3,5,5-trimethylhexanoic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on fertility were observed in three reliable studies in rats which were performed according to or similar to guideline and GLP. Effects on development (lower post implantation survival index and low live birth index) but not on fertility (including mating index, gestation length, estrous cycle and sperm parameters) were observed in an OECD 443 (EOGRTS; Covance, 2019) study at maternal toxic dose (120 mg/kg bw/d by gavage; five females were killed for animal welfare reasons during late gestation or early lactation).

No effects on fertility (including estrous cycle and sperm parameters) observed in rats in an OECD 422 study (Envigo, 2018) and a range-finding one-generation study (Exxon, 1998) at doses up to 100 mg/kg bw/d (gavage) or 0.5% in diet (NOAEL 289-477 mg/kg bw/d in males, 336-970 mg/kg bw/d in females), respectively.

Link to relevant study records

Reference

Reference 1

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:

From 21 September 2018 to 9 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Adopted 28 July 2011

Deviations:

yes

Remarks:

4 F0 males slightly higher bw on day 1 than requested by guideline (i.e. +/-20% of mean bw; here 22 or 23% difference from mean male bw); minor differences not considered significant, thus no adverse impact on the study integrity; food efficiency missing

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : males and females 10 weeks before pairing

- Basis for dose level selection
Dose levels of 5, 25 and 120 mg/kg/day were selected in conjunction with the Sponsor based on the result of a dose-range finding study in Wistar rats, where rats were dosed with 0, 10, 30 or 100 mg/kg bw/day (Envigo Study No. QX79YL reported in Envigo (+2018, OECD422, rat, RL2) ) and on the results of a 90-day toxicity study in which Han Wistar rats received Isononanoic acid at daily dose levels of 0, 5, 30 or 120 mg/kg bw/day in corn oil (BASF project No. 50C0166/07S047 as reported in BASF (+2013, 90 d, rat, RL1, KS) ).
• In the dose range-finding study there were slight effects on body weights and histopathological findings in male kidneys, consistent with the accumulation of alpha-2u globulin.
• In the 90-day study, effects at 120 mg/kg bw/day consisted of light brown discoloration of the kidneys (males: 2/10), slight to moderate eosinophilic droplets in kidneys (males: 10/10), slight to severe increase in basophilic tubules in kidneys (males: 5/10), minimal to severe granular casts in the kidneys (males: 9/10); minimal fat vacuoles in the tubules of kidneys (females: 5/10), increased absolute and relative kidney weight (males: 15%/17%), increased absolute (males: 17%, females: 30%) and relative (males: 19%, females: 24%) liver weights, minimal to slight fat vacuoles in the peripheral area of the liver (males: 8/10, females: 10/10), increased cyanide insensitive Palmitoyl CoA oxidation (males: 15%, females: 65%) and decreased albumin (females: 3%).
Based on these results, a high dose of 120 mg/kg bw/day was considered suitable for this study with low and intermediate dose levels of 5 and 25 mg/kg/day providing approximately 5-fold dose increments.

Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on the sexual maturation and estrous cycles.

- Inclusion/exclusion of extension of Cohort 1B: not included as no trigger according to REACH Annex IX, section 8.7.3 column 2

- Termination time for F2 : not applicable

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : not included as no trigger according to REACH Annex IX, section 8.7.3 column 2

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : not included as no trigger according to REACH Annex IX, section 8.7.3 column 2

- Route of administration : The oral gavage route of administration was chosen as it is a possible route of human exposure.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

RccHan™;WIST was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (F0) 27 to 33 days old; (F1) Nominally Day 21 of age
- Weight at study initiation:
(F0) Males 75 to 118 g; Females 74 to 109 g.
(F1) Males: x-x g; Females: x-x g

- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering lactation and maturation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Environmental enrichment during certain periods of time (i.e. Aspen chew block, plastic shelter, paper shavings as nesting material) was provided.
Please note that there was a deviation of the study plan for provision of papaer shavings:
Nesting paper shavings are normally provided to females/litters during lactation.
However, on 16-18 January 2019 paper shavings were not available and could not be given to the following females/litters:
16 January 2019: Group 2 nos. 240, 242, 244, 246, 230 and Group 4 nos. 283, 289, 302
17 January 2019: No nesting material given to females on Day 4 or 14 of lactation
18 January 2019: No nesting material given to females on Day 14 of lactation
From 19 January 2019 nesting material was available for all females/litters.
Impact Statement: This has no impact on study integrity as softwood bedding was available throughout this period.


- Number of animals per cage:
Pre-pairing (acclimatization and after selection): up to four animals of one sex
Pairing: one male and one female
Males to termination: up to four animals
Females after mating (from Day 0 after mating): one female
Females during littering (from Day 20 after mating): one female + litter
Females to termination (after weaning): up to four animals
Offspring maturation (from weaning until selection): litter

- Diet: ad libitum (but removed overnight before blood sampling for hematology, blood chemistry and biomarkers investigations) ; SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: ad libitum; Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: Five days before commencement of treatment.


Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES:
From: 01 October 2018 (F0 treatment commenced) To: 11 to 12 April 2019 (F1, females - Cohort B necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed and then mixed with the vehicle (approximately 40% of the final volume). The mixture was magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation
Group 2: Initially daily until sufficient homogeneity and stability had been established, and then weekly thereafter.
Groups 1, 3 and 4: Weekly, and may have been prepared in advance of the first day of dosing.

Storage of formulation: Refrigerated (2 to 8 °C).

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 500 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix as part of Envigo Study Number: MR96PY (Envigo (2019, OECD 414, rabbit, RL 1; KS as reported in section 7.8.2). In that study it was demonstrated for one day at ambient temperature (15 to 25°C) and 15 days refrigerated (2 to 8°C).

Subsequently homogeneity and stability of the dose formulation at 1.25 mg/mL was established for one day at ambient temperature (15-25°C) and 15 days when stored refrigerated (2 to 8°C).

Test material consumption has not been recorded; not applicable since test material was applied through gavage.

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups (sibling pairing was not permitted).
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray. Vaginal smear - examined for the presence of spermatozoa and the stage of the estrous cycle - referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no information
- After successful mating each pregnant female was caged (how): Alone in cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the gestation, littering lactation and maturation periods.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 of treatment (F0 and F1 generation), last week of treatment (F1 generation) were analyzed for achieved concentration of the test item. In addition a sample from the Group 2 formulation during Week 5 of the F0 generation was analysed to demonstrate accurate preparation.

The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M031/18) - see Annex 2 of Envigo Study Number: MR96PY (2019, OECD 414 in rabbits) reported in section 7.8.2.
The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 125 μg/mL.
All solutions injected on the GC contained tetradecane as an internal standard at a nominal concentration of ca. 45 μg/mL.
Calibration standards were shared with a related and con-current study (Envigo Study MR96PY (2019, OECD 414 in rabbits), reported in section 7.8.2).

The formulations for Week 1 (F0 generation), Week 5 (F0 generation), Week 1 (F1 generation) and Last week (F1 generation) were sampled. For all groups ((Group 2 only for Week 5 (F0 generation)), 4 × 1 mL (accurately weighed) was sampled from the middle of the formulation by Pharmacy personnel. The samples were dissolved using ultrasonic vibration in a suitable volume of acetone. The extract was diluted using acetone, where necessary, to provide a solution containing Isononanoic acid at an expected concentration within the range 20 µg/mL to 100 µ/mL.
Two samples from each group were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Contingency samples for Week 1 (F0 generation) Group 2 were analyzed for confirmation of the results. Samples were
disposed of once satisfactory results were achieved or confirmatory results were obtained.
Procedural recoveries were prepared as a quality control measure and were not used to correct for analytical results.

Duration of treatment / exposure:

F0 animals: For ten weeks before pairing until termination after litters are weaned.

F1 animals: From weaning until termination of respective cohort (details see below).

Oral gavage - Direct treatment of F1 offspring commences at weaning (Day 21 of age); although direct treatment starts at or soon after
weaning, all offspring have potential for exposure in-utero and via the milk during lactation.
Cohort 1A = General toxicity and pathology of the tissues of the male and female reproductive systems: Treated from weaning to 13 weeks of age
Cohort 1B = Spare Cohort: Treated from weaning to approximately 14 weeks of age.

Frequency of treatment:

once per day

Details on study schedule:

details of mating for F0 see above, no F1 mating performed

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

120 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

F0: 25
F1 Cohort 1A:20
F1 Cohort 1B: 25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels of 5, 25 and 120 mg/kg/day were selected in conjunction with the Sponsor based on the result of a dose-range finding study in Wistar rats, where rats were dosed with 0, 10, 30 or 100 mg/kg bw/day (Envigo Study No. QX79YL reported in Envigo (+2018, OECD422, rat, RL2) ) and on the results of a 90-day toxicity study in which Han Wistar rats received Isononanoic acid at daily dose levels of 0, 5, 30 or 120 mg/kg bw/day in corn oil (BASF project No. 50C0166/07S047 as reported in BASF (+2013, 90 d, rat, RL1, KS) ).

In the dose range-finding study there were slight effects on body weights and
histopathological findings in male kidneys, consistent with the accumulation of alpha-2uglobulin.

In the 90-day study, effects at 120 mg/kg bw/day consisted of light brown discoloration of
the kidneys (males: 2/10), slight to moderate eosinophilic droplets in kidneys (males:
10/10), slight to severe increase in basophilic tubules in kidneys (males: 5/10), minimal to
severe granular casts in the kidneys (males: 9/10); minimal fat vacuoles in the tubules of
kidneys (females: 5/10), increased absolute and relative kidney weight (males: 15%/17%),
increased absolute (males: 17%, females: 30%) and relative (males: 19%, females: 24%)
liver weights, minimal to slight fat vacuoles in the peripheral area of the liver (males: 8/10,
females: 10/10), increased cyanide insensitive Palmitoyl CoA oxidation (males: 15%,
females: 65%) and decreased albumin (females: 3%).

Based on the observed results, a high dose of 120 mg/kg bw/day was considered suitable for this study with low and intermediate dose levels of 5 and 25 mg/kg/day providing approximately 5-fold dose increments.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
during acclimatisation: at least once per day
during the study: at least twice daily for evidence of ill health or reaction to treatment
- Cage side observations and clinical signs checked in table 2,3, and 4 of attached results were included.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 males
Week 1 - daily
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week

F0 females
Week 1 - daily
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week
Gestation phase - Days 0, 7, 14 and 20 after mating
Lactation phase - Days 1, 7, 14 and 21

F1 generation
Week 1 - daily
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day


A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males and selected F1 generation: Once each week
F0 females:
Once each week until paired for mating
Gestation phase - Days 0, 5, 12, 18 and 20
Lactation phase - Days 1, 7, 14 and 21

A detailed physical examination was performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals were assessed for physical condition and behavior during handling. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal was recorded with respect to nature, and, where appropriate, degree of severity.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter
On the day of necropsy

F0 females
Before dosing on the day that treatment commenced (Week 0) and weekly until paired for mating
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating
Day 1, 4, 7, 14, 21 and 28 post partum
On the day of necropsy

F1 generation selected animals
From nominal four weeks of age, twice during Week 1 of the F1 generation and weekly thereafter
On the day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males: Each week until paired for mating
F0 females: Each week until paired for mating; Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17, 18-19 after mating; Days 1-3, 4-6, 7-13 and 14-20 of lactation
F1 generation selected animals: From nominal four weeks of age, twice during Week 1 of the F1 generation and weekly thereafter
From these records the mean consumption per animal (g/animal/day) or (g/animal/week) was calculated for each phase.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears: Smears were taken daily for 15 days before pairing, using cotton swabs moistened with saline.
Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.
For four days before scheduled termination (nominally Days 25 to 28 post-partum) daily vaginal smears were taken and used to determine the stage of the estrous cycle at termination.

Sperm parameters (parental animals):

Parameters examined in F0 as well as F1 Cohort 1A male animals:
testis weight, epididymis weight, sperm count in testis and epididymides animals from control (group 1) and high dose group (group 4; group 2 and 3 fixed samples were retained) enumeration of cauda epididymal sperm reserve, sperm motility (all animals), sperm morphology (animals from control (group 1) and high dose group (group 4; group 2 and 3 fixed samples were retained)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); surplus offspring subject to macroscopic examination

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, organ weights, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, sperm analysis (Cohort 1A malses), immunophenotyping,
Estrous cycle monitoring was also performed in the F1 generation:
Cohort 1A:
Dry smears: Smears were taken for two weeks from approximately Day 75 of age, using cotton swabs moistened with saline.
Wet smears (using pipette lavage): Following onset of vaginal opening until the 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.
Cohort 1B:
Wet smear (using pipette lavage): For at least three days prior to the start of the necropsy phase and on the day of termination.

GROSS EXAMINATION OF DEAD PUPS: No

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
F0 animals
- Male animals: All surviving animals after weaning of the F1 animals, after confirmation that no further mating required.
- Maternal animals: All surviving animals killed on day 28 post partum (i.e. after the litter weaning).
- F0 females failing to produce a viable litter: Terminated with first cohort of females with live litters.


GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in attached "T11269_Details on pathological procedures" were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
F1 selected animals:
Cohort 1A: approx. week 13 of age
Cohort 1B: approx. week 14 of age
Unselected offspring: On Day 4 and Day 22 of age

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in attached "T11269_Details on pathological procedures" were prepared for microscopic examination and weighed, respectively.

