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Effects on fertility

Description of key information

A NOAEL of 1000 mg/kg bw was observed in male and female Sprague-Dawley rats in a reproductive/developmental toxicity screening study according to OECD guideline 421 (Calvert Laboratories, Inc., 2010; Klimisch 1). Based on the available data and according to the CLP criteria, the test substance should not be classified as toxic to reproduction.


 


A NOAEL of 1000 mg/kg bw was observed in male and female Han Wistar rats in an extended one generation reproductive toxicity study according to OECD guideline 443 (Labcorp Early Development Laboratories Ltd., 2021; Klimisch 1). Based on the available data and according to the CLP criteria, the test substance should not be classified as toxic to reproduction.

Link to relevant study records

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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
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
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


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)
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.
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.
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
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: 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.
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
Key result
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
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.
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
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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.

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.
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-13 - 2010-11-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C8
- Physical state: dark liquid
- Storage condition of test material: at room temperature, 10 to 27 °C
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: (P) a minimum of 13 weeks old at initiation of cohabitation; records of dates of birth for animals used in this study will be retained in the Calvert archives
- Weight at study initiation: (P) Males: > or = 300 g; Females: > or = 200 g at initiation of cohabitation
- Fasting period before study: no data
- Housing: Upon arrival and until randomization, males and females are group-housed, sexes separate. Following randomization and until cohabitation, males and females will be individually housed. During cohabitation, one female will be placed with a male breeder from the same group. Following cohabitation, males and females will be housed individually. No later than gestation Day 17, mated female animals are plaec in totes with bedding. Animals will be housed in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". The room in which the animals are kept is documented in the study records. No other species are kept in the same room
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): All animals have access to Harlan Teklad Rodent Diet (certified) or equivalent ad libitum, unless otherwise specified. No contaminants are known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which will be maintained in the Calvert archives.
- Water (e.g. ad libitum): Water will be available ad libitum, to each animal via an automatic watering device. The water routinely analyzed for contaminants as per Calvert SOP's. No contaminants are known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis will be maintained in the Calvert archives.
- Acclimation period: Study animals will be acclimated to their housing for a minimum of 7 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26°C
- Humidity (%): 30 to 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights are turned on during the dark cycle to accomodate blood sampling or other study procedures

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test article and vehicle control preparations are prepared weekly or additionally as needed by diluting the test article in vehicle (w/v) to reach the proper concentrations.

Dose formulation samples:
On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1-mL samples are obtained from top, middle, and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test article in the vehicle. These samples are stored at room temperature, approximately 10 to 30°C.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
Animals are mated by placing one male and one female from the same dose group overnight in a breeding cage until evidence of copulation is noted, after which the male animal is separated. Animals remain in cohabitation for a maximum of three weeks. During this time, the study Director may elect to move certain females with other males from the same dose group, in an attempt to expedite mating. This procedure is documented in the study raw data records.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Males: The vehicle control or test article formulations are administered once daily for a minimum of four weeks (starting two weeks prior to cohabitation). Treatment continues during the same group cohabitation period and until the day before scheduled for euthanasia. Each animal received a mL/kg dose based upon its most recent body weight.
Females: The test/vehicle control or test article formulations are administered once daily for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed gestation days 0 through day 19 of gestation.
Procedure: Each animal receives a mL/kg dose based upon its most recent body weight.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control vehicle
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based upon previously conducted toxicity studyes
- Rationale for animal assignment (if not random): at random
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality: twice daily; once prior to scheduled sacrifice
- Cage side observations: each animal is observed for evidence of death or impending death (as per Calvert SOP VET-14)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, a minimum of once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: males: once weekly prior to initiation of cohabitation, during cohabitation and at terminal sacrifice. A final body weight is also obtained for all males sacrificed moribund; females: once weekly prior to initiation of cohabitation on gestation days 0, 4, 7, 10, 14, 17 and 20 (day 23 and 26 if required), and on Day 0 and 4 of lactation. A final body weight is also obtained for all females showing signs of premature delivery or sacrificed moribund.

