on PND 22 were unaffected by treatment.


Sperm motility, morphology or testicular or cauda epididymis sperm counts were unaffected by treatment in the F1 generation.


Corpora lutea count in the F1 generation at 800 mg/kg/day was slightly high, but this was considered due to chance.


At 800 mg/kg/day, total urinary protein was high in F0 generation males and urea concentration was high in F0 and F1 generation males and F1 females.


Kidney weight was high in F0 and F1 generation males at 250 or 800 mg/kg/day and there
was an accumulation of hyaline droplets in the tubules of males at these doses with associated
nephropathy, consistent with alpha-2u-nephropathy that was considered to be adverse in the
animals affected. However, the hyaline droplet accumulation and subsequent associated
pathology are considered irrelevant in man.


Liver weight was high in F0 generation females at 800 mg/kg/day and F1 generation females at 250 or 800 mg/kg/day, both correlated with vacuolation of hepatocytes in the periportal region that was considered non-adverse.

Secretory depletion of the acinar cells in the pancreas was present in occasional females given 250 and 800 mg/kg/day that was considered likely to be an adaptive change.



Conclusion
Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity relevant to human health, in the F0 and F1 generations and for reproductive performance of the F0 generation and survival and growth of the F1 offspring was 800 mg/kg/day.
The NOAEL for kidney changes in male rats, consistent with alpha-2u-nephropathy and considered to be adverse in the animals affected was 20 mg/kg/day.