Immunophenotyping Cohort 1A:
F1 - Cohort A animals (ten males and ten females per group) were selected for immunophenotyping. After the spleen was weighed a 3-5 mm mid transverse section was removed and retained for histopathological examination. The remaining spleen was weighed and then placed in to a vial of Hank’s Balanced Salt Solution (HBSS) and held on wet ice until processing for analysis. Samples were dispatched to Department of Bioanalysis,Biomarkers and Clinical Sciences, Covance. The analyses were performed by the Department of Bioanalysis, Biomarkers and Clinical
Sciences, Covance. Immunophenotyping was performed on T (including CD4+ and CD8+ T cell subsets), B, NK, monocyte and neutrophil cell populations by flow cytometry in rat spleen leukocytes.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles, pre-coital interval, mating performance for F0 adults and stage of estrous cycle at termination for F0, F1 - Cohort A and F1 - Cohort B females, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

For further details please go to the attached document "T11269_Statistical analysis".

Reproductive indices:

Estrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length and Index

Offspring viability indices:

Litter Size, Sex Ratio and Survival Indices

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs considered to be related to treatment in the F0 males or the F0 females which reared their litter to weaning.
For further details on male and female animals please see Table 1 in attachment "T11269_Results_Figures and summary tables".

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

At 120 mg/kg/day, 4 females were killed during late gestation and one other female was killed during early lactation for reasons of animal welfare.
One female from the control group was killed for reasons of animal welfare (swollen vaginal area, no evidence of vaginal opening; macroscopic findings included abnromal thin, brown fluid in the vagina and bilateral fluid distension of the uterus).
For details see Table 1 in attachment "T11269_Results_Figures and summary tables".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain of males and of females before pairing was unaffected by treatment.
During gestation, body weight gain of females receiving 120 mg/kg/day was slightly but statistically significantly low between Days 18 and 20. Overall body weight changes during lactation were unaffected by treatment, but females receiving 120 mg/kg/day lost less weight than Controls during Days 14-21.
For further details on male and female animals please see Figures 1-4 and result tables 5,6 and 7 in attachment "T11269_Results_Figures and summary tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of males and females before pairing was unaffected by treatment.
There was no effect of treatment on food consumption during gestation, but during lactation intake at 120 mg/kg/day was marginally low throughout with the difference attaining statistical significance for Days 4-7.
For details see Tables 8, 9, and 10 in attachment "T11269_Results_Figures and summary tables".

Effects on food consumption had no influence on test material consumption as test material was applied by gavage.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematocrit and red blood cell concentrations were slightly low among males at all doses of Isononanoic acid; hemoglobin levels were slightly low at 25 or 120 mg/kg/day; none of these differences showed a dose response and were therefore considered to be not related to treatment.
Platelet counts were slightly low among females receiving 120 mg/kg/day.
All other differences were minor and also lacked dose response or were only recorded in one sex.
For details see Table16 in attachment "T11269_Results_Figures and summary tables".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Among males receiving 120 mg/kg/day, the mean creatinine concentration was slightly but statistically significantly higher than in Controls.
All other differences were minor and also lacked dose response or were only recorded in one sex.
For details see Table17 in attachment "T11269_Results_Figures and summary tables".
There was no effect of treatment on serum TSH or T4 concentrations in F0 animals.

Urinalysis findings:

not examined

Description (incidence and severity):

No findings on urinalysis parameters observed in 90 d study, thus no urinalysis requiered in this study.

Behaviour (functional findings):

not examined

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 treatment with Isononanoic Acid were seen in the kidneys, liver and pancreas.

Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary revealed no abnormality.

Kidneys
Increased incidence and severity of hyaline droplet accumulation, basophilic tubules and interstitial inflammatory cell infiltrates, and/or granular casts were seen in males given 5, 25 or 120 mg/kg/day. Tubular vacuolation was seen in females given 120 mg/kg/day and occasional females given 5 or 25 mg/kg/day but not considered as adverse since these finding was not accompanied by degenerative changes.

Liver
Hepatocellular vacuolation was seen in males and females given 5, 25 or 120 mg/kg/day. This finding was considered as non adverse since it was not accompanied by degenerative changes.

Pancreas
Secretory depletion of the acinar cells was seen in females given 5, 25 or 120 mg/kg/day. This finding was considered as non-adverse since it was not accompanied by degenerative changes.

For details see Tables 33 and 34 in attachment "T11269_Results_Figures and summary tables". In addition, a summary is provided per organ with relevant findings in the attachment "T11269_F0_Summary of treatment related histopathological findings in the kidneys-liver-pancreas"

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles were unaffected by treatment. There was no effect of treatment on estrous cycles at termination.
For details see Tables 11 and 15 in attachment "T11269_Results_Figures and summary tables".

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There was no effect of treatment on sperm motility, counts or morphology.
For details see Tables 23, 24, and 25 in attachment "T11269_Results_Figures and summary tables".

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre coital interval, mating performance and fertility were unaffected by treatment.
For details see Tables 12, 13, and 14 in attachment "T11269_Results_Figures and summary tables".
Histopathology of the ovary did not reveal any abnormality.

Litter Size, Sex Ratio and Survival Indices

Females 285 and 288 in the 120 mg/kg/day group were killed for welfare reasons and their litters were killed with the dam.

There was no effect of treatment on the mean number of implantations.

Treatment at 120 mg/kg/day was associated with slightly lower post implantation survival and live birth indices compared with Controls (87.2% versus 95.4% in Controls); as a result,
the total litter size on Day 1 of age and live litter size on Days 1 and 4 of age were noticeably and statistically significantly lower than in Controls (Day 1: 8.9 versus 11.5 in Controls; Day 4: 8.7 versus 11.5 in Controls). The viability and lactation indexes and sex ratio were unaffected by treatment.

For details see Tables 18, 19, and 20 in attachment "T11269_Results_Figures and summary tables".

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: general systemtic toxicity as the only findings up to the highest dose were male rat specific effects

Key result

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Litter findings: Among offspring in the 120 mg/kg/day group, there was a higher incidence of pups abnormally cold to touch (total of 14 offspring of 3 litters, not observed in other dose groups) and/or with little or no milk present in the stomach compared with Control.

F1 Cohort A and B: There were no clinical signs considered to be related to treatment.
For details see Table 35 in attachment "T11269_Results_Figures and summary tables".

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Female No. 649, F1 female in the 25 mg/kg/day group was killed on Day 53 of the Treatment phase 2 for welfare reasons due to signs of decreased activity, irregular breathing, cold to touch, pale eyes, piloerection, hunched posture and whole body pallor. Macroscopic examination revealed masses in the liver and abnormal contents of the ileum, jejunum, cecum and abdominal cavity. At microscopic examination, findings were seen in the liver and reproductive tissues. The major factor contributory to death was considered as general poor clinical condition. This death was considered to be unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring body weight:
Mean bodyweights of male and female offspring on Day 1 of age were lower at 120 mg/kg/day (5.6 g versus 6.6 g in males; 5.4 g versus 6.2 g in females) and marginally lower at 25 mg/kg/day (6.2 g versus 6.6 g in males; 5.9 g versus 6.2 g in females); all differences attained statistical significance. At 120 mg/kg/day, body weights remained statistically significantly lower on Days 4 and 7 of age.
There was no effect of treatment on the overall weight gain of the offspring between Days 1 and 21 of age, although male and female offspring at 120 mg/kg/day group showed slightly lower weight gain between Days 4 and 7 of age when compared with Controls.
For details see Figures 5 and 6 and Table 22 in attachment "T11269_Results_Figures and summary tables".

F1 Cohort A and B:
Body weight gain of both sexes between Days 21 and 25 of age were marginally lower than in Controls.
Absolute mean body weights of selected F1 animals at the formal start of the F1 generation at 120 mg/kg/day were marginally lower but overall body weight gains were similar to Controls.
For details see Figures 7 and 8 and Table 36 in attachment "T11269_Results_Figures and summary tables".

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food intake.
For details see Table 37 in attachment "T11269_Results_Figures and summary tables".

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The mean prothrombin time in all groups of treated males was slightly but statistically significantly shorter than in Controls but there was no dose response so a relationship to treatment seems unlikely.
All other differences were minor and also lacked dose response or were only recorded in one sex.

For details see Table 43 in attachment "T11269_Results_Figures and summary tables".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean urea levels were slightly but statistically significantly higher in both sexes receiving 120 mg/kg/day, and in females receiving 25 or 5 mg/kg/day, but there was no dose response in females.
Mean creatinine levels were slightly higher in all groups of treated males with a dose response apparent. Mean glucose concentration was slightly lower in males receiving 120 mg/kg/day.
All other differences were minor and also lacked dose response or were only recorded in one sex.
For details see Table 44 in attachment "T11269_Results_Figures and summary tables".

Urinalysis findings:

not examined

Description (incidence and severity):

No findings on urinalysis parameters observed in 90 d study, thus no urinalysis requiered in this study.

Sexual maturation:

no effects observed

Description (incidence and severity):

F1 Cohort A and B:
- Sexual maturation:
There was no effect of treatment on mean age at vaginal opening.
The mean age at balano preputial separation for males receiving 120 mg/kg/day group was one day later than in Controls but as the animals are only examined once per day, for this magnitude of difference no effect of treatment is inferred.
For details see Table 38 in attachment "T11269_Results_Figures and summary tables".

- Vaginal Opening to First Estrus and Estrous Cycles
There was no effect of treatment on the interval between vaginal opening and first estrus.
Estrous cycles from Day 75 of age and at termination were unaffected by treatment.
For details see Tables 39, 40, 41, and 42 in attachment "T11269_Results_Figures and summary tables".

-Sperm analysis
There was no detrimental effect of treatment on sperm motility, counts or morphology. There were slightly more progressively motile sperm at 5 or 120 mg/kg/day but the highest value was at 5 mg/kg/day so the differences are concluded to be due to chance.
For details see Tables 45, 46, and 47 in attachment "T11269_Results_Figures and summary tables".

- Ovarian Follicle Counts and Corpora Lutea
There was considered to be no effect of treatment on ovarian follicle counts and corpora lutea counts.
For details see Table 48 in attachment "T11269_Results_Figures and summary tables".

Offspring parameters:
- Ano-Genital Distance
The adjusted mean ano-genital distance in male offspring only in the 120 mg/kg/day group was marginally but statistically significantly greater than in Controls. However, a higher
anogenital distance in males is not considered to be biologically or toxicologically relevant. Similaraly the significantly lower anogenital distance in females of the low dose group is
considered to be incidental and of no biological or toxicological relevance.
For details see Table 21 in attachment "T11269_Results_Figures and summary tables".

- Nipple counts:
There was no effect of treatment on nipple counts in male offspring; only two male pups had nipples and these were both in the 5 mg/kg/day group and thus judged to have occurred by chance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 litter:
There was no effect of treatment on the absolute or body weight relative brain, spleen or thymus weights.
For details see Table 30 in attachment "T11269_Results_Figures and summary tables".

F1 Cohort A and B:
Body weight relative mean liver weights were slightly but statistically significantly high for males and females receiving 120 mg/kg/day; mean relative kidney, brain and adrenal weights were also slightly higher in these males. Relative mean weights for the uterus/cervix were slightly higher in both cohorts of females receiving 120 mg/kg/day; at termination 10 % of Cohort 1A and 24% of Cohort 1B females at 120 mg/kg/day were in estrus compared with 20% and 24% in the Controls groups, so it is unlikely that the higher mean uterine weights were related to the stage of estrous.
For details see Tables 49, 50, 51, 52, 53, 54, 55, and 56 in attachment "T11269_Results_Figures and summary tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 litter: There were no findings considered to be related to treatment.

F1 Cohort A and B:
The macroscopic examination performed at approximately 13-14 weeks of age revealed the following changes in the kidneys.
Abnormal color (pale) and pale areas were seen in some males receiving 120 mg/kg/day (for each characteristisc 3 out of 45 animals) .
Dilated pelvis was also seen in some males receiving 5, 25 and 120 mg/kg/day (control and treatment groups consisted of 45 animals each; i.e. control: 1 male and 1 female; low dose group: 4 males, 2 females; mid dose group: 7 males and 2 females, high dose group: 4 males and 1 female)
The incidence and distribution of all other findings were considered incidental and unrelated to test item.
For details see Tables 57 and 58 in attachment "T11269_Results_Figures and summary tables".

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with Isononanoic Acid were seen in the kidneys, liver and pancreas of Cohort 1A animals and kidneys of Cohort 1B animals.

Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages.
No cell or stage specific abnormalities were noted.
The qualitative examination of the ovary with follicle and corpora lutea counts and staging of the estrus cycle revealed no abnormality.

Cohort 1A:
- Kidneys: Increased incidence and severity of hyaline droplet accumulation and basophilic tubules, and/or granular casts were seen in males given 5, 25 or 120 mg/kg/day. Tubular vacuolation was also seen in females given 5, 25 or 120 mg/kg/day, but not considered as adverse since these finding was not accompanied by degenerative change.
- Liver: Increased incidence and/ or severity of hepatocellular vacuolation was seen in males and females given 5, 25 or 120 mg/kg/day. This finding was considered as non adverse since it was not accompanied by degenerative changes.
- Pancreas: Increased incidence of secretory depletion of the acinar cells was seen in females given 5, 25 or 120 mg/kg/day. This finding was considered as non adverse since it was not accompanied by degenerative changes.

