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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:
2020-12-02 to final report date
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
- Basis for dose level selection
- Inclusion/exclusion of extension of Cohort 1B
- Route of administration

Test material

Constituent 1
Reference substance name:
Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
EC Number:
272-712-1
EC Name:
Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Cas Number:
68909-77-3
Molecular formula:
Not applicable (UVCB)
IUPAC Name:
2-(2-hydroxyethoxy)ethan-1-ol; 2-[1-(morpholin-4-yl)ethoxy]ethan-1-amine; 2-{2-[bis(2-hydroxyethyl)amino]ethoxy}ethan-1-ol; 4-{2-[2-(morpholin-4-yl)ethoxy]ethyl}morpholine; morpholin-3-one
Test material form:
liquid
Details on test material:
Name: Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Physical state: liquid
Appearance: brown liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test item identity: Ethanol, 2,2’-oxybis-, reaction products with ammonia, morpholine derivs, residues
- Appearance: Brown liquid
- Batch number of test material: 20/18097
- Stability/ Expiry date: 26 February 2022
- Purity: Not applicable (UVCB material)
- Correction factor: None


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25°C); protected from light and
moisture
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of a solution of the test item in the vehicle was demonstrated over a period of up to 21 days during refrigerated (2 to 8°C) storage, and for 24 hours during ambient (15 to 25°C) storage in Covance Study No. XM94DH.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): not applicable


FORM AS APPLIED IN THE TEST: liquid


Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by
regulatory agencies. The Han Wistar (RccHan™;WIST) strain was used because of the
historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: 106 male and 106 female RccHan™;WIST.rats, supplied by Envigo RMS Limited.

- Females (if applicable) nulliparous and non-pregnant: not indicated

- Age of the F0 animals at the start of treatment: 27 to 33 days old

- Weight of the F0 animals at the start of treatment: Males: 0.060 to 0.109 kg, Females: 0.060 to 0.098 kg.

- Fasting period before study: not indicated

- Housing: Cages comprised of a polycarbonate body with a stainless-steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily. 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. A soft white untreated wood block; provided to each cage throughout the study (except during late gestation and littering) and replaced when necessary. For F0 females, chew blocks were returned on Day 21 of lactation after weaning of offspring. Plastic shelter was provided to each cage throughout the study (except during pairing, late gestation and littering) and replaced at the same time as the cages. For F0 females, shelters were returned on Day 21 of lactation after weaning of offspring. From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.
- Number of animals per cage:
F0 generation (from acclimatization) and selected F1 maturation: 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 animal
Females during littering (from Day 20 of mating): one animal + litter
Females to termination (after weaning): up to four animals

- Diet: ad libitum, SDS VRF1 Certified, pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent (diet was removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones and during the period of urine collection).
- Water: ad libitum (except overnight during urine collection for F0 and F1 Cohort 1A animals), potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

- Acclimation period: Five days before commencement of treatment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12
- Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were considered not to have influenced the health of the animals and/or the outcome of the study

IN-LIFE DATES: F0: From: 2020-12-09 To: 2021-04-23 (males), 2021-04-27 (females)
F1:From: 2021-04-15 To: : 2021-06-24 (cohort 1A), 2021-07-01 (cohort 1B)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

- The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared in ascending order.
- The dosing formulations were prepared weekly and stored in a refrigerator (2 to 8°C). Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- Stability and homogeneity: The stability of a solution of the test item in the vehicle was demonstrated over a period of up to 21 days during refrigerated (2 to 8°C) storage, and for 24 hours during ambient (15 to 25°C) storage in Covance Study No. XM94DH. The validated concentration range was 2 to 200 mg/mL. Additional validation investigations were undertaken as part of Labcorp Study No. MR98KF, which confirmed the validity of the analytical method with respect to the determination of the specificity of analysis, limits of detection, linearity of detector response, repeatability, method accuracy and precision.


