[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 30 November 2016 and 29 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 30 November 2016 and 29 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD guideline 422 without deviation and in compliance with GLP guidelines.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 201522 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification : (µ(5-amino-1,3,3-trimethylcycloihexylamineN,N')hexafluorodiboron. The test item is also known as Aradur HZ 5933.
Physical State/Appearance : Clear colorless liquid
Purity : 100% UVCB substance
Batch Number : AEF0009100
Label : Aradur HZ 5933
Name of Test Article: [(µ(5-amino-1,3,3-trimethylcycloihexylamineN,N')]hexafluorodiboron Batch Number AEF0009100 29.08.2017 Net 2.01 KG
Date Received : 11 June 2016
Storage Conditions : Room temperature in the dark
Expiry Date : 29 October 2017

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST strain

Details on species / strain selection:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 303 to 369g, the females weighed 180 to 217g, and were approximately twelve weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose Administration
Animals were allocated to treatment groups as follows:

Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Male Female
Control 0 5 0 12 (1-12) 12(13-24)
Low 25 5 5 12 (25-36) 12 (37-48)
Intermediate 50 5 10 12 (49-60) 12 (61-72)
High 100 5 20 12 (73-84) 12 (85-96)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Distilled water.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken on six occasions and analyzed for concentration of (µ(5-amino-1,3,3-trimethylcycloihexylamine-N,N’)hexafluorodiboron at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given below. The results indicate that the prepared formulations were within ± 13% of the nominal concentration.

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Preparation of Calibration Standards
Stock solutions of test item in dilution solvent (see Section 2.2.2) were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters section 2.2.6.
To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.047 mg/mL by serial dilution covering the concentration range 0.05235 mg/mL to0.2094 mg/mL.

Preparation of Test Samples
The formulations received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of Accuracy and Precision Samples
Samples of Distilled Water were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples.

The concentration of Test Item in the final solution was quantified by GC using FID detection as detailed in the instrument parameters section.

Instrumentation Parameters

GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: Zb-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program: oven: 150°C, for 0 minutes with 15°C/minute to 300°C, for 0 minutes
Injection temperature: 130 ºC
Flame ionisation detector temperature: 200 ”C
Injection volume: 1 µL
Retention time: ~ 3.3 mins

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.

Validation of the Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The linearity of detector response over the calibration standard concentration range.
The method accuracy (recovery) and precision, by analyzing five recovery samples at nominal concentrations of 2 mg/mL and 100 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Homogeneity and Stability in Vehicle Formulations
The homogeneity and stability of Test Item in water formulations was assessed at nominal concentrations of 2 mg/mL and 100 mg/mL during storage.

Refrigerated storage (nominally +4ºC)
The formulations were analysed and refrigerated on receipt. On day 12; the formulations were removed from storage and equilibrated to ambient temperature. The formulations were mixed as stated in the mixing procedure (described in the main part of this study) and single samples were removed for analysis from the top, middle and bottom of the mixed formulation.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analyzed. Duplicate samples were analyzed in accordance with the analytical procedure. Samples were disposed of once satisfactory results were achieved.

RESULTS
Method Validation
The analytical procedure was successfully validated for Test Item in water with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision. Results are summarized below.
The specificity of the analytical method was demonstrated by the absence of a peak at the characteristic retention time for Test Item in the control sample chromatogram.
The calibration data for the calibration standards of the test item was found to have a linear correlation within the calibration range of 0.05235 mg/mL to 0.2094 mg/mL. The R² fit of the calibration curve to the data was 0.9999, and was considered to be acceptable.
A mean recovery value of 94% (CV=4.61%, n=5) was obtained for 2 mg/mL and 102% (CV=4.66%, n=5) was obtained for 100 mg/mL.
The limit of quantification (LOQ) was determined as the lowest standard concentration used during the study.

Homogeneity and Stability of Dose Formulations
The homogeneity and stability of Test Item in water formulations was assessed with respect to the level of concentration at nominal concentrations of 2 mg/mL and 100 mg/mL. Results are presented in Table 1 and Table 2 .
Homogeneity was confirmed at the initial stability time point. The mean analyzed concentration for the nine samples remained within 20% of the initial time zero value and the variation was less than 20%.

Concentration of Dose Formulations
The mean concentrations of Test Item in test formulations analyzed during the study and the results are presented in Table 3. The mean concentrations were within applied limits ±20%, confirming accurate formulation.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for Test Item in water formulations at nominal concentrations of 2 mg/mL and 100 mg/mL when stored at ambient for 4 hours.
The mean concentrations of Test Item in test formulations analyzed for the study were within ±20% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

Frequency of treatment:

Once a day for the duration of the study.