Cohort 1B:
- Kidneys: Hyaline droplet accumulation, basophilic tubules and granular casts were seen in some males of given 5, 25 or 120 mg/kg/day. These tissues were examined due to gross abnormalities
seen at necropsy.

All other microscopic findings were considered as incidental and unrelated to the test item.

For details see Tables 59 (cohort 1A) and 60 (cohort 1B) in attachment "T11269_Results_Figures and summary tables" as well as the respective summary tables in attachment "T11269_F1_Summary of treatment related histopathological findings in the kidneys-liver-pancreas".

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

The oral gavage administration of Isononanoic Acid had no observable effects on the immunophenotyping parameters measured in spleen leukocytes (i.e. T (including CD4+ and CD8+ T cell subsets), B, NK, monocyte and neutrophil cell populations from F1 - Cohort A animals (ten males and ten females per group) ).
For details see attachment "T11269_Spleen leucocytes-immunophenotyping.pdf"

Please note that Litter Size, Sex Ratio and Survival Indices of F1 litter was provided above in results of F0 animals.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL for offspring

Remarks on result:

other: It has to be mentioned that this difference occurred at a dose-level, which was not tolerated in five dams.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

120 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Analytical Verification of Dose formulation as presented in the "Formulation Analysis Contributing Report"

The mean concentrations of Isononanoic Acid in test formulations analyzed during the study and the deviation of the mean result from the nominal value are detailed in Table 1 (see attachment " T11269_Result for Analytical Verification of Dose formulation-Table1").

The mean concentrations were within 9% of the nominal concentration, confirming the accuracy of formulation, with the exception of Week 1 (F0 generation) Group 2. For Week 1 (F0 generation), Group 2 samples were outside of the acceptance criteria. Contingency analysis was performed which confirmed the original results. The contingency results have been reported alongside the original results. The difference from mean and coefficient of variation remained within 4%, confirming precise analysis.

Procedural recoveries remained within the validated range, confirming the continued robustness of the analytical methodology, with the exception of one procedural recovery prepared during Week 1 (F1 generation), which was excluded as an outlier as per SOP. Procedural recoveries were not prepared during Last week (F1 generation) in error. As the formulation samples were within the acceptance criteria and the procedural recoveries were not used to correct for the analytical results, it is considered to have no impact on the results.

The composition of the diet is documented in the attachment " T11269_food composition VRF1 (P) Batch 3604.pdf".

Conclusions:

Based on the results of this GLP conform, OECD testing guideline study no. 443 a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in male rats was not established at first.
The NOAEL would lie below 5 mg/kg/day due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts) - alpha- 2u-globulin nephropathy considered adverse at all dose levels. It is however acknowledged that this finding is male rat specific and generally considered to be of no relevance to humans.
Thus excluding this finding the NOAEL for male rats is 120 mg/kg/day.
The NOAEL for systemic and developmental reproductive toxicity in adult female rats was concluded to be 25 mg/kg/day based on poor clinical condition necessitating the humane kill of five females receiving 120 mg/kg/day during late gestation/early lactation. No effects on fertility were observed up to the highest dose tested (NOAEL fertility: 120 mg/kg bw/d).
A dose of 25 mg/kg/day is also concluded to be the NOAEL for the offspring based on lower post implantation and live birth survival indices resulting in a low total and live litter size at 120 mg/kg/day. It has to be mentioned that this difference occurred at a dose-level, which was not tolerated in five dams.

Executive summary:

The purpose of this study was to assess the influence of Isononanoic Acid on reproductive performance when administered continuously by oral gavage to Han Wistar rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on the sexual maturation, estrus cycles. Three groups of 25 male and 25 female rats received Isononanoic Acid at doses of 5, 25 or 120 mg/kg/day by oral administration. Males were treated daily for ten weeks before pairing until termination after litters are weaned. Females were treated daily for ten weeks before pairing, throughout pairing, gestation and until termination. Females were allowed to litter, rear their offspring and were killed on Day 28 post partum. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

For the F0 generation data were recorded on clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry 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 and 20 of age. Blood samples were collected from selected offspring on Day 4 and 22 of age for biomarker investigations.

The F1 generation comprised of two cohorts:

Cohort A: 20 male and 20 female progeny were selected from each dose group and continued to receive Isononanoic Acid at doses of 0 (Control), 5, 25 or 120 mg/kg/day from weaning until scheduled sacrifice at approximately Week 13 of age.

Cohort B: 25 male and 25 female progeny were selected from each dose group and continued to receive Isononanoic Acid at doses of 0 (Control), 5, 25 or 120 mg/kg/day from weaning until scheduled sacrifice at approximately Week 14 of age.

For F1 generation - Cohort A, data were recorded on clinical condition, body weight, food consumption, sexual maturation, vaginal opening and estrous cycles. Clinical pathology (hematology, blood chemistry and biomarkers), sperm assessment, ovarian follicle counts,

organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed.

For F1 generation - Cohort B, data was recorded on clinical condition, body weight, food consumption, estrous cycles, sexual maturation, organ weight, and a targeted set of macroscopic pathology investigations were performed.

Results

F0 adults and F1 offspring up to weaning

Treatment of F0 females at 120 mg/kg/day was not tolerated during late gestation and early lactation: three females were killed for welfare reasons during late gestation and two females were killed for welfare reasons during early lactation.

Microscopic pathology changes were seen in the liver, kidneys and pancreas of several of these females but were not considered to be responsible for the poor condition of these females.

There were no clinical signs considered to be related to treatment in F0 males or in the F0 females which reared their litters to weaning. Body weight gain of males and females before pairing was unaffected by treatment.

Females receiving 120 mg/kg/day showed low weight gain between Days 18 and 20 of gestation. Overall body weight during lactation was unaffected by treatment. Food consumption of both sexes before pairing and of females during gestation was unaffected by treatment. During lactation, intake of females receiving 120 mg/kg/day was marginally lower.

Hematology and blood chemistry investigations revealed that platelet counts were slightly low among females receiving 120 mg/kg/day and mean creatinine concentration was slightly higher among males receiving 120 mg/kg/day.

Sperm motility, counts and morphology were unaffected by treatment.

Kidney and liver weights were slightly but statistically significantly higher among males at 120 mg/kg/day; relative kidney weights were also slightly but significantly high among males receiving 25 mg/kg/day. Among females, liver weights were slightly but statistically significantly high at 120 mg/kg/day.

There were no test item related macropathology findings. Microscopic pathology changes related to treatment with Isononanoic Acid were seen in the kidneys, liver and pancreas. Increased incidence and severity of hyaline droplet accumulation, basophilic tubules and interstitial inflammatory cell infiltrates, and/or granular casts were seen in the kidneys of males given 5, 25 or 120 mg/kg/day. Tubular vacuolation was seen in the kidneys of females given 120 mg/kg/day and occasional females given 5 or 25 mg/kg/day. Hepatocellular vacuolation was seen in males and females given 5, 25 or 120 mg/kg/day. Secretory depletion of the acinar cells of the pancreas was seen in females given 5, 25 or 120 mg/kg/day. These findings were not considered as adverse, since they were not accompanied by degenerative changes.

At 120 mg/kg/day, there was no effect of treatment on the mean number of implantations but there was slightly low post implantation survival index and lower live birth index which resulted in a low total litter size and live litter size on Days 1 and 4 of age. There was a higher incidence of pups abnormally cold to touch or with little/no milk in stomach at 120 mg/kg/day compared with control. Mean bodyweights of male and female offspring on Day 1 of age were lower at 120 mg/kg/day and marginally lower at 25 mg/kg/day. Body weights remained lower in males at 120 mg/kg/day on Day 4 and Day 7. There was no effect of treatment on the overall weight gain of the offspring between Days 1 and 21 of age, although male and female offspring in the 120 mg/kg/day group showed slightly lower weight gain between Days 4 and 7 of age compared with Controls.

There was no effect of treatment on offspring ano-genital distance, nipple counts in males, organ weights or macropathology findings.

There was no effect of treatment on serum T4 or TSH levels in adults or offspring

Selected F1 offspring - Cohorts 1A and 1B

One female in the 25 mg/kg/day group was killed for welfare reasons due to poor clinical condition, this death was considered to be unrelated to treatment. There were no clinical signs considered to be related to treatment.

Body weight gain of both sexes between Days 21 and 25 of age was marginally lower than in Controls. Absolute mean body weights of selected F1 animals at the formal start of the F1 generation at 120 mg/kg/day were marginally low but overall body weight gains were similar to Controls.

Food consumption, age at sexual maturation, time between vaginal opening and first estrous and estrus cycles, ovarian follicle count and corpora lutea counts were unaffected by treatment.

Mean creatinine levels were slightly higher in all groups of Cohort 1A males and a dose response was apparent. Mean urea level was slightly higher in males receiving 120 mg/kg/day.

There was no effect of treatment on sperm motility, counts or morphology.

Relative mean liver weights were slightly but statistically significantly high for males and females receiving 120 mg/kg/day; relative kidney, brain and adrenal weights were also slightly high in these males. Relative weights for the uterus/cervix were slightly higher in both cohorts of females receiving 120 mg/kg/day.

Pale kidneys and pale areas on the kidneys were seen in some males receiving 120 mg/kg/day.

Microscopic pathology examination of the tissues from Cohort 1A animals revealed findings related to treatment with Isononanoic Acid in the kidneys, liver and pancreas.

Increased incidence and severity of hyaline droplet accumulation, basophilic tubules and/or granular casts were seen in the kidneys of males given 5, 25 or 120 mg/kg/day. Tubular vacuolation was seen in the kidneys of females given 5, 25 or 120 mg/kg/day. Increased incidence/severity of hepatocellular vacuolation was seen in males and females given 5, 25 or 120 mg/kg/day. Increased incidence of secretory depletion of the acinar cells of the pancreas was seen in females given 5, 25 or 120 mg/kg/day.

No test item related microscopic pathology changes were seen in the spleen of the F1A animals and there was no effect on the different populations of spleen leukocytes as evaluated by immunophenotyping.

There was no effect of treatment on serum T4 or TSH levels in F1A adults.

Conclusion

Based on the results of this study a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in male rats was not established and lies below 5 mg/kg/day due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts) - alpha-2µ-globulin nephropathy considered adverse at all dose levels. It is however acknowledged that this finding is male rat specific and generally considered to be of no relevance to man. Excluding this finding the NOAEL for male rats is 120 mg/kg/day.

The NOAEL for systemic and developmental reproductive toxicology in adult female rats was concluded to be 25 mg/kg/day based on poor clinical condition necessitating the humane kill of five females receiving 120 mg/kg/day during late gestation/early lactation. No effects on fertility were observed up to the highest dose tested (NOAEL fertility: 120 mg/kg bw/d). A dose of 25 mg/kg/day is also concluded to be the NOAEL for the offspring based on lower post implantation and live birth survival indices resulting in a low total and live litter size at 120 mg/kg/day. It has to be mentioned that this difference occurred at a dose-level, which was not tolerated in five dams.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 March 2018 to 10 September 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

restricted range of examinations

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The purpose of this study was to make an assessment of general systemic toxic potential in rats, including reproductive/development effects, with administration of the test substance by oral administration and to act as a preliminary study to assist with setting dose levels for the subsequent extended one generation reproductive toxicity study.

Species:

rat

Strain:

Wistar

Remarks:

RccHan:WIST

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 strain (RccHan™ ;WIST) was used because of the historical
control data available at the test facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo EMS (UK)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males: approximately 12 to 13 weeks old. Females: approximately 14 to 15 weeks old.
- Weight at study initiation: (P) Males: 309 to 353 g; Females: 193 to 239 g
- Fasting period before study: not specified
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Cage distribution: The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
- Bedding: Solid bottom cages contained softwood based bark-free
fiber bedding, which was changed at appropriate intervals
each week.

- Number of animals per cage:
Pre-pairing: up to four animals of one sex
Pairing: one male and one female
Males after mating: up to four animals
Gestation: one female
Lactation: one female + litter
F1 generation (Days 21-28 of age): one male and one female

- Diet: ad libitum, SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: ad libitum, Potable water from the public supply via polycarbonate
bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Males: six days before commencement of treatment. Females: 20 days before commencement of treatment.
ENVIRONMENTAL CONDITIONS
- Rodent facility: Limited access - to minimize entry of external biological
and chemical agents and to minimize the transference of
such agents between rooms.
- Air supply: Filtered fresh air which was passed to atmosphere and not
recirculated.
- Temperature (°C): Monitored and maintained within the range of 20-24ºC
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed and then mixed with the vehicle (approximately 40% of the final volume). The mixture was magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item. The frequency of preparation was weekly, and may have been prepared in advance of the first day of dosing.

Storage of formulation: refrigerated (2 to 8°C)

DOSING
- Constant doses in mg/kg/day
- Volume dose: 4 mL/kg bw

Details on mating procedure:

- pairing commenced after a minimum of two weeks of treatment.
- M/F ratio per cage:1:1, within the same treatment groups
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear, referred to as day 0 of gestation.
- Male/female separation: at the day when mating evidence 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

Details on analytical verification of doses or concentrations:

The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 µg/mL to 125 µg/mL. 1 mL test sample was dissolved using ultrasonic vibration in suitable volume of acetone. All solutions injected on the GC contained tetradecane as an internal standard at a nominal concentration of ca. 45 µg/mL.