VEHICLE
- Concentration in vehicle: 0, 30, 90, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
Details on mating procedure:
- F0 pairing commenced: after 10 weeks of treatment
- M/F ratio per cage: 1:1 from within the same treatment groups (sibling pairing was not permitted)
- Length of cohabitation: Up to two weeks (mating period). Male/female separation the day when mating evidence was detected.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear. referred to as day 0 of gestation (GD 0).
- After successful mating each pregnant female was caged (how): individually
- Pre-coital interval: calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Remarks:
Samples of each formulation prepared for administration in Weeks 1 and 7 and the last week of treatment of the F0 generation and the first and last week of treatment for the F1 generation were analyzed for achieved concentration of the test item.
Details on analytical verification of doses or concentrations:
The samples were analyzed in accordance with the validated Labcorp Analytical Procedure (DFA/M066/20). The analytical method involved extraction and dilution in acetonitrile/water 10/90 v/v followed by reverse phase high performance liquid chromatographic analysis with ELSD detector. Sample concentrations were determined with reference to external standards prepared in the concentration range 100 µg/mL to 1000 µg/mL.
For Week 1,7 and last week (F0 generation) and week 1 and last week (F1 generation), the formulations for Group 1, 2, 3 and 4 were sampled, 4 × 1 mL, from the middle of the formulation. Two of the samples were analyzed in duplicate in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved. For Week 1 and 7 (F0 generation) and week 1 (F1 generation), the formulations for Group 2 and 3 for homogeneity were sampled 2 × 1 mL from the top, middle and bottom of the formulation . One sample from the top, middle and bottom of the formulations from Groups 2 and 3 were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.
Results and conclusion: The mean concentrations were within 7% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis. The mean concentrations were within 10% of the nominal concentration, confirming the accuracy of the formulation. The coefficient of variation for the homogeneity samples remained within 3%. This is with exception to the week 1 (F0 generation) group 2 sample which was 10.1 % and week 1 (F1 generation) group 3 sample which was 5.5%.
Week 1 (F0 generation) were analyzed beyond the stability period and will be reported for information purpose only.
Duration of treatment / exposure:
F0 animals For ten weeks before pairing until termination after litters were weaned, for a total of 17-18 weeks.
F1 animals From weaning (Although direct exposure starts at weaning on Day 21 of age, all offspring had potential indirect exposure in-utero and through the milk during lactation) until termination of the respective cohort, ie from weaning on Day 21 of age for approximately 10-11 weeks.
- Unselected F1 offspring : Retention of brain, spleen, thymus and mammary tissue and
organ weights - no direct treatment, killed on Day 22 of age.
- Cohort 1A : Reproductive /developmental toxicity testing - treated from weaning to 13 weeks
of age.
- Cohort 1B : Reproductive /developmental toxicity testing - treated from weaning to 14 weeks
of age.
Frequency of treatment:
Once daily at approximately the same time each day. F0 females were not dosed if parturition was in progress at the scheduled time of administration.
Details on study schedule:
- F0 pairing commenced After ten weeks of treatment.
- F0 females failing to produce a viable litter Terminated with first cohort of females with live litters.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 generation: 24 male and 24 female rats/group
F1 generation (cohort 1A/1B): 20 male and 20 female animals/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Justification for route of exposure: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.
- Dose selection rationale: The doses used in this study (0, 150, 450 and 1000 mg/kg bw/day) were selected in conjunction with the Sponsor and were based on the results of a Preliminary Reproductive Performance Dose-Range Finding (DRF) Study in the Female Han Wistar Rat by Oral Gavage Administration (Labcorp Study No: 8440697). In that study, F0 females were dosed with 250, 500 or 1000 mg/kg bw/day of the test article by oral gavage from Day 6 of gestation to Day 20 of lactation; selected F1 generation animals were dosed at the same dose levels from Day 21 to Day 34 of age. A dosage of 1000 mg/kg bw/day did not result in any toxicologically significant effects for the pregnant or lactating F0 females, or for the preweaning and post-weaning F1 offspring that precluded this dose level from further assessment of reproductive toxicity. Effects on pre-weaning and post-weaning body weight gain and post-weaning food intake were observed for F1 offspring but there were no associated effects on survival or clinical condition.
- The high dose level for this main extended one generation study was therefore set at 1000 mg/kg bw/day. The low and intermediate dose levels were set at 150 and 450 mg/kg bw/day respectively, to achieve a dose response and/or aid in the determination of a No Observed Adverse Effect Level. (refer to 'any additional information on results' for a detailed description of the DRF study)


Selection of offspring to form F1 generation:
- Allocation (formal start of F1 generation): Nominally Day 28 of age (direct dose administration commenced on Day 21 of age).
- Method: The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals Where possible, two male and two female offspring were selected from each selected litter and were allocated to each of the two cohorts, with one of each sex assigned to Cohort 1A and Cohort 1B. If more were required, up to three males and three females were selected from each selected litter. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on Day 28±2 of age. Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after the formal F1 generation was fully established.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for evidence of ill-health or reaction to treatment.
- During the acclimatization period: at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
*Signs Associated with Dosing
Detailed observations were recorded at the following times in relation to dose administration: prior to dosing, one to two hours after completion of dosing and as late as possible in the working day.
- Time schedule:
- F0 generation: Week 1 - Daily. Weeks 2 to 4 - twice weekly (middle and end of the week).
Week 5 onwards - once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and
20 of lactation for F0 females).
- F1 generation: For selected F1 offspring, observations were performed daily from Day 21
of age until formal commencement of the F1 generation at nominal Week 4 of age. Formal
Week 1 - Daily. Weeks 2 to 4 - twice weekly (middle and end of the week). Week 5 onwards
- once each week.

*Clinical Signs
- F0 males and selected F1 generation: Weekly. After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14, 21 and 28 of lactation.
- F0 females: Weekly until paired for mating.
Days 0, 5, 12, 18 and 20 after mating. Days 1, 7, 14 and 21 of lactation.
- Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor
and abnormalities of gait or behavior.

BODY WEIGHT: Yes
- Time schedule for examinations:
- F0 males: Day that treatment commenced. Each week and before necropsy.
- F0 females: Day that treatment commenced. Each week until mating detected. Days 0, 2, 4,
6, 8, 10, 12, 14,16, 18 and 20 after mating. Days 1, 4, 7, 14, 21 and 28 of lactation and before
necropsy.
- F1 selected animals: From Day 21 of age to formal commencement of F1 generation
at nominal Week 4 of age: daily. Thereafter, from nominal Week 4 of age, twice during Week
1 of the F1 generation and weekly thereafter and before 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
- Time schedule for examinations: F0 males and females: Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 11) but recommenced for males in Week 12. For females after mating food consumption was performed to match the body weight recording: Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-20 after mating. Days 1-4, 4-7, 7-14, and 14-21 of lactation.
F1 selected animals: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

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

PARTITUTION AND GESTATION LENGTH: yes
- Duration of gestation: Time that elapsed between mating and commencement of parturition.
- Parturition observations: From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded, and any difficulties observed were noted.

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

HEMATOLOGY: yes
*Hematology, Peripheral Blood
- Fasting period before blood sampling for clinical biochemistry: diet was removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones and during the period of urine collection.
- Blood samples were collected after overnight withdrawal of food. Sampling was performed
on the morning after overnight collection of urine. The animals were, therefore, also deprived
of water overnight but had access to water for a minimum period of one hour prior to the
commencement of blood sampling procedures.
- Samples were collected at termination for F0 adults and F1 cohort 1A animals, 10male/10 female animals per group.
- Animals were held under light general anesthesia induced by isoflurane. Blood samples
(nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing
K2-EDTA anticoagulant and examined for the following characteristics using a Bayer Advia
120 analyzer: Hematocrit (Hct)*, Hemoglobin concentration (Hb), Erythrocyte count (RBC), Mean cell hemoglobin (MCH)*, Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt) (* Derived values calculated in ClinAxys).

* Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. Where necessary a manual count of the differential white blood cell parameters was performed. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of: Prothrombin time (PT), using IL PT Fibrinogen reagent., Activated partial thromboplastin time (APTT), using IL APTT reagent.


BLOOD CHEMISTRY: yes
- Fasting period before blood sampling for clinical biochemistry: diet was removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones and during the period of urine collection
- Samples were collected at termination for F0 adults and F1 cohort 1A animals, 10male/10 female animals per group
- Animals were held under light general anesthesia induced by isoflurane. Blood samples
(nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes
containing lithium heparin as anticoagulant. After separation, the plasma was examined using
a Roche Cobas 6000 Analyzer in respect of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Urea , Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb)
- Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: yes
- Fasting period before blood sampling for clinical biochemistry: diet was removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones and during the period of urine collection.
- Urine samples were collected (from 10 males and 10 females per group in F0 and F1 cohort 1A) at termination after overnight withdrawal of food and water at termination.
- The individual samples were examined for the following characteristics:
- Using manual methods: Clarity and Color (Appearance) (App), by visual assessment;
Volume (Vol) using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by
direct refractometry using a SG meter
- Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Glucose
(Gluc), Ketones (Keto), Bile pigments (Bili), Blood pigments (UBld)
- Using a Roche Cobas 6000 Analyzer: Protein - total (T-Prot) and concentration (Prot),
Sodium - total (T-Na) and concentration (U-Na), Potassium - total (T-K) and concentration
(U-K) Chloride - total (T-Cl) and concentration (U-Cl)
- A microscopic examination of the urine sediment was performed. An aliquot of the urine
sample was centrifuged, stained with Kova stain and the resulting deposit spread on a
microscope slide. The number of elements seen in nine high or low power fields (HPF or
LPF) was recorded in the raw data and entered onto the database and the number seen /HPF
or /LPF was derived from these data as described: Epithelial cells (Epi), Leucocytes (WBC)
Erythrocytes (RBC), Casts, Other abnormal components (A)
- The slide was also examined for abnormalities in spermatozoa and crystals.

THYRIOD HORMONE ANALYSIS: yes
- Fasting period before blood sampling for clinical biochemistry: diet was removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones and during the period of urine collection
- Blood samples were collected at termination.
- Blood volume: 1.0 mL.
- Parameter measured: TSH and T4
- Number of animals selected: Ten male and ten female animals per group.
- Blood sample site: Sublingual vein.
- Anesthetic: Isoflurane.
- Blood volume: 1.0 mL.
- Treatment of samples: Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
- Centrifugation conditions: At 2000g for ten minutes at 4°C.
- Number of aliquots: two per animal - Aliquot 1: 0.2 mL serum for T4; Aliquot 2: residual serum for TSH
- Storage conditions: Deep frozen (approximately -60°C to -90°C).
- The method of analysis:
- Using manual methods: Clarity and Color (Appearance) (App), by visual assessment;
Volume (Vol), using a measuring cylinder; pH, using a pH meter; Specific gravity (SG), by
direct refractometry using a SG meter.
- Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Glucose
(Gluc), Ketones (Keto), Bile pigments (Bili), Blood pigments (UBld)
- Using a Roche Cobas 6000 Analyzer: Protein, total (T-Prot) and concentration (Prot);
Sodium, total (T-Na) and concentration (U-Na); Potassium, total (T-K) and concentration (U-
K), Chloride, total (T-Cl) and concentration (U-Cl).
- A microscopic examination of the urine sediment was performed. An aliquot of the urine
sample was centrifuged, stained with Kova stain and the resulting deposit spread on a
microscope slide. The number of elements seen in nine high or low power fields (HPF or
LPF) was recorded in the raw data and entered onto the database and the number seen /HPF
or /LPF was derived from these data as described: Epithelial cells (Epi), Leucocytes (WBC), Erythrocytes (RBC), Casts, Other abnormal components (A).
- The slide was also examined for abnormalities in spermatozoa and crystals.

Oestrous cyclicity (parental animals):
- Dry smears: For 15 days before pairing, using cotton swabs.
- Wet smears (using pipette lavage): After pairing until evidence of mating confirmed.
- For four days before scheduled termination (nominally Days 25 to 28 post-partum). Females that failed to litter were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post-partum females started smearing and were then killed with that first batch of females.
Sperm parameters (parental animals):
- Immediately after scheduled sacrifice of each male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
- The following tests were performed:
- Sperm motility (all groups): A sample of sperm was expressed from the left vas deferens into
prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin
(BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by
capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS
II Computer Assisted Sperm Analyzer (CASA). The sample from F0 Group 3 animal 62 was
exposed to N.B.F and excluded from group mean values.
- Sperm morphology (Groups 1 and 4): A 200 µL aliquot of the sperm/medium mixture
(described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining
with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light
microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for
each male where possible (F0 Group 4M No’s. 74 and 82 and F1 Cohort 1A Group 4M No’s. 463
and 470, unable to assess 200 sperm on slide).
- Sperm morphology (Groups 2and 3): Fixed samples retained for possible future assessment.
- Sperm count (Groups 1 and 4): The left cauda epididymis of each male was weighed and then
the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of
0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared
IDENT stain tube before being assessed for sperm count using CASA.
- Sperm count (Groups 2 and 3): Samples frozen for possible future assessment.
- Homogenization-resistant spermatid counts (Groups 1 and 4): After removal of the tunica,
the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An
aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed
for homogenization-resistant spermatid count using CASA.
- Homogenization-resistant spermatid counts (Groups 2 and 3): Samples frozen for possible
future assessment
Litter observations:
- Clinical observations: Observed approximately 24 hours after birth and then daily for evidence of ill-health or reaction to treatment.
- Litter size: Daily on Days 1-21 of lactation. Litters culled to 8 (where possible 4 males and 4 females) on Day 4 of age. All offspring culled on Day 4 of age macroscopically examined (see Section 0), with thyroid hormone samples collected from 10 F1 litters per group
- Sex ratio of each litter: Days 1, 4 (before and after culling) and 21 of age.
- individual offspring body weights: All offspring: Days 1, 4, 7, 14 and 21 of age. Unselected F1: Day 22 of age.
- Weaning of offspring: Day 21 of age.
- Ano-genital distance: Offspring on Day 1 of age.
- Nipple count: Male offspring on Day 13 of age.
- Sexual maturation:
- Males Examined daily from Day 38 of age for the completion of balano-preputial separation.
Body weight recorded on day of completion of separation.
- Females Examined daily from Day 25 of age until vaginal opening occurs. Body weight
recorded on day of vaginal opening.