Details on study schedule:

The study was performed between 07 December 2016 and 29 May 2017. The in-life phase of the study was conducted between 08 December 2016 (first day of treatment) and 31 January 2017 (final day of necropsy).

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically

Remarks:

Doses / Concentrations:
25, 50 and 1000 mg/kg bw/day

No. of animals per sex per dose:

12 per sex per dose of test item plus 12 per sex in control group

Control animals:

yes, concurrent vehicle

Details on study design:

Route of Administration
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Distilled water

Rationale for Dose Level Selection
The dose levels have been chosen in consultation with the Sponsor and were based on the results from previous toxicity work including a fourteen day dose range-finding study in the rat (Envigo Study Number: YC36QB). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Positive control:

None

Parental animals: Observations and examinations:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week, at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 714 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week, at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity.
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Hematology,
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Oestrous cyclicity (parental animals):

Estrous Cycles
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
• For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
• After pairing until mating.
• For four days before scheduled termination (nominally Day 11 to 14 of lactation).

Litter observations:

Records Made During Littering Phase

Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring.

Postmortem examinations (parental animals):

Terminal Investigations
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown by * were weighed from all remaining animals:
Adrenals Pituitary (post-fixation)*
Brain Prostate and Seminal Vesicles*
Epididymides* Spleen
Heart Testes*
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries* Uterus (weighed with Cervix)*

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown by * were preserved from all remaining animals:
Adrenals Muscle (skeletal)
Aorta (thoracic) Ovaries*
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Pituitary*
Brain (including cerebrum, cerebellum and pons) Prostate*
Caecum Rectum
Coagulating gland* Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles*
Epididymides ♦* Skin
Esophagus Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions Spleen
Heart Stomach
Ileum (including peyer’s patches) Testes ♦*
Jejunum Thyroid/Parathyroid
Kidneys Trachea
Liver Thymus
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix*
Mammary gland* Vagina*

Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing. The tissues from five selected control and 100 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 100 mg/kg bw/day animals were also processed. In addition, sections of testes from all control and 100 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, 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, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of spleen (female only), liver (female only) and stomach (male and female) from animals in the low and intermediate groups.

Postmortem examinations (offspring):

Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)

Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre-implantation loss (%) = (No. Corpora lutea - No. implantation sites) / No. corpora lutea x 100

Post-implantation loss (%) = ( No. implantation sites - Tota; No. offspring born) / No. implantation sited x 100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live birth index (%) = Number of live offspring on Day 1 / Total number of offspring born x 100

Viability index (%) = Number of live offspring on Day 4 / Number live offspring on Day 1 after littering x 100

Sex Ratio
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Percentage males = Number of malesoffspring / Total number of offspring x 100

Statistics:

SEE "Any other information on materials and methods" below

Reproductive indices:

Reproductive Indices
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices

For each group the following were calculated:

Mating Index (%) = Number mated animals / Number of paired animals x 100

Pregnancy Index (%) = Number of pregnant females /Number of animals mated x 100


Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length

Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index

The following was calculated for each group:

Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs considered to be related to the systemic toxicity of the test item throughout the treatment period.
At 100 mg/kg bw/day, there were sporadic instances of increased post-dose salivation observed in males and females with one animal of either sex also showing some instances of noisy respiration. Isolated instances of increased post dose salivation were also evident in four males and one female from the 50 mg/kg bw/day dose group. Observations of this nature are often reported following the oral administration of an unpalatable or irritant test item formulation rather than evidence of true systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two unscheduled deaths during the study which were considered not to be test item related.
One male from the 50 mg/kg bw/day dose group was killed in extremis due to welfare reasons on Day 7 of dosing. Prior to death, clinical signs included respiratory distress, tiptoe gait, pilo-erection, hunched posture, staining around the snout and ptosis and macroscopic observations at necropsy included lungs and thoracic cavity filled with red brown fluid, hardened left lobe of the lung with white fibrous adhesions attached to the thoracic cavity wall, enlarged adrenals and gaseous distension of the intestines.
One female from the 100 mg/kg bw/day dose group was killed in extremis due to welfare reasons on Day 10 of dosing. Clinical signs prior to death included pilo-erection, hunched posture, staining around the snout, chromodacryorrhea and a mass under the left forelimb which, upon examination during necropsy, was found to contain straw colored liquid. Other macroscopic abnormalities for this female included enlarged adrenals and gaseous distension of the gastrointestinal tract.
The histopathology examination revealed findings in both animals that were indicative of mis-dosing and not related to treatment with the test item.
There were no further unscheduled deaths on the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the start of treatment, mean body weight gain in males treated with 100 mg/kg bw/day was statistically significantly lower than controls (p<0.05). Subsequent improvement was evident, but overall body weight gain in these males generally remained lower than controls which resulted in approximately 27% lower overall body weight gain.
There was no effect of treatment on body weight gain in females at any dose level during the pre-paring and gestation phases of the study. Females receiving the test item at 100 mg/kg bw/day showed lower body weight gain over Days 1 to 4 of lactation but the corresponding values for the remaining test item-treated females were comparable with controls