Duration of treatment / exposure:

F0: For a minimum of 15 days before pairing until termination.
F1: Day 21 to Day 27 of age.

Frequency of treatment:

Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.

Details on study schedule:

Scheduled sacrifices:
F0 Males: After successful littering by females
F0 females failing to produce a viable litter: Day 25 after mating
F0 females: Day 21 of lactation (following terminal blood sampling).
Unselected F1 offspring: Culls - Day 4 of age; Scheduled sacrifice - Day 21 of age
F1 offspring: Scheduled kill - Day 28 of age

The selection of offspring to form the F1 generation was made on Day 21 of age.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females for the F0 and for the selected F1 animals

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels for this study (0, 10, 30 and 100 mg/kg bw/day) were selected based on the results of a 90-day toxicity study, in which Han Wistar rats received the test substance at daily dose levels of 0, 5, 30 or 120 mg/kg bw/day in corn oil.

Effects at 120 mg/kg bw/day consisted of light brown discoloration of the kidneys (males: 2/10), slight to moderate eosinophilic droplets in kidneys (males: 10/10), slight to severe increase in basophilic tubules in kidneys (males: 5/10), minimal to severe granular casts in the kidneys (males: 9/10); minimal fat vacuoles in the tubules of kidneys (females: 5/10), increased absolute and relative kidney weight (males: 15%/17%), increased absolute (males: 17%, females: 30%) and relative (males: 19%, females: 24%) liver weights, minimal to slight fat vacuoles in the peripheral area of the liver (males: 8/10, females: 10/10), increased cyanide insensitive Palmitoyl CoA oxidation (males: 15%, females: 65%) and decreased albumin (females: 3%).

At 30 mg/kg bw/day, increased relative kidney weights (males: 8%), increased relative liver weights (males: 6%, females: 8%); slight to moderate eosinophilic droplets in kidneys (males: 9/10), slight to moderate increase in basophilic tubules in kidneys (males: 7/10), minimal to moderate granular casts in the kidneys (males: 7/10), minimal fat vacuoles in the tubules of kidneys (females: 2/10), minimal vacuoles in the peripheral area of the liver (females: 7/10) and increased cyanide insensitive Palmitoyl CoA oxidation (females: 28%) were observed.
No adverse effects were observed at 5 mg/kg bw/day.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule for examinations:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
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.

DETAILED CLINICAL OBSERVATIONS: yes,
- Time schedule for examinations: F0 males and selected F1 generation: Once each week
F0 females: Once each week until pairing
Gestation phase: Days 0, 5, 12, 18 and 20
Lactation phase: Days 1, 7, 14 and 21

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Twice weekly and on the day of necropsy.
F0 females: Twice weekly until mating detected. Days 0, 7, 14 and 20 after mating. Days 1, 4, 7, 10, 14, 17 and 21 of lactation. On the day of necropsy.
Selected F1 generation: Days 21, 24 and 28 of age.

FOOD CONSUMPTION: yes
- Time schedule
F0 animals: Twice weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Day 15 to 22), but recommenced for males from Day 22. For females after mating food consumption was performed to match the body weight recording: Days 0-6, 7-13 and 14-19 after mating Days 1-3, 4-6, 7-9, 10-13, 14-16 and 17-20 of lactation.
Selected F1 generation Days 21-23 and 24-27 of age.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each relevant phase.


OTHER:

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows: Dry smears From the beginning of treatment until animals were paired for
mating using cotton swabs. Wet smears Using pipette lavage during the following phases: For 14 days before treatment (all females including
spares); animals that failed to exhibit 4-5 day cycles were not allocated to study. After pairing until mating. For four days before scheduled termination.

Sperm parameters (parental animals):

Not performed

Litter observations:

Clinical observations All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
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 eight 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 (F1 generation) The selection of offspring to form the F1 generation was made on Day 21 of age. Selected F1 animals separated from littermates on Day 21 of age.

Postmortem examinations (parental animals):

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 following were recorded for F0 females (including those prematurely sacrificed, where
possible):
Uterus: Number of implantation sites.
Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection for non-pregnant females.

Histopathology: Necropsy and pathology of a wide range of organs as requested by the guideline of at least 5 males and 5 females per group; for the remaining animals only a reduced necropsy of sexual organs was performed and only organs with abnormalities were selected for pathology.

Postmortem examinations (offspring):

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.

Offspring were subject to a complete macroscopic examination. Additionally, the following procedures were applicable:
Premature deaths before weaning (excluding culled offspring): Missing offspring and any grossly autolyzed or grossly cannibalized could not be examined. All other remaining offspring dying before weaning were examined as detailed above; the examination also included an assessment for the presence of milk in the stomach, where this was possible. Abnormal tissues retained in appropriate fixative.

Culled offspring (Day 4): Culled offspring with clinical signs on Day 4 of age were subject to complete macroscopic examination with assessment of stomach for presence of milk, where this was possible. Abnormal tissues retained in appropriate fixative. Culled offspring with no clinical sign on Day 4 of age were killed and discarded without necropsy examination.

Unselected offspring at scheduled kill on Day 21 of age: Subject to an external examination. If an abnormality was detected the carcass was retained pending possible future examination.

Selected F1 generation on Day 28 of age: Subject to complete macroscopic examination. Any abnormal tissues and livers were retained/weighed at the discretion of necropsy staff.

Statistics:

see below at any other information on materials and methods

Reproductive indices:

Percentage mating
Conception rate
Fertility index
Gestation index

Offspring viability indices:

Post-implantation survival index
Live birth index
Viability index
Lactation index

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Group 1 Control male no 10 was killed for reasons of animal welfare on Day 9 of study, terminal signs included underactive behaviour, irregular/noisy breathing, piloerection and pallor. Macroscopic examination revealed that the oesophagus was perforated and that the thoracic content was abnormal; the cause of death was therefore attributed to dosing trauma.

Group 4 female no. 135 receiving 100 mg/kg/day was killed for welfare reasons on GD23 with evidence of dystocia; two offspring were observed at 09:00 with no further offspring apparent at 12:00; the dam was also observed to be pale with piloerection. Uterine examination revealed two placentae, three late resorptions and nine dead fetuses.

No signs were observed in association with dose administration and there were no signs at routine physical examination that could be attributed to administration of the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 2 mortalities not related to treatment. For details see clinical signs

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain for males during the treatment period and for females for the two week period prior to being paired for mating was unaffected by treatment with Isononanoic acid. During gestation body weight gain for females receiving Isononanoic Acid was slightly lower during the first week of gestation with the difference achieving statistical significance at 30 and 100 mg/kg/day (p<0.05); there was no dose response. Overall body weight and body weight gain for females during lactation showed no adverse effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for males during treatment and for females prior to pairing, during gestation and during lactation was unaffected by treatment with the test substance.

Food efficiency:

no effects observed

Description (incidence and severity):

The efficiency of food utilization was variable during the two week pre-pairing treatment period, however overall there was no adverse effect of treatment.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At termination of the male animals after 7 weeks of treatment there were no differences in the haematological parameters that were examined. On Day 21 of lactation females at 100 mg/kg/day showed higher red cell distribution width (p<0.01) and slightly longer activated partial thromboplastin time (p<0.05) when compared with Controls. Activated partial thromboplastin was also slightly longer at 30 mg/kg/day (p<0.05).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After 7 weeks of treatment males at 100 mg/kg/day showed higher creatinine levels (p<0.05) and males at 30 and 100 mg/kg/day showed higher sodium levels (p<0.05) when compared with Controls. On Day 21 of lactation urea, creatinine and chloride levels were higher for females that received 100 mg/kg/day (p<0.01, p<0.05 and p<0.05, respectively). Calcium levels for females that received the test item were lower when compared with Controls; p 0.05, p<0.05 and p<0.01 at 10, 30 and 100 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Decedents:
Male control animal No. 10 was euthanized for welfare reasons on Day 9 of treatment before pairing, due to general poor clinical condition (decreased activity, irregular breathing with rales, pallor of the whole body and piloerection). At necropsy, perforation of the esophagus, firm contents in the cecum, pale thick, and clear, fluid and adhesions in the thorax and edema of the thymus were seen. At microscopic examination, slight focal perforation of the esophagus, with minimal diffuse inflammatory cell infiltrate in the submucosa and adventitia and minimal diffuse hemorrhage in the submucosa accounted for the macroscopic finding in the esophagus and was considered the main factor contributing to the poor clinical condition and the major factory contributory to death. In the thoracic cavity, slight focal adhesions, which correlated with the macroscopic finding, slight focal necrosis of striated muscle, slight multifocal inflammation/foreign body reaction (plant material) and minimal focal hemorrhage were seen. Slight edema of the thymus correlated with the macroscopic change. Due to tissue processing, fluid in the thoracic cavity and the contents of the cecum could not be appreciated at microscopy. Other microscopic findings of note were minimal multifocal inflammatory cell infiltrate of the pleura of the lungs, minimal focal inflammatory cell infiltrate of the adventitia of the heart and minimal diffuse inflammatory cell infiltrate of the parasternal adipose tissue which were all considered secondary to the esophageal perforation.

Female animal No. 135, treated at 100 mg/kg/day, was euthanized on Gestation Day 23 for welfare reasons due to general poor clinical condition as a consequence of evidence of dystocia. At necropsy, pale kidneys, pale areas on the visceral surface of the liver and a small spleen were seen. At microscopic examination, slight bilateral epithelial microvacuolation of the cortical and outer medullary tubules of the kidneys was seen, that would have contributed, along with autolysis, to the pale appearance of the kidneys. Vacuoles in the renal tubules have been reported in treated females in a previous Han Wistar rat study with the test substance. Minimal multifocal hepatocellular necrosis, that accounted for the macroscopic pale areas of the liver, and slight generalized decreased cellularity of the spleen, which correlated with the small size, were also seen. Another finding of note was moderate bilateral luminal dilatation of the uterus. The main contributory factor to expected death was poor clinical condition as a consequence of evidence of dystocia.

F0 Males Killed After 7 Weeks of Treatment and F0 Females on Day 21 of Lactation:
Changes related to treatment with Isononanoic acid were seen in the kidneys of males: A minimal to moderate accumulation of hyaline droplets in cortical tubules was seen at a higher severity and/or incidence in males of all treated groups compared with control males. Minimal or slight basophilia of cortical tubules was seen in males of all treated groups. Minimal to moderate granular casts in tubules of the outer medulla were seen in males that received 30 or 100 mg/kg/day. All three findings demonstrated a dose-relationship.

In the spleen, increased extramedullary hemopoiesis (EMH), was seen in two control males and decreased EMH, compared with female controls, was seen in two females that received 100 mg/kg/day. As variation in the level of EMH in the spleen are acknowledged as a common change in rats, these variations in the levels between control and treated animals of both sexes were considered incidental and not related to treatment with the test substance.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cycles, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment.

Administration of the test substance to parental animals at 10, 30 or 100 mg/kg/day for two weeks before pairing, during pairing and up to termination of the F0 males after 7 weeks of treatment and F0 females on Day 21 of lactation and then to selected F1 animals from weaning up to Day 27 of age was generally well tolerated.
In the F0-F1 generation there were no adverse effects on clinical condition, body weight performance, food consumption, estrous cycles, mating performance, fertility, litter size, offspring survival or macropathology.

After 7 weeks of treatment the adjusted kidney weights for males that received 30 or 100 mg/kg/day were higher with the difference attaining statistical significance at 100 mg/kg/day.
Histopathological examination of the kidneys from males that received the test substance revealed an accumulation of hyaline droplets in cortical tubules, seen at a higher severity and/or incidence in animals of all treated groups when compared with Control males, basophilia of cortical tubules (all dose levels) and granular casts in tubules of the outer medulla (30 and 100 mg/kg/day) were also seen and all three findings exhibited a relationship to dose. These kidney findings are considered to have contributed to the high body weight adjusted kidney weight that was observed for males that received 30 or 100 mg/kg/day.

In female animals at scheduled termination on Day 21 of lactation there was no apparent difference in kidney weight and none of these microscopic findings were apparent. Hyaline droplets in the male kidneys is consistent with the accumulation of alpha-2u-globulin, and has been recognized as a common finding in untreated male rats. Hyaline droplet accumulation is both sex and species specific and is not generally considered to be of significance in man.
However, an increased incidence and severity of hyaline droplets in association with multifocal basophilic tubules and granular casts, known as alpha-2u-globulin nephropathy, were considered to be adverse in the animals affected at 30 and 100 mg/kg/day.

In the decedent female no. 135 that received 100 mg/kg/day, bilateral epithelial microvacuolation of the cortical and outer medullary tubules of the kidneys was apparent. Vacuoles in the renal tubules had been reported in treated females in a previous Han Wistar rat study with the test substance.

The slight differences observed in plasma creatinine, sodium and/or urea observed for males at 30 or 100 mg/kg/day and females at 100 mg/kg/day may also be related to changes in kidney function.

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Key result

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No signs were observed in association with dose administration and there were no signs at routine physical examination that could be attributed to administration of the test substance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring body weight on Days 1 and 4 of age were lower for offspring derived from the groups receiving 30 or 100 mg/kg/day; these differences were statistically significant for male offspring at 100 mg/kg/day and for female offspring at 30 or 100 mg/kg/day. At 100 mg/kg/day body weight gain for offspring from Day 1 to Day 4 of age was lower when compared with Controls; approximately 81% of Controls for male offspring and 82% of Controls for female offspring but these differences did not attain statistical significance. From Day 4 of age body weight gain for offspring at all dose levels was similar to Controls and at weaning on Day 21 of age absolute body weight was essentially similar across all groups.

