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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 September 2018 to 9 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5,5-trimethylhexanoic acid
EC Number:
221-975-0
EC Name:
3,5,5-trimethylhexanoic acid
Cas Number:
3302-10-1
Molecular formula:
C9H18O2
IUPAC Name:
3,5,5-trimethylhexanoic acid
Test material form:
liquid

Test animals

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)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed 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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs 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 / performance (P0)

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.

Details on results (P0)

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".

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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".

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

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"

Details on results (F1)

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

Effect levels (F1)

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.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

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

Any other information on results incl. tables

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".

Applicant's summary and conclusion

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.

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