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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
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: Two weeks. Animals of Cohort 1B are mated to produce the F2 generation and, thus, the premating exposure duration is 10 weeks for these Cohort 1B animals and the fertility parameters are covered allowing an evaluation of the full spectrum of effects on fertility in these animals.
- Basis for dose level selection: Based on the results obtained in a range-finding study of reproductive performance it was concluded that the high dose level for the main extended one-generation reproductive toxicity study (OECD TG 443) should be set at 1000 mg/kg/day (limit test concept).
- Inclusion/exclusion of extension of Cohort 1B: Inclusion of extension of Cohort 1B according to ECHA final decision TPE-D-2114460726-43-01/F.
- Termination time for F2: At weaning, according to OECD TG 443.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Inclusion of developmental neurotoxicity Cohorts 2A and 2B according to ECHA final decision TPE-D-2114460726-43-01/F.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Inclusion of developmental immunotoxicity Cohort 3 according to ECHA final decision TPE-D-2114460726-43-01/F.
- Route of administration: Oral route according to ECHA final decision TPE-D-2114460726-43-01/F. The oral route is the most appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction, and B-TEGME is a liquid.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The rat was selected as animal species because of the extent of background data and the comparability to general toxicity tests. The rat is the preferred species according to OECD TG 443 and ECHA final decision TPE-D-2114460726-43-01/F. Sprague Dawley rats were selected as this strain is accepted by regulatory agencies and historical control data are available.

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
EC Number:
250-418-4
EC Name:
Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Cas Number:
30989-05-0
Molecular formula:
C21H45BO12
IUPAC Name:
tris{2-[2-(2-methoxyethoxy)ethoxy]ethyl} borate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as test species because of the extent of background data and the comparability to general toxicity tests. The rat is the preferred species according to OECD TG 443 and ECHA final decision TPE-D-2114460726-43-01/F. Sprague Dawley rats were selected as this strain is accepted by regulatory agencies and historical control data are available in the test facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age of the F0 animals at the start of the treatment: Males 74 to 81 days old; Females 68 to 74 days old.
- Weight range of the F0 animals at the start of the treatment: Males 347 to 444 g; Females 209 to 278 g.
- Housing: The test facility has a policy of improvement of animal welfare which includes permitting social interaction through multiple housing of rats whenever possible. This policy was followed for this study, except after mating when females were housed singly to permit collection of food consumption data individually for pregnant females and during lactation, when adult females were singly housed with their litter for good husbandry practice. Animals were also housed singly (overnight duration) to permit in cage observation which were performed as part of the functional observation battery (Cohort 2A).
For details on animal accomodation see table. Grid bottomed cages were suspended above absorbent paper which were changed daily during pairing. Solid bottomed cages had bedding which was changed at appropriate intervals. Cages and cage-trays were changed at appropriate intervals.
- Diet (e.g. ad libitum): SDS VRF1 Certified, pelleted diet ad libitum (except overnight for blood sampling for hematology, blood chemistry, urinalysis and thyroid hormone investigations for F0 and F1 Cohort 1A animals). Food hoppers were changed at appropriate intervals. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum (except overnight for blood sampling for hematology, blood chemistry, urinalysis and thyroid hormone investigations for F0 and F1 Cohort 1A animals). Water bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Correction factor: A correction factor of 1.14 was used when calculating quantities of test item used during dose preparation. This calculation was used to correct for the test item purity.
- Frequency of preparation: Weekly, may have been prepared in advance of the first day of dosing.
- Storage of formulation: Refrigerated (2 to 8ºC).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): The test item is instable in water (hydrolyzes to 3x methyltriglycol + boric acid).
- Amount of vehicle (if gavage): 4 mL/kg