On Day 21 of age the mean body weight for selected F1 males and females at 100 mg/kg/day was slightly lower at approximately 90% of Controls; with the difference attaining statistical for the female animals (p<0.05) but not the males. At 100 mg/kg/day, subsequent weight gain for male animals up to termination on Day 28 of age was slightly lower, approximately 89% of Controls, and the absolute mean weight for males on Day 28 of age was statistically significantly low (p<0.05). For females there was no evidence of a dose related response in body weight gain from Day 21 of Day 28 of age, although statistical significance was attained at 100 m /kg/day. The absolute body weights for females on Days 24 and Day 28 of age were however low when compared with Controls (p<0.01; approximately 89/90% of Controls). Body weight at 10 or 30 mg/kg/day was unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first three days of direct administration F1 animals receiving the test substance at 100 mg/kg/day showed marginally but statistically low food consumption when compared with Controls (p<0.01); from Day 24 of age food consumption was the same as Controls. Food consumption at 10 or 30 mg/kg/day was unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Body weight adjusted liver weight for male animals at all dose levels and for female animals at 100 mg/kg/day were statistically significantly higher when compared with the Controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings for F1 animals at scheduled termination on Day 28 of age.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Administration of the test substance to F1 animals from weaning up to Day 27 of age was generally well tolerated.
There were no adverse effects on clinical condition, body weight performance, food consumption, litter size, offspring survival or macropathology.
Body weight on Days 1 and 4 of age was lower for offspring derived from the groups receiving 30 or 100 mg/kg/day and body weight gain at 100 mg/kg/day was marginally lower from Day 1 to Day 4 of age.

From Day 4 of age body weight gain for offspring at all dose levels was similar to Controls and at weaning on Day 21 of age absolute body weight was essentially similar across all groups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Administration of the test substance to parental animals at 10, 30 or 100 mg/kg/day for two weeks before pairing, during pairing and up to termination of the F0 males after 7 weeks of treatment and F0 females on Day 21 of lactation and then to selected F1 animals from weaning up to Day 27 of age was generally well tolerated. Adverse effects of treatment were limited to the F0 males that showed kidney pathology at 30 and 100 mg/kg/day (hyaline droplet accumulation, tubular basophilia and tubular granular casts); a dose response was apparent. In addition, there was evidence that the test item was acting as a peroxisome proliferator in the F0 generation male animals that received 100 mg/kg/day, as evidenced by its effects on cyanide-insensitive palmitoyl CoA oxidase activity.

However, within the context of this study there were no adverse effects of treatment on clinical condition, reproductive performance, fertility, offspring survival or development at doses up to and including 100 mg/kg/day.

Executive summary:

The purpose of this study was to make an assessment of general systemic toxic potential in rats, including reproductive/development effects, with administration of the test substance, by oral administration. A further purpose is for this to act as a preliminary study to assist with setting dose levels for the subsequent extended one generation reproductive toxicity study.

For the F0 generation, three groups of ten male and ten female rats received the test substance at doses of 10, 30 or 100 mg/kg/day by oral gavage administration for two weeks before pairing until termination. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

The F1 generation comprised of ten male and ten female progeny from each group, and they received direct treatment with the test substance from Day 21 to 27 of age, until termination on Day 28 of age. During the study, clinical condition, body weight, food consumption, hematology  peripheral blood), blood chemistry, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. Offspring clinical condition, body weight, litter size, survival and sex ratio were assessed and macroscopic observations were undertaken.

Results

The mean concentrations of the test substance in test formulations analyzed during the study were within 4% of the nominal concentration, confirming the accuracy of formulation.

F0 generation

There were two mortalities (one Control male and one high dose female) but these were considered to be incidental and unrelated to administration of the test substance. No signs were observed in association with dose administration and there were no signs at routine physical examination that could be attributed to administration of the test substance. Body weight gain and food consumption for males during the treatment period and for females for the two week period prior to being paired for mating were unaffected by treatment with the test substance. Overall the efficiency of food utilization during the two week pre-pairing period was unaffected by treatment for both male and female animals. During gestation body weight gain for females receiving the test substance was slightly lower during the first week of gestation; food consumption was unaffected during this phase. Overall body weight gain and food consumption for females during lactation showed no adverse effect of treatment. Estrous cycles, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by treatment.

On Day 21 of lactation females at 100 mg/kg/day showed higher red cell distribution width and longer activated partial thromboplastin time (p<0.05) when compared with Controls. Activated partial thromboplastin was also slightly longer at 30 mg/kg/day. After 7 weeks of treatment males at 100 mg/kg/day showed higher creatinine levels and males at 30 and 100 mg/kg/day showed elevated sodium levels. On Day 21 of lactation urea, creatinine and chloride levels were higher for females that received 100 mg/kg day and calcium levels for females that received the test substance were lower at all dose levels. Administration of the test substance to F0 male rats for 15 days prior to pairing at the high dose level of 100 mg/kg/day produced a statistically significant (p<0.05) increase in mean cyanide-insensitive palmitoyl CoA oxidase activity. Adjusted liver weights for males at 30 and 100 mg/kg/day and for females at 100 mg/kg/day were higher when compared with Controls. Adjusted kidney weights for males at 30 and 100 mg/kg/day were also higher. Macroscopic examination of males after 7 weeks of treatment and females on Day 21 of lactation did not reveal any findings that could be related to treatment. A minimal to moderate accumulation of hyaline droplets in cortical tubules was seen at a higher severity and/or incidence in males of all treated groups compared with control males. Minimal or slight basophilia of cortical tubules was seen in males of all treated groups. Minimal to moderate granular casts in tubules of the outer medulla were seen in males that received 30 or 100 mg/kg/day. All three findings demonstrated a dose-relationship.

F1 litter responses

There was no clear effect of parental treatment on general condition of the offspring, litter size, offspring survival and sex ratio. Offspring body weight on Days 1 and 4 of age were lower for offspring derived from the groups receiving 30 or 100 mg/kg/day and body weight gain at 100 mg/kg/day was marginally lower from Day 1 to Day 4 of age. From Day 4 of age body weight gain for offspring at all dose levels was similar to Controls and at weaning on Day 21 of age absolute body weight was essentially similar across all groups. There were no macroscopic observations for offspring that died prior to scheduled termination that could be attributed to parental treatment and there were no macroscopic findings for unselected offspring on Day 21 of age.

F1 generation

Following treatment of F1 male rats at 100 mg/kg/day between Day 21 to Day 27 of age, a decrease was observed in the mean 3,000 g supernatant protein concentrations (mg/g liver). No signs were observed in association with dose administration and there were no signs at routine physical examination that could be attributed to administration of the test substance.

On Day 21 of age the mean body weight for selected F1 males and females at 100 mg/kg/day was slightly low. At 100 mg/kg/day, subsequent weight gain up to termination on Day 28 of age and the absolute mean weight on Day 28 of age was lower. During the first three days of direct administration F1 animals at 100 mg/kg/day showed lower food consumption; from Day 24 of age food consumption was the same as Controls. Body weight adjusted liver weight for male animals at all dose levels and for female animals at 100 mg/kg/day were higher. There were no macroscopic findings for selected F1 animals at scheduled termination on Day 28 of age.

Conclusion

Administration of the test substance to parental animals at 10, 30 or 100 mg/kg day for two weeks before pairing, during pairing and up to termination of the F0 males after 7 weeks of treatment and F0 females on Day 21 of lactation and then to selected F1 animals from weaning up to Day 27 of age was generally well tolerated. Adverse effects of treatment were limited to the F0 males that showed kidney pathology at 30 and 100 mg/kg/day (hyaline droplet accumulation, tubular basophilia and tubular granular casts); a dose response was apparent. In addition, there was evidence that the test item was acting as a peroxisome proliferator in the F0 generation male animals that received 100 mg/kg/day, as evidenced by its effects on cyanide-insensitive palmitoyl CoA oxidase activity.

However, within the context of this study there were no adverse effects of treatment on clinical condition, reproductive performance, fertility, offspring survival or development at doses up to and including 100 mg/kg/day. It was therefore concluded based on this preliminary study and a 90 day study that a dose slightly higher than 100 mg/kg/day but no greater than 120 mg/kg/day would suitable as the high dose in an extended one generation study

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was designed similar to guideline OECD 415 and was performed according to GLP guidelines, but was designated mainly to select dose levels for a subsequent definite reproduction study. No histological examination of the reproductive or other organs were performed.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

no histological examinations

Principles of method if other than guideline:

one generation range-finding study similar to guideline OECD 421 and performed according to GLP guidelines. No histological examination of the reproductive or other organs were performed.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CDBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Inc., males: Raleigh, NC, USA, females: Stone Ridge, NY, USA
- Age at study initiation: (P) approx. 7 wks
- Weight at study initiation: (P) Males: 221-266 g; Females: 168-201 g
- Fasting period before study:
- Housing: individual, except the mating and post partum periods
- Diet: PMI Certified Rodent Diet Meal 5002 ad libitum
- Water: ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24,4
- Humidity (%): 40-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: undiluted test substance used

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (first 5 weeks), twice weekly (therafter
- Mixing appropriate amounts with (Type of food): basal diet
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug and/ or sperm in vaginal smear, referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All meal samples were within 10% of nominal concentration; stable for at least 4 days at 0.06% concentration, for at least 15 days at higher concentrations, details given in Appendix W

Duration of treatment / exposure:

P1 males and females were both exposed for at least 10 weeks proir to mating, through the mating period. Males were further exposed until their sacrifice, females throughout gestation and lactation until day 28 post partum. F1 pups were exposed from day 28 post partum until their sacrifice (postnatal day 42 (females) to 49 (males)).

Frequency of treatment:

daily, via feed

Dose / conc.:

0 other: % (nominal in diet)

Dose / conc.:

0.06 other: % (nominal in diet)

Dose / conc.:

0.12 other: % (nominal in diet)

Dose / conc.:

0.25 other: % (nominal in diet)

Dose / conc.:

0.5 other: % (nominal in diet)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cageside observations were performed daily on all P1 adults and after weaning for all F1 offspring, except the days clinical observations were performed.

VIABILITY: Yes
-Tiime schedule: All animals were examined for viability at least twice daily Monday through Friday, and at least once daily on weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A clinical examination was given to each male prior to P1 selection, on the first day of dosing, and at least weekly thereafter until euthanized. Females received a clinical examination prior to P1 selection, on the first day of dosing, and at least weekly thereafter until confirmation of mating, then on GD 0, 7, 14, and 21, and on PPD 0, 4, 7, 14 and 21.


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded for all females pretest, at initiation of dosing (Day 0), on Days 4 and 7, and weekly until confirmation of mating or the end of the mating period. Body weights were recorded for confirmed-mated females on GDs 0, 7, 14, and 21, on PPDs 0, 4, 7, 14, and 21 and weekly after Postpartum Day 21. After the mating period, body weights were recorded weekly for females not confirmed mated until they were sacrificed. Confirmed-mated females which did not deliver by GD 26 were weighed weekly after GD 26 until sacrificed. Body weights also were recorded on the day of sacrifice for all females.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured on Days 1, 2, 3, and 7, and then concurrently with body weight after Day 7, except during the mating period and on GD 0 and PPD 0 for the females when food consumption was not measured.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


OTHER:
- signs of parturition were examined at least twice daily beginning on gestational day (GD) 21. The duration of gestation was calculated and any difficulties occurring at parturition were noted. The date of parturition was recorded as the dam's Postpartum Day 0 (PPD 0).

POSTNATAL EXAMINATION: Yes
Each morning and afternoon during the postnatal period, the litters were checked for dead offspring and unusual conditions, and the dams were examined for viability, nesting, and nursing behavior.
Dead pups were removed from the litter immediately after their discovery. If intact, dead pups were examined externally and internally for anomalies. Dead pups discovered on PND 0 also were examined internally to determine whether they were stillborn.
On PND 0, 1, 4, 7, 14, 21, and 28 the offspring were counted, sexed, and each live pup was weighed. Pups were counted and examined externally on a daily basis during the postnatal period. All animals were weighed on PND 35, 42, and 49 (males only were weighed on PND Day 49).
On PND 4, after counting, weighing, and examining the pups, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Partial adjustment (e.g., 5 males and 3 females) was permitted whenever there were not enough pups to obtain 4 per sex per litter. Litters of eight pups or less were not adjusted.
Culled pups were sacrificed. Culled pups that appeared normal received only an external examination and tissues were not saved. Culled pups that appeared abnormal were subjected to a visceral examination.
The pups from each litter were examined daily for pinna detachment (starting PND 1), hair growth (starting PND 3), righting reflex (starting PND 3), incisor eruption (starting PND 7), and eye opening (starting PND 11). The examinations continued for an individual landmark until the criterion for that landmark was attained. Additionally, beginning on PND 29, all surviving female offspring were examined daily for vaginal opening. Beginning on PND 35 all surviving male offspring were examined daily for preputial separation. The examinations continued until all animals reached criteria.

Oestrous cyclicity (parental animals):

Vaginal smears were performed on each female on their day of sacrifice to determine its stage in the estrous cycle. The stage of the estrous cycle was recorded, but not used for estrous cycle calculations.