FOOD CONSUMPTION:
Males and females: frequency: full feeder weights and/or feeder weigh backs are recorded once weekly prior to cohabitation, on gestation days 0-4, 4-7, 7-10, 10-14, 14-17, 17-20 (20-23 and 23-26 if required), and on Day 0 and 4 of lactation. During cohabitation food consumption is not recorded

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

Oestrous cyclicity (parental animals):
Estrous cycle evaluation is performed daily for 2 weeks prior to cohabitation and daily during the treatment and cohabitation periods. Day 0 of gestation is determined by evidence of copulation which is determined by the examination of vaginal smears made daily to determine if sperm are present in a smear of vaginal contents or by the presence of a copulatory plug in situ. Examination of vaginal smears is performed at approximately the same time each day (early morning) throughout the cohabitation period.
Postmortem examinations (parental animals):
SACRIFICE
a) Method of euthanasia:
All adult animals are sacrificed by CO2 asphyxiation. All male animals are euthanized 2 weeks post-mating and all females are euthanized on Day 4 of lactation. All pups are euthanized by an intrathoracic injection of a barbiturate overdose.
b) Unscheduled deaths of males and females:
For all males and females sacrificed moribund or found dead prior to scheduled sacrifice, a complete gross necropsy is performed. The necropsy includes the examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. The ear tag, all gross lesions, ovaries, uterus, cervix, vagina, seminal vesicles and prostate are retained in 10% neutral buffered formalin for possible histopathological evaluation.
Additionally, for male rats, the testes and epididymides are retained in modified Davidson's fixative for possible histopathological evaluation. Where applicable, the total number of implantation sites and the total number of corpora lutea for each ovary are recorded. Where applicable, the number of viable and non-viable fetuses are also recorded. All fetuses are discarded.
c) Terminal sacrifice of males:
all surviving male animals are euthanized 2 weeks post-mating and a macroscopic examination are performed. The necropsy includes the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
d) Terminal sacrifice of females:
All surviving females are euthanized on Day 4 of lactation and a macroscopic examination is performed. The necropsy includes the examination of the external body surface, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The total number of implantation scars are also determined.

GROSS NECROPSY
A complete gross necropsy is performed by Calvert personnel on all adult animals that are sacrificed or found dead during the study. The necropsy includes examination of:
- the external body surface
- all orifices
- the cranial, thoracic and abdonimal cavities and their contents
All abnormalities are described completely and recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
For all male animals at scheduled sacrifice at 14 days post cohabitation, the following organs are weighed before fixation, after dissection of excess of fat and other excess tissues. Organ weights are not recorded for animals found dead or sacrificed moribund: epididymides, testes. Organ to body weight ratios are calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.
Tissue collection and preservation: all tissues for all adult animals are examined. For all animals necropsied, the tissues are preserved in 10% neutral buffered formalin (except for the epididymides and testes that will be retained in modified Davidson's fixative for optimum fixation). Tissues collected: ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vescicles. Special emphasis on stages of spermatogenesis and histopathology of the interstitial testicular structure are given.
Postmortem examinations (offspring):
SACRIFICE
All pups are euthanized by an intrathoracic injection of a barbiturate overdose on Day 4 of lactation. All neonates are sexed, weighed, examined as soon as possible after delivery for litter seize, still births, live births and any gross anomalies. Neonates are not retained. All neonate malformations are photographed.

Statistics:
Statistical analysis will be performed on in-life and necropsy data when 3 or more animals are present in 2 or more dose groups. Statistical analysis is not performed if N<3 animals per group. For in-life parameters, the homogeneity of the data is determined by Bartlett's Test. If the data is homogeneous, a one-way analysis of variance is performed to assess statistical significance. If statistically significant differences between the meand are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If the data is non-homogeneous, the Kruskal-Wallis non-parametric analysis is performed to assess statistical significance. If statistically significant differences between the means are fround (p<0.05, p<0.01), the Mann-Whitney U-Test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups are present for evaluation, the Mann-Whitney U-Test is used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means is made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups are present for evaluation, the Mann-Whitney U-test is used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means is made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test is used to determine the degree of significance from the control means (p<0.05 and p<0.01).
Reproductive indices:
Pre-coital interval (in days): sum of days until successful copulation/number of presumed pregnant animals
Copulation index (%): (number of presumed pregnant animals/number of paired animals) x 100
Fertility index (%): (number of pregnant animals/number of presumed pregnant animals) x 100
Preimplantation loss (%): (no. of corpora lutea - number of implantations/number of corpora lutea) x 100
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal was found dead on lactation day 2. All other females survived until scheduled sacrifice.
All male animals survived until scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in bw or bw gain (in females and males)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No biologically relevant effects on food consumption during study (in females and males)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic toxic effects observed at 1000 mg/kg/dose (males and females)
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
no data
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
no data
Reproductive performance:
no effects observed
Description (incidence and severity):
In Life: One animal was non-gravid, another female did not mate. All males appeared normal.
Post mortem: no definitive effects on percent gravidity. No effects on pre-coital interval, copulation index and duration of gestation period were noted.
no statistically significant findings in the number of gravid animals, corpora lutea/dam, total implantations and pre-implantation loss.
no treatment-related differences in litter viability parameters.
no statistically significant changes in fetal sex ratios.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Incidence of neonates born alive/found dead, stillborn or missing between lactation days 0-4 was comparable among study groups.
One female (1000 mg/kg) was found dead on lactation day 2, and all neonates were stillborn.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related differences in litter viability parameters.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the neonates were statistically significantly increased. The biological significance of this finding is unknown.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related malformations were observed for neonates.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant changes in fetal sex ratios.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
No treatment-related differences in litter viability parameters.