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food intake and food conversion efficiency across all test item-treated dose groups of males was generally similar to controls.

There did not appear to be any detrimental effect of treatment with the test item on food consumption or food conversion efficiency in females during the pre-pairing, gestation or lactation phases of the study.

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

not examined

Description (incidence and severity):

Visual inspection of water bottles did not indicate any differences in water intake levels for the animals given the test item when compared with controls.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day, females showed statistically significantly lower group mean hemoglobin, erythrocyte count, hematocrit and mean corpuscular hemoglobin concentration in relation to controls (p<0.01) with the difference for the latter extending to females treated with 50 mg/kg bw/day (p<0.05) in a dose-related manner. Mean corpuscular volume in females from the 100 mg/kg bw/day dose group was statistically significantly higher than controls (p<0.05). Although the majority of individual values were within historical control ranges, these findings are likely to be associated with the histopathological changes in hematopoiesis in the spleen and liver of females in the 50 and 100 mg/kg bw/day dose groups and a direct effect of the test item can not be ruled out.
Females treated with 100 mg/kg bw/day showed a statistically significant increase in lymphocytes (p<0.05). The majority of individual values were within historical control range and in isolation was considered not to be of toxicological significance.
Males treated with the test item at all doses showed statistically significantly lower mean corpuscular volume values than controls (p<0.05) with the 50 and 100 mg/kg bw/day males also showing statistically significantly lower mean corpuscular hemoglobin (p<0.05). The majority of individual values were within historical control ranges and in the absence of any associated histopathological correlates in this sex, the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
Animals of either sex treated with 100 mg/kg bw/day and males treated with 50 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase in relation to controls (p<0.05-p<0.01). In the absence of any associated histopathological correlates, the intergroup differences were considered of no toxicological significance.
At all dose levels, albumin/globulin ratio was statistically significantly higher than controls (p<0.01), but there was no dose-relationship. Group mean total protein values in males from the 100 mg/kg bw/day dose group was statistically significantly lower than controls (p<0.05). The majority of individual values were within historical control ranges and in the absence of any associated histopathological correlates, the intergroup diffences were considered of no toxicological significance.
At 100 mg/kg bw/day, other statistically significant intergroup differences for the females included higher potassium, phosphorus and creatinine concentrations when compared with controls (p<0.05). The majority of individual values were within historical control ranges and in the absence of any associated histopathological correlates, the intergroup differences were considered to be of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Performance Tests
There were no treatment-related changes in the functional performance.
Females treated with 50 or 100 mg/kg bw/day showed a statistically significant increase (p<0.01) in the second forelimb grip strength test. A true dose related response was not evident and in the absence of any supporting clinical observations to suggest any neurotoxic effect, the intergroup differences were considered not to be of toxicological significance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Premature Decedents
One 50 mg/kg bw/day male (No.55) and one 100 mg/kg bw/day female (No.92) were sacrificed early during the study, the changes noted were indicative of mal-dosing and not related to treatment with the test item.
Terminal Sacrifice Spleen
There was an increase in incidence and severity of hematopoiesis in the spleen of 50 and 100 mg/kg bw/day females and this correlated with the increased weight noted at necropsy. Liver
Hematopoiesis was also present in the liver of one female treated with 25 mg/kg bw/day, two females treated with 50 mg/kg bw/day and three females treated with 100 mg/kg bw/day at a mild severity. Stomach
Foveolar hyperplasia in the glandular region (with eosinophilic globule cells), minimal or mild, was present in all animals treated with the test item.
Inflammatory cell infiltration in the glandular region, minimal or mild was present in all males treated with the test item and four females each from the 25 and 50 mg/kg bw/day dose groups and three females from 100 mg/kg bw/day dose group.
Mucous cell hypertrophy, of minimal or mild severity, in the glandular region was present in two males treated with 25 mg/kg bw/day, three males and females treated with 50 mg/kg bw/day and four males and three females treated with 100 mg/kg bw/day. It occurred in a dose related pattern.
Paneth cell metaplasia was present in one 100 mg/kg bw/day male.
No other changes were noted which could be related to the administration of the test item.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating
There were no treatment-related effect on mating performance.
The majority of females mated within four days after pairing. One female treated with 50 mg/kg bw/day mated 13 days after pairing.