GROSS EXAMINATION OF DEAD PUPS: no, no mortality occurred.
Postmortem examinations (parental animals):
SACRIFICE
- Time of Necropsy: F0 males After approximately 18 weeks of treatment, following weaning of the F1 animals, after confirmation that no further mating required. F0 females Day 28 post-partum (after approximately 17 weeks of treatment). F0 females failing to produce a viable litter Terminated with first cohort of females with live litters.
- Method: Animals 14 days and older: Carbon dioxide asphyxiation with
subsequent exsanguination.

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

HISTOPATHOLOGY
The tissues indicated below were prepared for microscopic examination and weighed, respectively: Adrenals , Brain (cerebellum, cerebrum, midbrain) , Cecum, Colon , Duodenum , Epididymides, Esophagus , Eyes , Femurs (femorotibial joint and including bone marrow), Heart (including auricular and ventricular regions) , Ileum , Jejunum , Kidneys , Liver (section from two lobes) , Lungs (section from two major lobes including bronchi) , Optic nerves , Ovaries , Pancreas , Pituitary , Prostate (dorsolateral and ventral combined) , Rectum , Sciatic nerves, Seminal vesicles (with coagulating gland) , Skeletal muscle, Skin with mammary glands (inguinal area) , Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels) Spleen , Sternum - bone marrow , Stomach, Testes , Thymus, Thyroid with parathyroids, Trachea
Urinary bladder , Uterus with cervix and oviducts , Vagina , Vas Deferens

ORGAN WEIGHTS
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of the Testes (modified Davidson’s fluid) and eyes (Davidson’s fluid).
- Histology: Wet tissues were dispatched to the Test Site (Labcorp Harrogate, UK) for processing.
- Histopathology:
- All animals of Groups 1 and 4: Adrenals, Brain (cerebellum, cerebrum, midbrain),
Epididymides, Heart (including auricular and ventricular regions), Kidneys, Liver, ovaries,
Pituitary, Prostate (dorsolateral and ventral combined),
Seminal vesicles (with coagulating glands), Spleen, Testes, Thymus, Thyroid with
parathyroids (Weighed after partial fixation), Uterus with cervix and oviducts.
- All animals of Groups 2 and 3: Abnormalities only
- All F0 animals of Groups 2 and 3 with suspect fertility* Reproductive organs only:
*Testes: A detailed qualitative examination was made, taking into account the tubular
stages of the spermatogenic cycle. The examination was conducted in order to identify
treatment related effects such as missing germ cell layers or types, retained spermatids,
multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell- or stage-specificity of testicular findings was noted.
*Ovaries F0 - Qualitative evaluation of one section from each ovary
*Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology
(and appearance of the uterus and endometrial glands).
- Where paired organ weights were weighed separately, these were summed for the presentation of group mean values. For adults and offspring organ weights, group mean values and SD were calculated for absolute weights and also for weights expressed relative to body weight (%) using the body weight recorded on the day of necropsy.
Postmortem examinations (offspring):
SACRIFICE
- Unselected offspring Culled on Day 4 and Day 22 of age. Cohort 1A animals At approximately 13 weeks of age. Cohort 1B animals At approximately 14 weeks of age.
- Method: F0 generation, F1 Cohorts 1A and 1B Animals 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination. Animals less than 14 days of age: Intraperitoneal injection of sodium pentobarbitone.

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

HISTOPATHOLOGY
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
- Cohort A: Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain) , Cecum , Colon, Duodenum , Epididymides, Esophagus, Eyes, Femurs (femorotibial joint and including bone marrow), Heart (including auricular and ventricular regions), Ileum, Jejunum , Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes - mesenteric
- left axillary , Optic nerves, Ovaries , Pancreas , Pituitary , Prostate (dorsolateral and ventral combined), Rectum, Sciatic nerves , Seminal vesicles (with coagulating glands) , Skeletal muscle , Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical,, thoracic and lumbar levels), Spleen , Sternum - bone marrow , Stomach , Testes , Thymus ,Thyroid with parathyroids , Trachea , urinary bladder , Uterus with cervix and oviducts , Vagina, Vas Deferens.
- Cohort B: Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femurs (femorotibial joint and including bone marrow), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes (mesenteric and left axillary), Lungs (section from two major lobes including bronchi), Optic nerves, Ovaries, Pancreas, Pituitary, Prostate (dorsolateral and ventral combined), Rectum, Sciatic nerves, Seminal vesicles (with coagulating glands), Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum - bone marrow, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix and oviducts, Vagina, Vas Deferens, Target organs.