Neotnate Observation
Incidence of neonates born alive/found dead, stillborn or missing between lactation days 0-4 was comparable among study groups.
One female (1000 mg/kg) was found dead on lactation day 2, and all neonates were stillborn.
No treatment-related malformations were observed for neonates.
No statistically significant changes in fetal sex ratios.

BODY WEIGHT (OFFSPRING)
Neonate bw were statistically significantly increased. the biological significance of this finding is unknown.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
In a screening reproductive / development toxicity test (performed according to OECD guideline 421), rats were exposed to 1000 mg test substance/kg bw/d orally (via gavage). No adverse effects were observed. A NOAEL of 1000 mg/kg bw/d was derived. The test substance is not to classified as reproductive toxicant according to the criterial laid down in the CLP Regulation.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A Reproductive / Development Toxicity Screening Testing in Rats was performed according to OECD guideline 421 (Calvert Laboratories, Inc., 2011, Klimisch 1). In this GLP-compliant study, male and female Sprague-Dawley rats (10 animals/sex/dose) were exposed to 1000 mg/kg bw/day. Control animals received concurrent vehicle (water). The rats were exposed on a daily basis, starting two weeks prior to cohabitation until the day before the scheduled euthanasia (minimum of 4 weeks) for males and starting for a minimum of 15 days prior to cohabitation, during cohabitation and from presumed gestation days 0 through day 19 of gestation for females. One female animal was found dead on lactation day 2. All other females survived until scheduled sacrifice. No treatment-related effects were observed in clinical signs, mortality, body weight (gain), food consumption, gross pathology, organ weights or histopathology in the parental animals. There was no treatment-related effect on reproductive performance or in reproductive parameters like for instance number of gravid animals, corpora lutea per dam, total implantations, litter viability or fetal sex ratios. The incidence of neonates born alive/found dead, stillborn or missing between lactation days 0 -4 was comparable among study groups. No treatment-related malformations were observed for neonates. The body weight of neonates was statistically significantly increased with an unknown biological significance for this finding. A NOAEL of 1000 mg/kg bw/day was established.


 


An extended one generation reproductive toxicity study in rats was performed according to OECD guideline 443 (Labcorp Early Development Laboratories Ltd., 2021; Klimisch 1). In the F0 generation, three groups of 24 male and 24 female rats were exposed on a daily basis to dose levels of 150, 450 or 1000 mg/kg bw/day at a volume dose of 5 mL/kg body weight by gavage. In the F1 generation, 40 males and 40 females were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. At weaning, the F1 generation was split into two cohorts: cohort A and cohort B. A control group received purified water at the same volume dose and throughout the same relevant periods. The test item was administered 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. There were no premature deaths or signs associated with dose administration, or test item-related changes in general clinical condition observed during the study. 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 not adverse. Estrous cycle regularity of the F0 and the F1 Cohort 1A females, pre-coital interval, mating performance, and conception rate and fertility index of the F0 animals were unaffected. Duration between vaginal opening and first estrus, ovarian follicle counts and corpora lutea counts for F1 Cohort 1A females, or the stage of estrus at termination of the F0 or the F1 Cohort 1A and 1B females were similarly unaffected. Litter size, offspring survival, sex ratio, offspring body weight gain and ano-genital distance were also unaffected at all dose levels investigated. Examination of the F1 male offspring did not reveal any evidence of nipple retention. There was also no effect on the attainment of sexual maturation among F1 Cohort 1A and 1B animals at any dose level investigated. Macroscopic and subsequent microscopic examination for the F0 and F1 Cohort 1A animals 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. No effect was obsrved on serum TSH concentrations in F0 or F1 Cohort 1A adult animals, or in the F1 offspring. 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 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. Based on these observations, a NOAEL of 1000 mg/kg bw/day was established 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.