Fertility
There was no effect of treatment on fertility.
Female 22 (paired with Male 10) from the control group and Female 63 (paired with Male 51) from the 50 mg/kg bw/day dose group were found to be non-pregnant following positive evidence of mating. Histopathology examinations revealed no histological changes to account for the lack of pregnancy.

Gestation Length
Gestation lengths were between 22 and 23½ days with their distribution appearing to be evenly distributed across all dose groups.
Litter Responses
In total 11, 12, 11 and 11 females from the control, 25, 50 and 100 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
There was no adverse effect of maternal treatment on the number of corpora lutea and implantations, pre and post implantation losses, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 25, 50 or 100 mg/kg bw/day. Sex ratio (percentage male offspring) was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development
There was no detrimental effect of treatment with the test item indicated by offspring body weight, body weight gain, litter weights or surface righting on Day 1 post partum at 25, 50 and 100 mg/kg bw/day.
The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 25, 50 and 100 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive and developmental toxicity

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 25, 50 and 100 mg/kg bw/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no detrimental effect of treatment with the test item indicated by offspring body weight, body weight gain, litter weights or surface righting on Day 1 post partum at 25, 50 and 100 mg/kg bw/day

Gross pathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Litter Size, Survival Indices, Sex Ratio and Viability
There was no adverse effect of maternal treatment on the number of corpora lutea and implantations, pre and post implantation losses, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 25, 50 or 100 mg/kg bw/day. Sex ratio (percentage male offspring) was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Treatment related:

no

Relation to other toxic effects:

not specified

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations		Dose Level (mg/kg bw/day)
		0 (Control)	25	50	100)
Paired animals	n	12	12	12	11
Females showing evidence of copulation	n	12	12	12	11
Pregnant females	n	11	12	11	11
Conception Days 1-4	n	12	12	11	11
Conception Day 9	n	0	0	1	0
Gestation = 22 Days	n	4	7	3	1
Gestation = 22 ½ Days	n	5	3	5	8
Gestation = 23 Days	n	1	1	3	1
Gestation = 23 ½ Days	n	1	1	0	1
Dams with live young born	n	11	12	11	11
Dams with live young at Day 4post partum	n	11	12	11	11
Corpora lutea/dam	x	13.5	14.1	14.5	13.5
Implants/dam	x	13.3	13.3	13.9	13.1
Live offspring/dam at Day 1post partum	x	12.5	12.4	12.3	12.4
Live offspring/dam at Day 4post partum	x	12.2	12.3	12.2	12.3
Sex ratio: % males at Day 1post partum	x	53.8	43.5	52.1	44.4
Sex ratio: % males at Day 4post partum	x	53.7	43.1	51.5	44.7
Litter weight (g) at Day 1post partum	x	70.45	69.43	69.15	67.77
Litter weight (g) at Day 4post partum	x	98.59	99.33	100.64	95.14
Male offspring weight (g) at Day 1post partum	x	5.69	5.91	5.75	5.66
Male offspring weight (g) at Day 4post partum	x	8.22	8.60	8.55	7.94
Female offspring weight (g) at Day 1post partum	x	5.50	5.48	5.44	5.37
Female offspring weight (g) at Day 4post partum	x	8.06	8.19	7.99	7.61
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)
0	n	8	9	8	7
1	n	3	1	2	3
2	n	0	1	0	1
4	n	0	0	1	0
6	n	0	1	0	0
Pre-natal (implantations minus live births)
0	n	6	7	3	7
1	n	3	3	5	2
2	n	1	0	2	0
3	n	1	1	1	2
4	n	0	1	0	0
Post natal (live births minus offspring alive on Day 4post partum)
0	n	7	10	10	10
1	n	4	1	0	1
2	n	0	1	0	0
7	n	0	0	1	0

Conclusions:

The oral administration of (µ(5-amino-1,3,3-trimethylcycloihexylamineN,N')hexafluorodiboron to rats for a period of up to seven weeks (including two weeks prepairing, gestation and early lactation for females) at dose levels 25, 50 and 100 mg/kg bw/day resulted in microscopic stomach changes in animals of either sex from all treatment groups, microscopic spleen and liver changes together with hemotogical changes in females treated with 100 and 50 mg/kg bw/day and reduced body weight gains in males treated with 100 mg/kg bw/day. The stomach changes identified in all treatment groups may be considered to be an adverse effect of treatment, however they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. In terms of risk assessment, these findings would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 50 mg/kg bw/day for males and 25 mg/kg bw/day for females.
The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 100 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 25, 50 and 100 mg/kg bw/day.  A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.  