Sperm parameters (parental animals):

Samples of sperm from the left distal cauda epididymis (or proximal vas deferens) were collected at necropsy and evaluated for the percentage of progressively motile sperm and sperm morphology. Also, the entire left cauda epididymis was minced in saline to enumerate the total number of sperm (cauda reserves).

Litter observations:

Each morning and afternoon during the postnatal period, the litters were checked for dead offspring and unusual conditions, and the dams were examined for viability, nesting, and nursing behavior.
Dead pups were removed from the litter immediately after their discovery. If intact, dead pups were examined externally and internally for anomalies. Dead pups discovered on PND 0 also were examined internally to determine whether they were stillborn.
On PND 0, 1, 4, 7, 14, 21, and 28 the offspring were counted, sexed, and each live pup was weighed. Pups were counted and examined externally on a daily basis during the postnatal period. All animals were weighed on PND 35, 42, and 49 (males only were weighed on PND Day 49).
On PND 4, after counting, weighing, and examining the pups, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4 females per litter. Partial adjustment (e.g., 5 males and 3 females) was permitted whenever there were not enough pups to obtain 4 per sex per litter. Litters of eight pups or less were not adjusted.
Culled pups were sacrificed. Culled pups that appeared normal received only an external examination and tissues were not saved. Culled pups that appeared abnormal were subjected to a visceral examination.
The pups from each litter were examined daily for pinna detachment (starting PND 1), hair growth (starting PND 3), righting reflex (starting PND 3), incisor eruption (starting PND 7), and eye opening (starting PND 11). The examinations continued for an individual landmark until the criterion for that landmark was attained. Additionally, beginning on PND 29, all surviving female offspring were examined daily for vaginal opening. Beginning on PND 35 all surviving male offspring were examined daily for preputial separation. The examinations continued until all animals reached criteria.sul

Postmortem examinations (parental animals):

Gross necropsies were performed on all adult animals that were found dead. Body weight was recorded on the day of necropsy. The uterus of each female used for mating, but failing to deliver, was examined grossly for evidence of implantations and these data were recorded.
A gross necropsy was performed on all adult animals surviving to termination. Body weights were recorded on the day of necropsy. The uterus of each female was examined grossly for evidence of implantation and the number of implantation sites was recorded.

The following tissues and organs of all males surviving to termination and all females were weighed prior to fixation:
ovaries (individual) uterus
testes (individual) prostate
liver
right epididymis (total and cauda)
seminal vesicles (with coagulating glands and their fluids)

The following organs and tissues of all adults were preserved in 10% neutral buffered formalin:
coagulating gland right epididymis
seminal vesicles prostate
testes* uterus
liver ovaries
*:The right testis was preserved in Bouin's solution. The right testis remained in Bouin's solution for approximately 24 hours. The right testis was then rinsed with tap water and stored in 70 percent Ethyl Alcohol. The left testis was frozen for enumeration of homogenization resistant spermatids.

Abnormal tissues were preserved in 10% neutral buffered formalin at the discretion
of the Study Director or designee for possible future microscopic examination.

Postmortem examinations (offspring):

Intact dead pups or pups sacrificed in moribund condition on PND 0 were examined by fresh visceral dissection. Dead pups and pups sacrificed as moribund after PND 0 were examined externally for anomalies and internally for gross visceral abnormalities. Culled pups (PND 4) with external abnormalities were subjected to a visceral examination at the discretion of the Study Director or his designee.

Statistics:

Group means and standard deviations were calculated.

Reproductive indices:

mean male fertility and mating indices and female fertility, fecundity and gestational indices

Offspring viability indices:

offspring survival

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Significant increases in body weight gain in males of the 0.12 and 0.5% dose groups were considered incidental and unrelated to treatment with the test material (absence of a clear consistent response over the test period).
Statistically significantly decreased mean body weights when compared with controls were observed in the 0.5% females on Gestation Day (GD) 7 (9%) and on GD 21 (11%) and on Postpartum Days (PPD) 4, 7, and 14 (17%, 12%, and 10%, respectively). Also, there was a corresponding statistically significant decrease in mean body weight change for the 0.5% group females at the PPD 0-4 interval (209%). There also was a statistically significantly decreased mean body weight compared with controls in the 0.25% females on PPD 4 (10%).
There also were statistically significant increases in mean body weight change in the 0.25 and 0.5% group females at the PPD 4/7 (470% and 567%, respectively), PPD 14/21 (154% and 235%, respectively), and in the 0.25% females on PPD 0/21 intervals (112%).
There were statistically significant decreases in mean food consumption for the 0.5% group males at Days 1 (2 1%) and 2 (2 1%). There were statistically significant decreases in mean food consumption in the 0.25% group females at Day 1 (30%), Day 3 (18%), and Weeks 1 (13%), 2 (11%), 3 (21%), 4 (13%), and 6 (12%). There also were statistically significant decreases in mean food consumption for the 0.5% group females at Day 1 (47%) and Weeks 1 (15%), 2 (13%), 4 (11%), and 6 (10%). These decreases were considered to be due to reduced palatability of the diet mixtures.
There were no statistically significant differences in food consumption during the gestation period. However, statistically significant decreases in food consumption were noted in the 0.5% group females during the PPD 0/4 (46%), 7/14 (15%), and entire postpartum period (PPD 0-28) (17%).


ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no statistically significant changes in the mean absolute or mean relative organ weights for the reproductive organs weighed during the study. There were statistically significant increases in the mean absolute and mean relative liver weights of the 0.5% males (15% and 21%, respectively) and 0.5% females (21% and 24%, respectively) and the 0.25% females mean absolute and relative liver weights (15% and 14%, respectively). The significance of the increases in the liver weights could not be determined because histopathology was not performed on the tissues.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
There was a statistically significant decrease in the mean percent live offspring (6%) and the corresponding increase in the mean percent dead offspring (700%) in the 0.5% dose group.

Key result

Dose descriptor:

LOAEL

Effect level:

165 - 500 mg/kg bw/day

Sex:

female

Basis for effect level:

other: decreases in body weight, increased liver weight; 0.25% in diet, calculated by the authors on the basis of food consumption and body weights

Key result

Dose descriptor:

NOAEL

Effect level:

79 - 228 mg/kg bw/day

Sex:

female

Basis for effect level:

other: 0.12% in diet, calculated by the authors on the basis of food consumption and body weights

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
There were statistically significant decreases in 0.5% group offspring compared with the control offspring for the live birth index (6.0%), the Day 1 survival index (14%), and the Day 4 survival index (2 1%).

BODY WEIGHT (OFFSPRING)
There were statistically significant decreases in mean offspring body weights in the 0.25% group males and females at PND 0, 1, 4, 7, 35 and 42. There also were statistically significant decreases in mean offspring body weights in the 0.25% group males at PND14, 28, and 49.

SEXUAL MATURATION (OFFSPRING)
There was a statistically significant advance for preputial separation for the 0.06% group males (1.2 days) when compared with the controls. Due to the small size of this advance and the absence of a dose response, this difference was not considered biologically significant. There also was a statistically significant retardation of preputial separation for the 0.5% group males (2.1 days) compared with the control male offspring. In the females, the 0.5% group exhibited a statistically significant retardation (1.8 days) for vaginal patency compared with controls. These two findings are not biologically significant but rather reflect the normal maturation of these animals with the delays due to somewhat smaller body weights.

OTHER FINDINGS (OFFSPRING). DEVELOPMENTAL LANDMARKS
There were statistically significant retardations in the male eye opening for the 0.5% group (0.7 days later), and the male and female pinna detachment in the 0.5% group (1.0 day later each). These findings are not biologically significant but rather reflect the normal maturation of these animals with the delays due to somewhat smaller body weights.

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

0.25 other: % in diet

Sex:

male/female

Basis for effect level:

other: reduced fetal weights, no calculation of body doses stated

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

0.12 other: % in diet

Sex:

male/female

Basis for effect level:

other: no calculation of body doses stated

Reproductive effects observed:

not specified

BODY DOSES BASED ON FOOD CONSUMPTION AND BODY WEIGHTS

	DOSE (%)	WEEK 1 (mg/kg/day)	WEEKS 2-9 (mg/kg/day)	WEEK 10 (mg/kg/day)	Gestation	Postpartum
M A L E S	0.06	59	33-51	32	Not Applicable	Not Applicable
	0.12	117	66-101	66
	0.25	247	147-215	145
	0.5	477	289-435	290
F E M A L E S	0.06	57	40-56	40	37-42	57-126
	0.12	116	79-112	81	79-86	109-228
	0.25	231	168-224	167	165-185	241-500
	0.5	450	347-453	347	336-382	311-970

MEAN OFFSPRING BODY WEIGHT - F1 (PREWEANING)

}Group	MALE PND 0	MALE PND 1	MALE PND 4	MALE PND 7	MALE PND 14	MALE PND 21	MALE PND 28
0%	6.71	7.09	9.54	15.58	32.32	51.18	91.53
0.06%	6.82	7.31	10.13	16.70	34.12	53.06	93.88
0.12%	6.39	6.85	9.01	14.44	30.72	48.88	90.23
0.25%	5.76**h	5.95**h	7.98*h	12.55**h	28.64*h	46.23	81.65**
0.5%	5.66**h	5.73**h	7.67**h	11.07**h	23.31**h	39.18**h	72.63**
Historical Control	6.35-7.02	6.68-7.49	8.53-11.43	13.64-18.74	28.81-37.09	44.89-62.34	98.34
Group	FEMALE PND 0	FEMALE PND 1	FEMALE PND 4	FEMALE PND 7	FEMALE PND 14	FEMALE PND 21	FEMALE PND 28
0%	6.36	6.89	9.33	14.72	30.50	47.77	82.46
0.06%	6.51	7.00	9.67	15.35	31.89	49.18	83.84
0.12%	5.92h	6.40	8.78	13.81	28.79	46.18	81.83
0.25%	5.46**h	5.65**h	7.83**h	12.77*h	28.89	45.33	77.09
0.5%	5.65**h	5.65**h	7.55**h	11.10**h	23.47**h	38.59**h	69.34**
Historical Control	5.96-6.74	6.30-7.16	8.32-11.05	13.33-17.69	27.22-35.89	42.39-61.19	90.68

MEAN OFFSPRING BODY WEIGHT - F1 (POSTWEANING)

{ }Group	MALE PND 35	MALE PND 42	MALE PND 49
0%	147.7	205.8	266.3
0.06%	151.0	211.4	272.4
0.12%	144.3	205.1	266.3
0.25%	128.4**	183.4**	242.1**
0.5%	119.8**	170.9**	227.6**
Group	FEMALE PND 35	FEMALE PND 42	FEMALE PND 49
0%	124.8	158.3	NA
0.06%	126.5	161.4	NA
0.12%	123.1	157.2	NA
0.25%	114.9**	148.6*	NA
0.5%	109.3**	140.9**	NA

NOTE: All weights are in grams

* Mean significantly different from control mean (p0.05)

** Mean significantly different from control mean (p0.01)

NA not applicable

Conclusions:

Under the conditions of this study administration of the test substance resulted in evidence of maternal toxicity and findings in offspring that may have been secondary to maternal toxicity. Maternal toxicity was demonstrated by decreased body weight and food consumption during the gestation and postpartum periods, and increased absolute and relative liver weights in the 0.25% and 0.5% groups (79-228 and 165-500 mg/kg bw and day). Paternal effects were limited to increased absolute and relative liver weights in the 0.5% group. The differences between male and female adults in this study may be due to the somewhat higher dose rates observed in the females, especially during the postpartum period. Coincident with the maternal effects, offspring body weights were decreased in both sexes in the 0.25% and 0.5% groups, and offspring survival was decreased in the 0.5% dose group. There were also some small delays in developmental landmarks in the high dose offspring. These delays are likely secondary to the decreased offspring growth in this group.
There was no evidence of adverse effects on mating behavior, fertility, sperm parameters, or reproductive organ weights in the parental animals. Thus, the test material does not effect fertility at the dose levels tested. There were also no malformations observed in the offspring in this study. There were indications of offspring toxicity that may be related to maternal toxicity in the two highest doses. The apparent NOAEL for both maternal and offspring effects in this study is the 0.12% level.

Executive summary:

In this one-generation range-finding study (performed similar to OECD guideline 415) the test substance was administered to male and female rats in the diet (10 per sex and dose; 0.06, 0.12, 0.25 and 0.5% in diet) for at least ten weeks prior to mating and during the mating period. The dams were exposed during the gestation and postpartum periods, until weaning of the offspring on Postpartum Day (PPD) 28. The parental male animals were sacrificed at the end of the mating period, the females and the offspring after weaning.

Effects in the P-generation:

There were no treatment-related deaths, clinical signs or effects on male or female reproductive organ weights or reproductive parameters. Statistically significant decreases were observed in food consumption of the 0.5% group females at various postnatal timepoints. The body weights of the parental generation were reduced in females of the 0.25% group at PPD 4 as well as in 0.5% females at Gestation Days (GD) 7 and 21 and at PPD 4, 7, and 14. Liver weights were increased in males at 0.5%, in females at 0.25% and above.

Effects in offspring:

Reduced Live Birth Index and survival indices were observed in the 0.5% group. Postnatal offspring body weights of both sexes of the 0.25% and 0.5% groups were reduced compared with the controls.