Effects on developmental toxicity

Description of key information

A prenatal development toxicity study in rabbits was performed according to OECD guideline 414 (Renaut, 2019). It was concluded that the NOEL was 250 mg/kg/d for both maternal toxicity and embryo-fetal development.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Species:
rat
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-27 - 2019-11-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Huntsman Europe BVBA and 17/19961
- Expiration date of the lot/batch: 2019-11-22
- Purity test date: Not applicable (UVCB)

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 under storage conditions: Not specified
- Stability under test conditions: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2and 200mg/mL were analyzed to assess the stability of the test item in the liquid matrix.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Not specified
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixed to vehicle (purified water)
- Final preparation: 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.
- Frequency of preparation: Weekly, in advance of dose administration.
- Storage of formulation: Refrigerated at 2 to 8°C

OTHER SPECIFICS
- Physical State/appearance: Brown liquid
- other information: No correction factor

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS.
- Age at study initiation (day 0 of gestation): 19 to 23 weeks old
- Weight at study initiation: 2.82 to 4.61 kg
- Fasting period before study: No
- Housing: Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also fitted with a plastic resting platform.
Number of animals per cage: acclimatization one female, during mating one stock male and one female, during gestation one female.
- Diet (e.g. ad libitum): Teklad 2930 Diet, the diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Availability was restricted (initially 150g/animal/day during acclimatization up to one week prior to the onset of mating and 200g/animal/day thereafter). Should an individual show a significant non-treatment related reduced food consumption, moistened diet (50 g pelleted diet moistened with 20 to 50 mL of water) was offered and the consumption was recorded. In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

- Water (e.g. ad libitum): Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were offered to individuals during acclimatization.
- Acclimation period: 19 days before commencement of mating.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21°C
- Humidity (%): 45 - 70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: 2018-07-04 To: 2018-08-24
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
-The test item was prepared at the appropriate concentrations as a solution in Distilled Water.
- The stability of formulations was demonstrated over a period of up to 21 days at 2 to 8°C and one day at ambient temperature (15 to 25°C). Formulations were therefore prepared weekly and stored at approximately 2 to 8 ºC.
VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable (vehicle is water)
- Concentration in vehicle: 0, 25, 50,100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability was confirmed for Amine C8 in Distilled Water formulations at nominal concentrations of 2 mg/mL and 200 mg/mL when stored refrigerated for 21 days and at room temperature in the light for 24 hours.
Samples of test item formulations were taken on two occasions and analyzed for concentration and the results indicate that the prepared formulations were within 100-112% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 6 days
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: Expelled uterine contents were identified and examined, as appropriate
- Any other deviations from standard protocol: No applicable
Duration of treatment / exposure:
Day 6 to 28 after mating
Frequency of treatment:
Once daily at approximately the same time each day.
Duration of test:
29 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: not specified
- Rationale for animal assignment (if not random): Where possible only females mating at least twice were allocated.
Method to group and cage position in the sequence of mating: Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded weekly during acclimatization, on Days 0, 3 and 6-29 after mating

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

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

POST-MORTEM EXAMINATIONS: Yes
- 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.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead).
Fetal examinations:
- External examinations: Yes [half per litter]
- Soft tissue examinations: Yes [ half per litter]
- Skeletal examinations: Yes [half per litter]
- Head examinations: Yes: [ half per litter ]
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon1945) were made. For all other analyses the/The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs were observed at either routine physical examination or in association with dose administration that could be related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female dosed at 125 mg/kg/day was euthanized prematurely with evidence of abortion. Uterine examination revealed a single implantation that was resorbing. This was therefore considered to be a resorption rather than abortion and as there was a solely a single implant it is considered to relate to biological efficiency rather than an effect of treatment.