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum.  Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.  

Adult males were terminated on Day 43 or 44, followed by the termination of all surviving females and offspring on Day 5 post partum.  Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses

Mortality

There were two unscheduled deaths during the study which were considered not to be test item related.

Clinical Observations

There were no clinical signs considered to be related to the systemic toxicity of the test item throughout the treatment period.

Behavioral Assessment

There were no treatment-related effects detected.

Functional Performance Tests

There were no treatment-related changes in the functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Males treated with 100 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment.  Overall body weight gain for these males was 27% lower than controls.  No such effect was evident in males treated with 50 or 25 mg/kg bw/day.  There was no adverse effect on body weight development for females during pre-pairing or gestation, however, females treated with 100 mg/kg bw/day showed lower body weight gains between days 1 and 4 of lactation.  

Food Consumption

There were no adverse effects on food consumptions or food conversion efficiency at any dose level during the study.

Water Consumption

There were no treatment-related effects detected for water consumptions at any dose level during the study.

Reproductive Performance

Mating

There were no treatment-related effects on mating performance.

Fertility

There was no effect of treatment on fertility.

Gestation Lengths

Gestation lengths were between 22 and 23½ days with their distribution appearing to be evenly distributed across all dose groups.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum was comparable to controls. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gains and litter weights on Days 1 and 4 post partum were unaffected by treatment.  Surface righting in treated litters was comparable to controls.  The clinical signs apparent for offspring on the study were typical for the age observed.

Laboratory Investigations

Hematology

At 100 mg/kg bw/day, females showed statistically significantly lower group mean hemoglobin, erythrocyte count, hematocrit, mean corpuscular volume and  mean corpuscular hemoglobin concentration in relation to controls.  The effect on mean corpuscular haemoglobin concentration also extended to females treated with 50 mg/kg bw/day.  These findings are likely to be associated with the histopathological changes of hematoiesis in the spleen and liver in females treated with 50 and 100 mg/kg bw/day.  

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.  

Pathology

Necropsy

One fermale treated with 100 mg/kg bw/day had an enlarged spleen at necropsy.  No such effects were detected in males treated with 100 mg/kg bw/day or in animals of either sex treated with 50 or 25 mg/kg bw/day.  

Organ Weights

There was an increase in spleen weights for females treated with 50 and 100 mg/kg bw/day. No such effects were detected in males treated with 100 or 50 mg/kg bw/day or animals of either sex treated with 25 mg/kg bw/day.  

Histopathology

There was an increase in incidence and severity of hematopoiesis in the spleen of 50 and 100 mg/kg bw/day females and in the liver of one female treated with 25 mg/kg bw/day, two females treated with 50 mg/kg bw/day and three females treated with 100 mg/kg bw/day at a mild severity.  

The changes in the glandular stomach: foveolar hyperplasia, mucous cell hypertrophy, Paneth cell metaplasia and inflammatory cell infiltrate are indicative of an irritant effect of the test item and occurred in all treatment groups.

Conclusion

The oral administration of  (µ(5-amino-1,3,3-trimethylcycloihexylamineN,N')hexafluorodiboron to rats for a period of up to seven weeks (including two weeks prepairing, gestation and early lactation for females) at dose levels 25, 50 and 100 mg/kg bw/day resulted in microscopic stomach changes in animals of either sex from all treatment groups, microscopic spleen and liver changes together with hemotogical changes in females treated with 100 and 50 mg/kg bw/day and reduced body weight gains in males treated with 100 mg/kg bw/day.  The stomach changes identified in all treatment groups may be considered to be an adverse effect of treatment, however they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity.  In terms of risk assessment, these findings would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 50 mg/kg bw/day for males and 25 mg/kg bw/day for females.  

The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 100 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 201522 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

EC Number:

289-348-4

EC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Cas Number:

87788-32-7

Molecular formula:

C10H22B2F6N2

IUPAC Name:

[μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N')]hexafluorodiboron

Results and discussion

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

haematology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no