ORGAN WEIGTHS
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of the Testes (modified Davidson’s fluid), F1 adults only, and eyes (Davidson’s fluid).
- Histology: Wet tissues were dispatched to the Test Site (Labcorp Harrogate, UK) for processing.
- Histopathology: F1 Cohort 1A:
- All animals of Groups 1 and 4: Adrenals, Brain (cerebellum, cerebrum, midbrain),
Epididymides, Heart (including auricular and ventricular regions), Kidneys, Liver, Lymph
nodes (mesenteric and left axillary), Pituitary, Prostate (dorsolateral and ventral combined),
Seminal vesicles (with coagulating glands), Spleen, Testes, Thymus, Thyroid with
parathyroids (Weighed after partial fixation), Uterus with cervix and oviducts.
- All animals of Groups 2 and 3 Abnormalities only.
- Immunophenotyping of Spleen Leucocytes: Ten males and ten females per group from F1 Cohort 1A were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter. The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portion of the spleen was then weighed, placed into a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held on wet ice until processing for analysis. Samples were sent via courier to the Department of Immunology and Immunotoxicology (I&I), Labcorp. A copy of the whole spleen and partial spleen weights were provided to I&I.
- For bilateral organs, left and right organs were weighed together, unless specified in the
relevant pathology procedures table. Requisite organs were weighed for animals killed at
scheduled intervals. For unselected F1 offspring on Day 22 of age, organs were weighed from ten males and ten females per sex per group from as many litters as possible.
Statistics:
See section Any other information on materials and methods incl. tables
Reproductive indices:
Percentage mating (%) = Number of animals mating/Animals paired x 100
Conception rate (%) = Number of animals achieving pregnancy/animals mate x 100
Fertility index (%) = Number of animals achieving pregnancy/animals paired x 100
Gestation index (%) = Number of live litters born/ animals paired x 100
A/G Ratio = Albumin concentration/Total protein - albumin concentration