One female dosed at 500 mg/kg/day prematurely delivered six live offspring, one dead offspring and one late resorption within the animal facility. The live offspring were euthanized within the animal facility and dispatched to necropsy for subsequent examinations. Macroscopic examination of female no 72 and offspring did not reveal any findings that could be attributed to treatment. As this was an isolated incidence it was considered to be unrelated to administration of Amine C8.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
As treatment progressed weight gain for females receiving 500 mg/kg/day was low when compared with Controls and overall the bodyweight gain from GD6 to GD29 bodyweight gain was 17% of Controls (p<0.01). The overall body weight gain for females at 125 or 250 mg/kg/day were similar to the Controls and showed no adverse effect of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption for females receiving 500 mg/kg/day was slightly but consistently low from Day 6 to Day 27 of gestation when compared with Controls, with the total consumption over the treatment period at approximately 79% of the Control value.
At 250 mg/kg/day there were occasions of slightly low consumption however overall the food consumed was similar to Control values.
Food consumption at 125 mg/kg/day was unaffected by treatment with Amine C8.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The maternal body weight loss following adjustment for the gravid uterine weight was slightly greater than Controls for females that received 500 mg/kg/day; however this difference did not attain statistical significance. The maternal weight loss for females at 125 or 250 mg/kg/day were similar to the Controls and showed no adverse effect of treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females did not reveal any findings that could be attributedto treatment with Amine C8.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The post-implantation loss (%) for females at 500mg/kg/day was slightly high when compared with Controls and the resultant mean litter size was low when compared with Controls (p<0.05). However, the live litter size and percentage of post implantation loss were within the historical control data range so no effect of treatment is inferred.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One control female and 3 females treated at 250 mg/kg/day were found to be not pregnant at macroscopic examination. There was no clear effect of treatment on offspring survival and litter size at 125 or 250 mg/kg/day.
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Group mean placental weights, male or fetal weight and overall fetal weight were essentially similar to Controls at dose levels up to and including 500 mg/kg/day.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not specified
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter weight for females that received 500 mg/kg/day was slightly but significantly low when compared with Controls; this was attributed to the low live litter size rather than the mean fetal weight at this dose level.
Changes in postnatal survival:
not examined
External malformations:
not specified
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The range and incidence of major abnormalities at 125 and 500 mg/kg/day showed no relationship to treatment. At 500 mg/kg/day there was a slight increased incidence of full supernumerary 13th ribs and unilateral caudal shift of pelvic girdle when compared with both the Concurrent Controls and the historical control data range. The combination of these skeletal variants at 500 mg/kg/day indicates a slight shift in rib/vertebral configuration which is not considered adverse.
Visceral malformations:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
no
Relation to maternal toxicity:
not specified
Relevant for humans:
not specified
Conclusions:
Oral administration of the test substance to New Zealand White rabbits from Day 6 to Day 28 of gestation at dose levels of 125, 250 or 500 mg/kg/day was associated with low maternal body weight gain and food consumption at 500 mg/kg/day.
Embryo-fetal survival, fetal and placental weight were unaffected by treatment and the fetal pathology findings at 500 mg/kg/day were minor skeletal variants which were not considered adverse.
It was therefore concluded that 250 mg/kg/day was the no observed effect level (NOEL) for both maternal toxicity and embryo-fetal development.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

Prenatal development study in rabbit (key, K1):


The effect of the test substance on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White rabbit were assessed. Three groups of 22 females received the test substance at doses of 125, 250 or 500 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating. A similarly constituted control group received the vehicle, purified water, at the same volume dose as treated groups. Animals were euthanized on Day 29 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination. 


The mean concentrations of test substance in test formulations analysed for the study were within +/-12% of nominal concentrations, confirming accurate formulation. There were no signs observed at either routine physical examination or in association with dose administration that could be related to treatment. 


At 500 mg/kg/day the overall body weight gain and food consumption of females was low; approximately 17% and 79% of controls, respectively. Maternal body weight and food consumption at 125 or 250 mg/kg/d showed no adverse effects of treatment. Macroscopic examination of females on GD29 did not reveal any findings that could be attributed to treatment with the test substance. On Day 29 of gestation, mean numbers of implantations embryo-fetal survival, litter size and sex ratio were considered to be unaffected by treatment with the test substance. Group mean placental weights, male or fetal weight and overall fetal weight were essentially similar to controls at dose levels up to and including 500 mg/kg/day. The range and incidence of major abnormalities at 125 and 500 mg/kg/day showed no relationship to treatment. At 500 mg/kg/day there was a slight increased incidence of full supernumerary 13th ribs and unilateral caudal shift of pelvic girdle when compared with both the concurrent controls and the historical control data range. The combination of these skeletal variants at 500 mg/kg/day indicates a slight shift in rib/vertebral configuration which is not considered adverse. 


In conclusion, oral administration of the test substance to New Zealand White rabbits from Day 6 to day 28 of gestation at dose levels of 125, 250 or 500 mg/kg/day was associated with low maternal body weight gain and food consumption at 500 mg/kg/day. Embryo-fetal survival, fetal and placental weight were unaffected by treatment and the fetal pathology findings at 500 mg/kg/day were minor skeletal variants which were not considered adverse. It was therefore concluded that 250 mg/kg/day was the NOEL for both maternal toxicity and embryo-fetal development.


A prenatal development study in rat will be considered after data interpretation and final reporting of the EOGRTS study in rat (ongoing).

Justification for classification or non-classification

Based on the available data and the criteria laid down in the CLP regulation, the test substance is considered not to be classified as reproductive toxicant.

Additional information