No treatment-related clinical signs were noted for the offspring. A postnatal developmental delay was obvious in the 0.5% dose group offspring in form of delayed onset of eye opening in male offspring, pinna detachment in both sexes, retardation of preputial separation and vaginal patency. These delays were considered to be treatment-related and secondary to reduced offspring body weight.

The NOAEL for reproductive toxicity was 0.5% (highest tested dose, 289-477 mg/kg bw/day in males, 336-970 mg/kg bw/d in females for P generation). The LOAEL for systemic parental (female) and offspring toxicity was 0.25% (165 -500 mg/kg/bw/day for P generation), the NOAEL was 0.12% (79 -228 mg/kg bw/day for P generation) (Exxon, 1998).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

reliable and qualified guideline studies available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An Extended One-Generation Reproductive Toxicity Study (EOGRTS) three groups of 25 male and 25 female rats received Isononanoic Acid at doses of 5, 25 or 120 mg/kg/day by oral administration (gavage). Males were treated daily for ten weeks before pairing until termination after litters are weaned. Females were treated daily for ten weeks before pairing, throughout pairing, gestation and until termination. Females were allowed to litter, rear their offspring and were killed on Day 28 post partum. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups. The F1 generation comprised of two cohorts. Treatment of F0 females at 120 mg/kg/day was not tolerated during late gestation and early lactation: three females were killed for welfare reasons due late gestation and two females were killed for welfare reasons during early lactation. Microscopic pathology changes were seen in the liver, kidneys and pancreas of several of these females but were not considered to be responsible for the poor condition of these females. Treatment of F0 females at 25 or 5 mg/kg/day, and of F0 males at all doses, was well tolerated during the in life phase of the study.

At 120 mg/kg/day, there was no effect of treatment on the mean number of implantations but there was slightly low post implantation survival index and low live birth index which resulted in a low total litter size on Day 1 of age and low live litter sizes on Days 1 and 4 of age. There was a higher incidence of pups abnormally cold to touch or with little/no milk in stomach at 120 mg/kg/day compared with control. Mean body weights of male and female offspring on Day 1 of age were lower at 120 mg/kg/day and marginally lower at 25 mg/kg/day; all differences attained statistical significance. Body weights remained lower in males at 120 mg/kg/day on Day 4 and Day 7. There was no effect of treatment on the overall weight gain of the offspring between Days 1 and 21 of age, although male and female offspring in the 120 mg/kg/day group showed slightly lower weight gain between Days 4 and 7 of age compared with Controls. Body weight gain of both sexes was marginally low between Days 21 and 25 of age.

Treatment at 5, 25 or 120 mg/kg/day was well tolerated in the selected F1 offspring (Cohorts 1A and 1B) during the in life phase of the study. Macroscopic examination of the F0 adults did not reveal any test item related changes but examination of the selected F1 animals did reveal abnormal color (pale) and pale areas in the kidneys of some males receiving 120 mg/kg/day. Microscopic examination of a full list of tissues revealed test item related changes in the kidneys, liver and pancreas of F0 and F1A animals and in the kidneys of F1B males.

A No Observed Adverse Effect Level (NOAEL) for systemic toxicity in male rats was not established and lies below 5 mg/kg/day due to hyaline droplets with associated degenerative changes (basophilic tubules and granular casts) - alpha2u-globulin nephropathy considered adverse at all dose levels. It is however acknowledged that this finding is male rat specific and generally considered to be of no relevance to man. Excluding this finding the NOAEL for male rats is 120 mg/kg/day. The NOAEL for systemic and developmental reproductive toxicology in adult female rats was concluded to be 25 mg/kg/day based on poor clinical condition necessitating the humane kill of five females receiving 120 mg/kg/day during late gestation/early lactation. A dose of 25 mg/kg/day is also concluded to be the NOAEL for the offspring based on lower post implantation and live birth survival indices resulting in a low total and live litter size at 120 mg/kg/day (Covance, 2019).

In a reliable one-generation range-finding study, the test substance was administered to rats in the diet in concentrations of 0.06, 0.12, 0.25 and 0.5% for at least ten weeks prior to mating, during the mating gestation and postpartum periods until weaning of the F1 offspring. The male P1 animals were sacrificed at the end of the mating period, the females and the offspring after weaning.

No effects were oberserved with respect to fertility (NOAEL 0.5%, 289-477 mg/kg bw/day in males, 336-970 mg/kg bw/d in females).

Other effects in the P generation were decreases in food consumption in the 0.5% group dams during the postpartum period, decreases in body weights in females of the 0.25% group at Postpartum Day (PPD) 4 as well as in 0.5% females at Gestation Days (GD) 7 and 21 and at PPD 4, 7, and 14. Liver weights were increased in dams at dietary concentrations of 0.25% and above, in males at 0.5%.

The LOAEL for systemic parental (female) effects was 0.25% (165-500 mg/kg bw/d for females), the NOAEL 0.12% (79 -228 mg/kg bw/d for females)

(Exxon, 1998).

Addtionally, a Reproduction/Developmental Screening Test in rats according to OECD 422 is available. Gavage administration of the test substance to parental animals at 10, 30 or 100 mg/kg day for two weeks before pairing, during pairing and up to termination of the F0 males after 7 weeks of treatment and F0 females on Day 21 of lactation and then to selected F1 animals from weaning up to Day 27 of age was generally well tolerated. Adverse effects of treatment were limited to the F0 males that showed kidney pathology at 30 and 100 mg/kg/day (hyaline droplet accumulation, tubular basophilia and tubular granular casts); a dose response was apparent. In addition, there was evidence that the test item was acting as a peroxisome proliferator in the F0 generation male animals that received 100 mg/kg/day, as evidenced by its effects on cyanide-insensitive palmitoyl CoA oxidase activity. Within the context of this study there were no adverse effects of treatment on clinical condition, reproductive performance, fertility, offspring survival or development at doses up to and including 100 mg/kg/day (Envigo, 2018).

Effects on developmental toxicity

Description of key information

In a reliable range-finding one-generation rat study the offspring showed reduced body weights, developmental delays and increased fetal mortality at doses which produced maternal toxicity (NOAEL 0.12% diet, 79-228 mg/kg bw/d for dams; LOAEL 0.25%, 165-500 mg/kg bw/d for dams. No developmental defects have been observed in litters in which no maternal toxicity occurred (OECD 414, BASF SE 2013) -> NOAEL 60 mg/kg/d.

No effects have been observed in a developmental toxicity study in rabbits at doses up to 250 mg/kg bw/day, the highest dose tested (OECD 414, Envigo, 2019).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-13 weeks
- Weight at study initiation:
- Housing: 1 rat per cage
- Diet: Ground Kliba maintenance diet ad libitum
- Water : filtered tap water ad libitum
- Acclimation period: From GD 0 (day of supply) to the beginning of administration (GD6), the animals were accustomed to the environmental conditions and to the diet

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 airchanges per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

4 mL/kg body weight; for test substance preparation, the specific amount of test substance will be weighed, topped up with corn oil in a calibrated beaker and intensely mixed with a magnetic stirrer; during administration, the preparations were kept homogenous with a magnetic stirrer; preparations are stored at room temperature

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Animals paired by the breeder (time-mated animals) were supplied at noon on the day of evidence of mating; this day is referred to as GD0

Duration of treatment / exposure:

Animals are treated once daily from GD6-GD19

Frequency of treatment:

Once daily

Duration of test:

Dams are sacrificed on day 20

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, abnormalities and changes are documented

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Bw is recorded on day 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consuption is recoreded from GD0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19, 19-20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
Examinations included:
- Gross-pathological examination
- Weight of the unopened uterus
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Site of implantations in the uterus

Fetal examinations:

After removal of fetuses from the uterus, the following examinations have been made:
- weight of each fetus
- sex
- weight of placentas
- gross pathological examination of fetuses after dissection from uterus
- half of the fetuses of each dam is skined, fixed in ethyl alcohol and the skeleton and cartilage stained (method by Kimmel and Trammell)
- the other half of the fetuses is fixed in Harrisons fluid and soft tissues examined

Statistics:

Dunnett's test, Fishers exact test and Wilcoxon test

Historical control data:

Results were compared to historical control data for Wistar rats that were available in the test facility

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

200 mg/kg bw/day group: 2 dams died shortly before term

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day group: Decreased net body weights (8% below control) at the end of gestation, distinctly reduced net body weight gain during treatment (47% below control)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day group: Decreased food consumption towards the end of gestation (13-23% below control)

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day group: Increased red blood cell (RBC) counts, hemoglobin and hematocrit values, decreased relative reticulocyte counts, increased absolute and relative monocyte and large unstained cell (LUC) counts

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased urea levels, decreased total protein, albumin, globulin, cholesterol, triglyceride and calcium levels

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day group: Increase in liver weight (absolute +13%, relative +23%)

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

mortality

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day group: Reduced fetal weights (14% below control), at 60 mg/kg bw/day: only minimal weight reduction (5% below control), not considered as adverse: there was no no effects on the degree of fetal maturity and the reduction was within the historical control range

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg bw/day group: Morphological changes indicating a delay or disturbance of development, such as increased
incidences of supernumerary ribs (73.9 % affected fetuses per litter) and wavy
ribs (14.4%), incompletely ossified Skulls 43.7%, unossified sternebra (31.5%), incomplete
ossification of metacarpal (7.3%), incomplete ossification of pubis (4.7%), incomplete
ossification of ischium (3.3%)
Increased incidence of severely altered rib cages (multiple rib findings like wavy, bent or
knobby) in 9.4% fetuses per litter, resulting in a skeletal malformation rate of 11.1% affected
fetuses per litter

60 mg/kg bw/day group: skull ossification was decreased in mid-dose animals; however, these decreases were still within historical control data

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A yellow discoloration of fetal livers was noted in almost all high-dose litters (200 mg/kg bw/day) and approximately half of the mid-dose litters (60 mg/kg bw/day). The rate of affected fetuses per litter was about 60 and 20%, respectively. The dose-relationship suggests an association to the treatment, but the cause of this discoloration is not known. The shape and size of those livers were completely unchanged. As there is no frank morphological change observable, the level of concern for this finding is rather low. It is not considered as evidence for an adverse effect.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Test Group 3 (200 mg/kg/d)
• Reduced fetal weights (14% below control)
• Morphological changes indicating a delay or disturbance of development, such as increased incidences of supernumerary ribs (73.9 % affected fetuses per litter) and wavy ribs (14.4%), incompletely ossified Skulls 43.7%, unossified sternebra (31.5%), incomplete ossification of metacarpal (7.3%), incomplete ossification of pubis (4.7%), incomplete ossification of ischium (3.3%)
• Increased incidence of severely altered rib cages (multiple rib findings like wavy, bent or knobby) in 9.4% fetuses per litter, resulting in a skeletal malformation rate of 11.1% af-fected fetuses per litter

Test Group 2 (60 mg/kg/d)
• No test substance-related adverse effects on fetuses

Test Group 1 (20 mg/kg/d)
• No test substance-related adverse effects on fetuses

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: developmental effects at maternal toxic doses

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: pelvic girdle

Description (incidence and severity):

supernumary and wavy ribs, altered rib cages, other localisations: incomplete ossification

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of I-Nonanoic acid to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity at a dose of 200 mg/kg bw/d, such as mortality, reduced body weights/weight gain, hematological alterations, dysregulated liver cell metabolism and dose-relatedly increased liver and adrenal weights. An increase in liver weight in the mid dose group was without histopathological correlate and hence not considered adverse. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 60 mg/kg bw/d.
Pups of the high-dose group showed signs of developmental defecets as depicted by reduced body weights, severely altered rib cages (eading to an increased malformation rate), wavy ribs, decreased ossification of skulls, sternebrae, sacral arch, metacarpal, pubis and ischium. Considering strong systemic toxicity observed in dams, these effects are most likely due to maternal toxicity and not a direct effect of the substance on development. In the mid dose-group (60 mg/kg/d), fetal body weights were statistically significantly reduced (5%). Since there were no effects on the degree of fetal maturity at 60 mg/kg bw/d, the slight weight reduction in this group is not considered to be an adverse, toxicologically relevant effect of the test substance. Moreover, the decrease in fetal body weight was within the historical control range. Accordingly, skull ossification was decreased in mid-dose animals; however, these decreases were still within historical control data, indicating that this effects is not considered adverse. A yellow discoloration of fetal livers was noted in almost all high-dose litters (200 mg/kg bw/d) and approximately half of the mid-dose litters (60 mg/kg bw/d). The rate of affected fetuses per litter was about 60 and 20%, respectively. The dose-relationship suggests an association to the treatment, but the cause of this discoloration is not known. The shape and size of those livers were completely unchanged. As there is no frank morphological change observable, the level of concern for this finding is rather low. It is not considered as evidence for an
adverse effect.
Since no effects have been noted in low-dose animals and effects observed in mid-dose rats are not considered to be an adverse, toxicologically relevant effect of the test substance, the NOAEL for developmental toxicity is set at the mid dose, i.e. 60 mg/kg/d.
Since morphological changes in offspring were only observed at 200 mg/kg bw/d, a dose level which caused distinct maternal toxicity, including mortality isononanic acid does not need to be classified with respect to teratogenic effects.