Offspring viability indices:
A/G Ratio = Albumin concentration/Total protein - albumin concentration
Post implantation survival index (%) = Total number of offspring born/ Total number of uterine implantation sites x 100
Live birth index (%) = Number of offspring on Day 1 after littering/ total number of offspring born x 100
Viability index (%) = Number of live offspring on Day 4 before culling/Number of live offspring on Day 1 after littering x 100
Lactation index (%) = Number of live offspring on Day 21 after littering/Number of live offspring on Day 4 (after culling)
Percentage males = Number of males in litter/Total number of offspring in litter
x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Oral administration of the test substance to the F0 generation animals at dose levels up to and
including 1000 mg/kg bw/day was well tolerated; there were no premature deaths, no signs
observed in relation to dose administration and no test item-related changes detected in the
general clinical condition of the animals at routine weekly physical examination.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No premature deaths observed.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of test substance administration on mean body weight gain for the
treated F0 males and females at any dose level investigated.
In Weeks 4 and 6 of treatment, all groups of treated males showed slightly, but statistically
significantly low mean body weight gain when compared to Controls. There was, however,
no dose response apparent, in other study weeks intermittent periods of slightly superior body
weight gain compared to Controls was observed, and overall mean body weight gain over the
18-week dosing period was unaffected at all dose levels (≤5% lower than Controls in all
groups), therefore the minor differences observed in Weeks 4 and 6 were considered to be of
no toxicological significance.
During the 10-week pre-pairing period, females given 1000 mg/kg bw/day showed
statistically significantly increased mean body weight gain when compared to Controls,
primarily as a consequence of marginally increased body weight gain during Weeks 2 and 9
of dosing. In view of the extent of the difference from Control and the direction of change
(9% increased), and in the absence of any similar increases during the gestation and lactation
periods, this slight increase in mean body weight gain during the pre-pairing period was
considered to be incidental and of no toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on mean food consumption for males at any dose
levels investigated. For males receiving 1000 mg/kg bw/day, mean food intake was
statistically significantly lower than Control during Week 4-7 of the 10-week pre-pairing
treatment period; males given 150 or 450 mg/kg bw/day also showed statistically
significantly low mean food intake during Week 7. The differences from Control were,
however, small and overall mean food consumption during the 18-week treatment period was
unaffected, therefore these transient minor differences in food consumption were considered
to be of no toxicological significance
Mean food consumption for females was unaffected by the test substance during the 10-week prepairing
treatment period, and during gestation and lactation.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of test substance administration on food conversion efficiency of the F0
males and females during the 10-week pre-pairing treatment period or of the F0 females
during gestation.
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):
Analysis of hematological parameters at scheduled termination of the F0 animals did not
reveal any changes which were attributable to test substance administration. There were no
statistically significant differences apparent, all differences from control were minor, limited
to one sex and/or lacked a dose response relationship, and were therefore attributed to normal
biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical analysis of the plasma at scheduled termination of the F0 animals did not reveal
any changes which were clearly attributable to test substance administration.
All differences from control, including those which attained statistical significance were
minor, limited to one sex, showed a different direction of change between the sexes and/or
lacked a dose response relationship, and were therefore attributed to normal biological
variation. These primarily included differences in electrolyte concentrations, comprising a slight increase in chloride concentration in males at 1000 mg/kg bw/day, minor changes in
calcium concentrations in both sexes given 1000 mg/kg bw/day and in phosphorus
concentrations in females given 150 or 450 mg/kg bw/day and both sexes given
1000 mg/kg bw/day, and minor differences in sodium concentrations in females given 150 or
1000 mg/kg bw/day. In addition, there were slight decreases in urea concentration in all
groups of treated females and a slight increase in total protein concentration in females given
1000 mg/kg bw/day.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis of the clarity/colour and composition of the urine and microscopy of the urine
sediment of F0 animals prior to scheduled termination did not reveal any test item-related
changes.
In females given 450 or 1000 mg/kg bw/day, urinary pH was statistically significantly
increased when compared with Controls, with a dose response apparent. Males and females
given 1000 mg/kg bw/day showed, when compared to Controls, statistically significantly
increased specific gravity.
Females given 1000 mg/kg bw/day, showed increased total and urinary sodium concentration
when compared to Controls, with statistical significance attained for urinary sodium
concentrations.
Males given 1000 mg/kg bw/day showed statistically significantly low total protein (32%
lower than Controls); although slightly lower than Control (15%) differences in urinary
protein for these males did not attain statistical significance and the difference in total protein
was considered to reflect the lower urine output of these males.
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, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic findings detected among the F0 generation
animals. All microscopic findings were considered spontaneous and/or incidental because
they occurred at a low incidence, were randomly distributed across groups (including
concurrent Controls), and/or their severity was as expected for Han Wistar rats of this age.
They were, therefore, considered not test item related.
In males, the testes revealed normal progression of the spermatogenic cycle and the expected
cell associations and proportions in the various stages of spermatogenesis were present.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of test item administration on estrous cycle regularity during Weeks 9
and 10 of the pre-pairing treatment period, or on pre-coital interval; with the exception of one
mating pair in the Control group, two pairs in the 450 mg/kg bw/day group and one pair in
the 1000 mg/kg bw/day group, all mating pairs showed positive evidence of mating within
four days of pairing (i.e. at the first estrous opportunity). These four mating pairs showed
positive mating evidence within the permitted 14-day pairing period. Estrous cycles at termination were unaffected by treatment at all dose levels investigated,
with all females in all groups showing an estrus smear during the 4-day period prior to
necropsy
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
At scheduled termination, the motility and motion of the sperm, testicular and cauda
epididymal sperm counts and sperm morphology for the F0 males were unaffected by
treatment with test item administration at all dose levels investigated.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance and fertility were unaffected by treatment at all dose levels investigated,
with conception rate and fertility index at 96% in all treated groups, compared with 88% in
Controls.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed among the F1 offspring that were considered to be
related to parental treatment with the test item.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Oral administration of the test substance to the F1 generation animals at dose levels up to and
including 1000 mg/kg bw/day was well tolerated; there were no premature deaths and no
signs observed in relation to dose administration at any dose level investigated.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of test item administration on mean body weight gain for the
treated F1 males and females at any dose level investigated.
Following the onset of dosing, females given 450 mg/kg bw/day and both sexes given
1000 mg/kg bw/day showed marginally, but statistically significantly, low mean body weight
gain during Days 21-25 of age when compared to Controls (mean of 1-2 grams lower than
Control, representing 7-12%). Thereafter, mean body weight gain in all groups of treated
males and females was essentially similar to Control and considered unaffected by test substance
administration; occasional differences in weekly mean body weight gain attained statistical
significance, however differences were minor, varying between marginally higher than and
marginally lower than Control and as such were considered incidental.
Overall mean body weight gain from Day 21 of age to termination was considered unaffected
by the test item in all groups of treated males and females; overall mean body weight gain for
females given 1000 mg/kg bw/day was marginally, but statistically significantly higher than
Control; differences were small (6% higher than Control), and in view of the direction of
change this difference was considered incidental and of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
When compared to Controls, there was no effect of test substance administration on mean food
consumption for treated F1 males and females at any dose level investigated.
Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of test item administration on food conversion efficiency of the F1
males and females.
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):
Analysis of haematological parameters at scheduled termination of the Cohort 1A animals
revealed, when compared to Controls, a slight but statistically significant increase in
erythrocyte concentration, associated with a statistically significant decrease in mean cell
haemoglobin for females given 1000 mg/kg bw/day. All groups of treated females showed
slightly low mean cell volume when compared to Controls, with statistical significance
attained at 450 or 1000 mg/kg bw/day.
All groups of treated females showed a statistically significant increase in neutrophil
concentrations when compared to Controls, although in the absence of a dose response
relationship. As a consequence, total white blood cell concentrations were also slightly
increased when compared to Controls in all groups of treated females, with statistical
significance attained in the 1000 mg/kg bw/day group.
A non-dose-dependent, but statistically significant, decrease in mean platelet concentration
was apparent in all groups of treated females.
There were no statistically significant differences in haematological parameters amongst
treated males at any dose level investigated.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical analysis of the plasma at scheduled termination of the Cohort 1A animals
revealed when compared to Controls, statistically significantly decreased creatinine and
phosphorus concentrations in all groups of treated males although in the absence of a dose
response relationship. Males given 450 or 1000 mg/kg bw/day also showed a non-dosedependent,
but statistically significant increase in total protein concentration, with an
associated decrease in albumin/globulin ratio.
Females given 1000 mg/kg bw/day showed statistically significantly increased cholesterol
concentrations when compared to Controls, and all groups of treated females showed
statistically significantly increased potassium concentrations.
All other biochemical differences from Controls observed at scheduled termination were
minor, limited to one sex and/or lacked a clear dose relationship, and were therefore
attributed to normal biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared to Control, urinary changes observed in the Cohort 1A animals comprised a
statistically significant reduction in chloride (total and concentration) in females given 450 or
1000 mg/kg bw/day, sodium and potassium (total) in females given 1000 mg/kg bw/day and sodium and potassium (concentration) in females given 450 or 1000 mg/kg bw/day; there was
no clear dose response apparent for these differences.
Urinary parameters were not clearly affected in treated males. It was noted that urinary
protein (total) values were statistically significantly decreased in males given 150 or
1000 mg/kg bw/day; total values were unaffected at 450 mg/kg bw/day and concentration
values were unaffected in all treated groups, therefore the differences in total values were
considered to be of unlikely relationship to treatment.
Sexual maturation:
no effects observed
Description (incidence and severity):
The age and body weight at attainment of sexual maturation, as assessed by the timing of
balano pre-putial separation and vaginal opening, were considered unaffected by the test substance
at all dose levels investigated.
Males in the 1000 mg/kg bw/day group attained balano pre-putial separation at a slightly
lower body weight than Controls, with the difference attaining statistical significance; the
difference was small, and since the age of attainment of sexual maturation was unaffected,
this difference in body weight was considered to be incidental.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of parental test item administration at any dose level investigated on
ano-genital distance of male or female offspring on Day 1 of age.
It was noted that the mean ano-genital distance of female offspring in the 450 mg/kg bw/day
group was slightly, but statistically significantly increased when compared to Controls. In the
absence of an effect at 1000 mg/kg bw/day, this minor difference was considered incidental
and unrelated to parental treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
All F1 male offspring were assessed on Day 13 of age for the presence of nipples; no nipples
were observed.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was considered to be no effect of parental test item administration on the weights of
the brain, spleen or thymus of the unselected F1 offspring killed on Day 22 of age.
In all treated groups, female offspring showed slightly decreased absolute and body weightrelative
brain weights when compared to Controls, with statistical significance attained in
absolute weight terms in females in the 450 or 1000 mg/kg bw/day groups. Differences were
small, there was no dose response apparent, and no brain weight changes were observed in
male offspring on Day 22 of age or in the F1 (Cohorts 1A and 1B) animals. Therefore, the
minor differences in the female offspring brain weights were considered to be fortuitous. At scheduled termination of the Cohort 1A animals, analysis of organ weights revealed, when
compared to Controls, increased absolute and body weight-relative kidney weights in males
given 1000 mg/kg bw/day and in all groups of treated females; the majority of these
differences attained statistical significance. Liver weights were increased in absolute and
body weight-relative terms in females given 450 or 1000 mg/kg bw/day, with body weightrelative
values also increased in males given 1000 mg/kg bw/day. Ovary weights were
similarly increased, in absolute and body weight-relative terms, in females given 450 or 1000
mg/kg bw/day. All groups of treated males showed an increase in body weight-relative testes
weights when compared to Controls, although in the absence of a dose response relationship.
At scheduled termination of the Cohort 1B animals, there were no clear test item-related
differences in organ weights at any dose level investigated. It was noted that absolute testes
weights and body weight-relative epididymides and testes weights were statistically
significantly higher than Control in males at 1000 mg/kg bw/day. Mean values in the
150 mg/kg bw/day were, however, similarly increased although in the absence of statistical
significance, whilst values at 450 mg/kg bw/day were essentially similar to Control. These
differences in the 1000 mg/kg bw/day group were therefore considered fortuitous and of no
toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled termination of Cohort 1A animals at approximately 13 weeks of age and of
Cohort 1B animals at approximately 14 weeks of age, there were no item-related macroscopic
abnormalities detected at any dose level investigated. All macroscopic findings were considered spontaneous and/or incidental because they
occurred at a low incidence, were randomly distributed across groups (including concurrent
Controls), and/or were as expected for Han Wistar rats of this age. They were, therefore,
considered not test item related.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic findings detected among the F1 Cohort 1A
animals. All microscopic findings were considered spontaneous and/or incidental because
they occurred at a low incidence, were randomly distributed across groups (including
concurrent Controls), and/or their severity was as expected for Han Wistar rats of this age.
They were, therefore, considered not test item related.
In the F1 Cohort 1A males, the testes revealed normal progression of the spermatogenic cycle
and the expected cell associations and proportions in the various stages of spermatogenesis
were present.
Other effects:
no effects observed
Description (incidence and severity):
Estrous cyclicity:
There was no effect of test item administration on the duration between vaginal opening
and the first estrus occurring in the Cohort 1A females.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
Analysis of immunophenotyping parameters measured in spleen leukocytes of the F1 Cohort
1A animals revealed, fluctuations were observed in the percentages (%) of all cell
populations analyzed across all groups in both genders. Variations were also observed in the
cell counts (cells/spleen) of the different cell populations in all treatment groups. However,
these changes were within the ranges of the control group and were therefore considered
likely to be due to biological variation, rather than an effect of test item administration.
When compared to Controls, a statistically significant decrease in WBC % and monocyte cell
count (cells/spleen) was apparent in males given 1000 mg/kg bw/day; decreases in
monocytes, both in % and cells/spleen terms, were also apparent in all other groups of treated
males and females, although differences were small and did not attain statistical significance.
Decreased NK cells % were evident as observed in females given 450 or 100 mg/kg bw/day
test substance animals, with statistical significance attained for these differences when compared
to Controls. In addition, a statistically significant decrease NK cells cells/spleen was apparent
in both sexes at 1000 mg/kg bw/day, when compared to Controls.
CD4+ T cell % was statistically significantly increased in all groups of treated females,
however values were within the Control range and although observed in males, this was
considered not to be a significant finding based on the small extent of the change.
In female given 150 or 450 mg/kg bw/day, there was a statistically significant increase in B
cells % when compared to Controls; a similar increase was observed for B cells/spleen at
450 mg/kg bw/day, although statistical significance was not attained. In the absence of
similar increases in the 1000 mg/kg bw/day group, these increases were considered incidental
and unrelated to the test item.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Summary of the DRF


In the F0 generation, three groups of six female rats received the test substance at doses of 250, 500 or 1000 mg/kg/day by oral (gavage) administration from Day 6 after mating to Day 20 of lactation at a volume dose of 5 mL/kg body weight.


In the F1 generation, six males and six females were treated from weaning to Day 34 of age at the same dose levels and volume dose as the F0 generation. Similarly constituted Control groups (for each generation) received the vehicle, purified water, at the same volume dose as treated groups over the same treatment periods.


During the study, for F0 generation females, clinical condition, body weight, food
consumption, gestation length and parturition observations, and macroscopic pathology investigations were undertaken. For F1 generation animals, clinical signs, body weight, food consumption and macroscopic pathology investigations were undertaken at necropsy. The clinical condition, litter size and survival, sex ratio and body weight for all offspring were also assessed.


Results
Treatment of F0 females at dose levels up to 1000 mg/kg/day was well tolerated with no deaths or changes in the clinical condition of the animals throughout the study. There was no adverse effect of treatment on body weight gain or food consumption during pregnancy or lactation. Gestation length was also unaffected by treatment. No macroscopic abnormalities were detected at necropsy of F0 females at scheduled termination.


Implantation counts, litter size, offspring survival, sex ratio and clinical condition of the offspring were unaffected by maternal treatment at up to 1000 mg/kg/day. A slight reduction in offspring body weight gain was observed in the 250, 500 or 1000 mg/kg/day groups but mean overall gain to weaning did not attain statistical significance or show a clear dose response. Lower mean body weight gain, when compared to the control group, did attain statistical significance for offspring during Days 14 to 17 of age at 1000 mg/kg/day.


Macroscopic abnormalities observed for decedent offspring or offspring terminated at weaning were unaffected by maternal treatment at dose levels up to 1000 mg/kg/day. Mean body weights of male offspring that received 1000, 500 or 250 mg/kg/day and female offspring that received 1000 mg/kg/day were slightly lower than control on Day 21 of age, when direct treatment of the selected F1 offspring commenced. Lower overall mean body weight gains were evident throughout the treatment period for F1 selected offspring that received 250, 500 or 1000 mg/kg/day, achieving statistical significance for females that receiving 500 mg/kg/day and both sexes receiving 1000 mg/kg/day. Lower mean body weights for males that received 1000 mg/kg/day achieved statistical significance, when
compared with control, throughout the treatment period, however, no statistical significance was apparent for body weights of females treated at 1000 mg/kg/day or for either sex treated at 500 or 250 mg/kg/day. Lower food consumption observed for treated offspring, but food intake did not show a consistent dose relationship and only occasionally achieved statistical significance. The lower body weight was not associated with any changes in the clinical condition of the offspring or macroscopic findings at terminal necropsy.


Conclusion: Based on the results of this preliminary reproductive performance dose-range finding study, there were no effects apparent at 1000 mg/kg/day for the pregnant or lactating F0 female, or for the pre-weaning and post-weaning F1 offspring that precluded this dose level from further assessment of reproductive toxicity. It is advised that the high dose level in the main extended one-generation reproductive toxicity study (OECD443) study should be 1000 mg/kg/day.


 


Discussion of the EOGRTS main phase


The purpose of this study was to assess the effects of repeated oral administration of the test substance on the reproductive performance of male and female Han Wistar rats, including gonadal function, estrous cycles, mating behavior, conception, gestation, parturition, lactation, weaning and the growth and development of offspring. Cohorts of F1 animals were used to assess the potential for systemic toxicity. The study was also designed to provide information on potential prenatal and postnatal development.


Oral administration of the test substance at dose levels up to and including 1000 mg/kg bw/day to F0 generation animals for a total of 17-18 weeks and to selected F1 generation animals from weaning on Day 21 of age for approximately 10-11 weeks was well tolerated. There were no premature deaths or signs associated with dose administration, or test item-related changes in general clinical condition during routine physical examination in either the F0 or F1 generation. Whilst some transient periods of minor variations in body weight gain and/or food consumption were evident in all groups of treated males, differences from Control were small, and there were considered to be no adverse effects on body weight performance, food intake or food conversion efficiency at any dose level investigated throughout the F0 and F1 generations.


Estrous cycle regularity of the F0 and F1 Cohort 1A females were unaffected by test substance administration, and in the F0 generation there was no effect on pre-coital interval, mating performance, conception rate or fertility index.  Similarly, there was no effect of treatment on the duration between vaginal opening and first estrus in the F1 Cohort 1A females or the stage of estrus at termination of the F0 or the F1 Cohort 1A females. Ovarian follicle counts and corpora lutea counts for F1 Cohort 1A females given 1000 mg/kg bw/day were similarly unaffected


Litter size, offspring survival, sex ratio, offspring body weight gain and ano-genital distance were unaffected at all dose levels investigated. Examination of the F1 male offspring on Day 13 of age did not reveal any evidence of nipple retention. There was also no effect on the timing of attainment of sexual maturation among F1 Cohort 1A and 1B animals at any dose level investigated.


Macroscopic and subsequent microscopic examination of a full list of retained tissues for the F0 and F1 Cohort 1A animals at scheduled termination did not identify any target organs of toxicity.  Computer-assisted analysis of the sperm of F0 and F1 Cohort 1A males did not identify any changes in sperm motility, sperm counts or sperm morphology, and subsequent histopathological evaluation of the testes revealed normal progression of the spermatogenic cycle. Evaluation of red and white blood cell parameters, clotting factors, biochemical analysis of the plasma and urine analysis of F0 and F1 Cohort 1A animals at scheduled termination did not reveal any adverse test item-related changes at any dose level investigated.  Some minor differences in individual clinical pathology parameters were evident in the treated groups when compared to Controls, however differences were small and often limited to one sex, and in the absence of any histopathological correlates these minor sub-clinical differences were attributed to normal biological variation and therefore non-adverse.  Similarly, in the F1 generation at scheduled termination some differences in kidney, liver, ovary and testes weights were observed in F1 Cohort 1A animals given 450 or 1000 mg/kg bw/day; no histopathological changes were identified in these organs, and therefore the slight differences in organ weights were considered to be non-adverse.


There was no effect of test substance administration at any dose level investigated on serum TSH concentrations in F0 or F1 Cohort 1A adult animals, or in the F1 offspring on Day 22 of age. T4 results to be added when results available.


Immunophenotyping of the spleen leukocytes of the F1 Cohort 1A did not reveal any test item-related changes in the cell populations in either percentage or cells/spleen terms.

Applicant's summary and conclusion

Conclusions:
The purpose of this study was to assess the effects of repeated oral administration of Amine C8 on the reproductive performance of male and female Han Wistar rats, including gonadal function, estrous cycles, mating behavior, conception, gestation, parturition, lactation, weaning and the growth and development of offspring. Cohorts of F1 animals were used to assess the potential for systemic toxicity. The study was also designed to provide information on potential prenatal and postnatal development.Based on the results obtained in this study it was concluded that the No-Observed-Adverse-
Effect-Level (NOAEL) for general systemic toxicity in the F0 and F1 generation, for
reproductive performance in the F0 generation and for prenatal and postnatal development of
the F1 generation was 1000 mg/kg bw/day.