Executive summary:

i-Nonanoic acid was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an emulsion in corn oil to groups of 25 time-mated female Wistar rats by gavage at doses of 20, 60, and 200 mg/kg body weight/day (mg/kg bw/day) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

The following test substance-related adverse effects/findings were noted:

Test group 3 (200 mg/kg bw/day):

Dams

􀁸 Mortality in 2 dams shortly before term

􀁸 Decreased food consumption towards the end of gestation (13-23% below control)

􀁸 Decreased net body weights (8% below control) at the end of gestation, distinctly reduced net body weight gain during treatment (47% below control)

􀁸 Increased red blood cell (RBC) counts, hemoglobin and hematocrit values

􀁸 Decreased relative reticulocyte counts

􀁸 Increased absolute and relative monocyte and large unstained cell (LUC) counts

􀁸 Increased urea levels

􀁸 Decreased total protein, albumin, globulin, cholesterol, triglyceride and calcium levels

􀁸 Increase in liver weight of absolute +13% and relative +23%

Fetuses

􀁸 Reduced fetal weights (14% below control)

􀁸 Morphological changes indicating a delay or disturbance of development, such as increased incidences of supernumerary ribs (73.9 % affected fetuses per litter) and wavy ribs (14.4%), incompletely ossified Skulls 43.7%, unossified sternebra (31.5%), incomplete ossification of metacarpal (7.3%), incomplete ossification of pubis (4.7%), incomplete ossification of ischium (3.3%)

􀁸 Increased incidence of severely altered rib cages (multiple rib findings like wavy, bent or knobby) in 9.4% fetuses per litter, resulting in a skeletal malformation rate of 11.1% affected fetuses per litter

Test group 2 (60 mg/kg bw/day):

􀁸 No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 1 (20 mg/kg bw/day):

􀁸 No test substance-related adverse effects on dams, gestational parameters or fetuses

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 60 mg/kg bw/day.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/day, based on reduced fetal weights, as well as a delay of development and a slightly increased malformation rate at 200 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Aug 2018 to 22 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

"current" (presumably Version of 22 January 2001 due to study initiation in March 2018)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS
- Age at study initiation: 18-22 weeks
- Weight at study initiation: 2.6 to 4.6 kg
- Fasting period before study: no
- Housing: individually, during mating with one male
- Diet: Teklad 2930 Diet, initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter
- Water (ad libitum): Potable water from the public supply
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21
- Humidity (%): 45-70
- Photoperiod (hrs dark / hrs light): 10/14

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: weekly


VEHICLE
- Concentration in vehicle: 0-500 mg/mL
- Amount of vehicle (if gavage): 0.5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for the first and last dosing occasion were analyzed for achieved concentration of the test item.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until natural mating observed

- Proof of pregnancy: day of mating referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 to 28 after mating

Frequency of treatment:

Daily

Duration of test:

total: 29 days

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

80 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on findings from a preliminary embryo-fetal development study (QC63YF, T11230) with 100 and 250 mg/kg bw/day, leading to slight weight loss and reduced weight loss gain in treated animals in comparison to controls (details see under "Any other information on results")

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on days 0, 6, 12, 18, 24 and 29

BODY WEIGHT: Yes
- Time schedule for examinations:weekly during acclimatization, on the day of mating (Day 0), on Day 3 and then daily from Day 6 after mating

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Recorded daily from Day 1 after mating

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: full macroscopic examination of the tissues, all external features and orifices were examined visually

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made.For all other analyses the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.

Indices:

not calculated

Historical control data:

Presented in attachment

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs at routine physical examination or in association with dose administration that could be related to treatment with Isononanoic acid.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On GD7 one female no 72 receiving 250 mg/kg/day was found dead in the afternoon following dose administration in the morning. No signs were observed prior to death and no macroscopic abnormality was detected at necropsy; in the absence of any effect on clinical condition in the high dose group this was considered to be an incidental death and unrelated to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight gain during the treatment period GD6-28 was unaffected by administration of Isononanoic acid at dose levels up to and including 250 mg/kg/kg/day.
Maternal body weight gain after adjustment for the gravid uterine weight showed no adverse effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption data was highly variable across all groups including the Controls and as such differences were considered to relate to administration of corn oil rather than the test item.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of females on Day 29 of gestation did not reveal any findings that could be related to administration of Isononanoic acid.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: see below

Key result

Dose descriptor:

LOAEL

Effect level:

> 250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: see below

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
At 25, 80 and 250 mg/kg/day there was a slightly higher incidence of incompletely ossified 1st -4th and 6th sternebrae when compared with the concurrent control; but the incidences were within the historical control range.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see below

Key result

Dose descriptor:

LOAEL

Effect level:

> 250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see below

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Rationale for dose selection, results from dose range finding test

Dose levels of 0, 25, 80 and 250 mg/kg/day were selected in conjunction with the Sponsor based on findings from a preliminary embryo-fetal development study in the New Zealand white rabbit that was performed at this laboratory (Envigo Study No. QC63YF, T11230). In the preliminary study, Isononanoic acid was administered at dose levels of 100 and 250 mg/kg/day. There was no effect on clinical condition, food consumption or macropathology with effects limited to an initial loss in body weight (GD6-7/8) and low overall weight gain (GD6-29) at 100 and 250 mg/kg/day; the maternal weight loss when adjusted for the gravid uterine weight was also slightly greater at 250 mg/kg/day.

The vehicle corn oil was selected as this was used on a previous prenatal developmental toxicity study in rats (BASF Project No. 30R0166/07R048) in which pregnant rats were treated at dose levels of 0, 20, 60 or 200 mg/kg/day; at 200 mg/kg/day 2/25 died during the treatment period whilst at 60 mg/kg/day no adverse effects were apparent.

The maximum tolerated volume dose for corn oil in the rabbit is deemed to be 0.5 mL/kg/day (reference the Pilot rabbit study with Isononanoic acid Envigo No. MX20NN, T11224). Therefore the maximum practical dose level was 250 mg/kg/day when administering 500 mg Isononanoic acid/mL at 0.5 mL corn oil/kg.

A high dose of 250 mg/kg/day was therefore selected based on the preliminary studies with a low dose of 25 mg/kg/day and an intermediate dose of 80 mg/kg/day to achieve approximate 3-fold dose intervals.

Conclusions:

Under the conditions of this study, no maternal or developmental effects were produced in rabbits at doses up to 250 mg/kg bw/day

Executive summary:

Three groups of 22 females received Isononanoic Acid at doses of 25, 80 or 250 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Oral administration of 25, 80 and 250 mg/kg/day was well tolerated with no adverse effects on maternal condition, body weight or food consumption. In addition embryo-fetal survival and development was unaffected by maternal treatment at dose levels up to and including 250 mg/kg/day.

Therefore the high dose of 250 mg/kg/day was considered to represent the no observed effect level (NOEL) for both maternal toxicity and embryo-fetal survival and development.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two reliable developmental toxicity studies, performed in rats and rabits

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

In a one-generation range-finding study, the test substance was administered to rats in the diet in concentrations of 0.06, 0.12, 0.25 and 0.5% for at least ten weeks prior to mating, during the mating gestation and postpartum periods until weaning of the F1 offspring. The male P1 animals were sacrificed at the end of the mating period, the females and the offspring after weaning.

Reduced live birth index and survival indices were noted in the offspring of the 0.5% group. Offspring body weights of both sexes of the 0.25% and 0.5% groups were reduced compared with the controls during the postnatal period. A postnatal developmental retardation was obvious in the 0.5% dose group offspring, based on the delay of onset of eye opening in male offspring, pinna detachment in both sexes, retardation of preputial separation and vaginal patency. While treatment related, these delays were considered secondary to reduced offspring body weight.

The LOAEL for developmental effects was 0.25% (165-500 mg/kg bw/d for dams), the NOAEL 0.12% (79 -228 mg/kg bw/d for dams) (Exxon, 1998; key study, RL2).

 

Developmental toxicity:

Rats:

In a prenatal developmental toxicity study according to OECD414 the test compound i-Nonanoic acid was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity (BASF SE, 2013).

Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle.

Generally, clinical observations revealed no toxicologically relevant difference between the dams receiving 20 or 60 mg/kg bw/d i-Nonanoic acid and controls. Most dams receiving the highest dose of 200 mg/kg bw/d and 2 dams of the mid-dose (60 mg/kg bw/d) showed transient salivation after treatment. This salivation persisted in the respective females for a few minutes immediately after each administration. It is considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.

The high-dose of the test substance (200 mg/kg) bw/d produced distinct adverse effects in gestating female Wistar rats. Two dams of the high-dose group (200 mg/kg) died shortly before term, without having shown any preceding signs of intoxication or illness. Anatomic and microscopic pathology did not reveal the cause of these fatalities. However, these investigations also did not furnish evidence for misgavage or any other cause of death unrelated to treatment. Thus these deaths are assumed to be associated with the exposure of these two dams to the test substance. In addition, food consumption (15-30% below control) and body weights (9% below control on GD 20) were significantly reduced towards the end of gestation. Corrected (net) body weight gain was substantially reduced (47% below control) in these animals.

Regarding clinical pathology, changes were observed exclusively in the dams of test group 3 (200 mg/kg bw/d): The measured red blood cell parameters (RBC counts, hemoglobin and hematocrit values) were increased indicating hemoconcentration, most likely because of stress. Relative reticulocyte counts were decreased because of higher RBC counts, whereas the absolute count of reticulocytes was most likely unchanged. Higher stress was confirmed by increased monocyte counts. Because activated monocytes were measured by the hematology analyzer in the fraction of the large unstained cells (LUC), this cell portion was also increased. Higher urea and lower total protein, albumin and globulin levels in the dams of the mentioned test group were due to an increased protein metabolism. Because of decreased transport protein levels total calcium levels were also lower. Decreased cholesterol and triglyceride levels were most probably also caused by an altered liver cell metabolism.

Regarding pathology, there was a treatment - related decrease in terminal body weight (-8%) and an increase in liver weights (increase of relative weights +4%, +14%, +23% in test groups 1, 2, and 3 respectively, and in absolute weights in test groups 2 and 3 by +15% and +13%). As the weight changes were not accompanied by histopathological findings, only test group 3 was assessed as adverse because of clinical pathology findings. It cannot be ruled out that the weight increases of the liver of test groups 1 and 2 are a treatment – related effect as well but they were neither accompanied by adverse clinical pathology findings nor a histopathological correlate.

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

There was no evidence for any impairments of gestational parameters at any dose. Reproductive parameters such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss were not influenced by the treatment. Similarly, no influence of the test compound on sex distribution of the fetuses was noted at any dose.

In the mid- and high-dose groups lower fetal body weights (5% and 14% below control, respectively) were noted. Since there were no effects on the degree of fetal maturity at 60 mg/kg bw/d, the slight weight reduction in this group is not considered to be an adverse, toxicologically relevant effect of the test substance.

Only in the high-dose group (200 mg/kg bw/d) lower fetal weights were associated with morphological changes indicating a delay or disturbance of development. In addition to incomplete ossification of various bones, severely altered rib cages (multiple rib findings like wavy, bent or knobby) were noted in 9.4% fetuses per litter, resulting in a skeletal malformation rate of 11.1% affected fetuses per litter.

 

Taken together, these findings suggest that the test item caused developmental toxicity at dose levels of 200 mg/kg bw/d, a dose at which strong maternal toxicity including mortality has been noted. Since no developmental defects have been noted at doses that do not induce maternal toxicity, it can be concluded that isononanic acid does not affect development and does hence not require classificiation with respect to developmental toxicity.

Rabbits:

Three groups of 22 females received Isononanoic Acid at doses of 25, 80 or 250 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination. Oral administration of 25, 80 and 250 mg/kg/day was well tolerated with no adverse effects on maternal condition, body weight or food consumption. In addition embryo-fetal survival and development was unaffected by maternal treatment at dose levels up to and including 250 mg/kg/day. Therefore the high dose of 250 mg/kg/day was considered to represent the no observed effect level (NOEL) for both maternal toxicity and embryo-fetal survival and development.

These data indicate that rats are more susceptible for toxic effects induced by isononanoic acid than rabbits, since 250 mg/kg bw/d was well tolerated in an OECD 414 rabbit study, whereas 200 mg/kg bw/d led to pronounced toxicity including mortality in an OECD 414 rat study. The responses in rats revealed an increased liver/general maternal toxicity compared to rabbits. The observed differences in liver toxicity may be explained by induction of peroxisome proliferation; a specific effect with high susceptibility in rodents only. For female rats, the induction of peroxisome proliferation was observed already at doses ≥ 30 mg/kg bw/d in a 90 -day study with isononanoic acid.

 

Mode of Action Analysis / Human Relevance Framework

In the absence of information on a species specific activity the effects are regarded as relevant for humans.

Justification for classification or non-classification

No effects on fertility were observed in three different rat studies (OECD 422, OECD 415 dose-range-finder, OECD 443) at all dose groups including doses which caused systemic toxicity. Therefore, no classification for effects on fertility is required according to the criteria of Regulation (EC) No 1272/2008.

Effects on development (reduced body weights, developmental retardation, defects in bone development) were observed in rats only at maternally toxic (lethal) doses (200 mg/kg bw/d). The effects on the offspring are regarded as secondary to maternal toxicity and not as relevant for classification. No effects on development were observed in rabbits up to the highest dose tested (250 mg/kg bw/d).

Therefore, no classification for effects on reproductive development is required according to the criteria of Regulation (EC) No 1272/2008.