ADMINISTRATION:
- Route: Oral gavage.
- Treated at: Constant doses in mg/kg/day.
- Volume dose: 4 mL/kg body weight.
- Individual dose volume: Calculated from the most recently recorded scheduled body weight.
- Control (Group 1): Vehicle at the same volume dose as treated groups.
- Frequency: Once daily at approximately the same time each day.
- Formulation: A daily record of the usage of formulation was maintained based on weights before and after dosing. Thie difference between actual and expected usage was monitored as a check of correct administration. After filling the dose equipment for each animal, the semi-ridged cannula was wiped dry and dipped in corn oil before administration. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Details on mating procedure:
- Time schedule: F0 pairing: After 2 weeks of treatment. F1 Cohort 1B pairing: After 10 weeks of formal treatment (nominal Week 14 of age).
- M/F ratio per cage: 1:1 (sibling pairing was not permitted).
- Length of cohabitation: Up to 2 weeks.
- Proof of pregnancy: Ejected copulation plugs and sperm within vaginal smear; referred to as Day 0 of gestation.
- M/F separation: Day when mating evidence detected.
- Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of formulations during storage were determined as part of another study, Covance Study Number MQ60WK. In that study formulations in the range 2 to 250 mg/mL were determined to be stable for 24 hours at ambient temperature (15 to 25°C) or 15 days when stored refrigerated (2 to 8°C). Samples of each formulation prepared for administration in Week 1 of the F0 and F1 generation and the last week of treatment were analyzed for achieved concentration of the test item. The analytical method involved dilution with acetone followed by further dilution with water:methanol (80:20 v:v) prior to quantitation performed using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). Sample concentrations were determined with reference to external calibration standards freshly prepared in the concentration range 0.5 ng/mL to 50 ng/mL.
Duration of treatment / exposure:
F0 animals: Males: 2 weeks pre-pairing to necropsy after selection of the F1 generation receiving at least 10 weeks of treatment; Females: 2 weeks pre-pairing to necropsy on or soon after Day 28 of lactation following selection of the F1 generation.
F1 animals: Direct treatment of F1 offspring per gavage commenced at weaning (Day 21 of age) until termination of respective cohort (all offspring had potential indirect exposure in-utero and via milk during lactation):
- Unselected F1 offspring: No direct treatment; killed on Day 22 of age, organs retained as specified.
- Cohort 1A: Primary assessment of effects upon reproductive systems and of general toxicity; treated from weaning to approximately 13 weeks of age (Day 90).
- Cohort 1B: Follow-up assessment of reproductive performance; treated from weaning to 21/22 weeks of age following breeding at ca. 14 weeks of age.
- Cohort 2A: Developmental neurotoxicity testing (neurobehavioral testing followed by neurohistopathology assessment as adults); treated from weaning up to approximately Day 75 of age.
- Cohort 2B: Developmental neurotoxicity testing; no direct treatment, assigned to neurohistopathology assessment at weaning (Day 21 or Day 22 of age).
- Cohort 3: Developmental immunotoxicity testing; treated from weaning up to Day 60 of age.
F2 animals: No direct treatment; F2 animals had potential indirect exposure in-utero and via milk during lactation.
Frequency of treatment:
Once daily, at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
F0: 24 animals per sex per dose;
F1, Cohort 1A: 20 animals per sex per dose;
F1, Cohort 1B: 24 animals per sex per dose;
F1, Cohort 2A: 10 animals per sex per dose;
F1, Cohort 2B: 10 animals per sex per dose;
F1, Cohort 3: 10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study have been selected based on the findings of the Preliminary Reproductive/Development Toxicity Screening Study in Sprague Dawley Rat by Oral Gavage Administration (Covance Study Number: 8436521).
In that study, F0 males and females were dosed with 100, 300 or 1000 mg/kg/day of the test item by oral gavage for 15 days before pairing until termination either after all litters had been born for the males or at weaning of F1 generation for the females; selected F1 generation animals were dosed at the same dose levels from Day 21 to Day 28 of age. A dosage of 1000 mg/kg/day did not result in any toxicologically significant effects 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. Effects on post-weaning body weight gain was observed for directly dosed F1 offspring but there were no associated effects on survival or clinical condition.
The high dose level for this main extended one generation reproductive toxicity study (EOGRTS; OECD 443) was therefore set at 1000 mg/kg/day. The low and intermediate dose levels were set at 100 and 300 mg/kg/day respectively, to achieve a dose response and/or aid in the determination of a No Observed Adverse Effect Level (NOAEL).

- Rationale for animal assignment (if not random):
F0: Allocation after acclimatization period; By sex; After exclusion of animals showing signs of ill-health; Animals at the extremes of the body weight range were not selected if alternatives were available. At commencement of the study the weight variation did not exceed 20% of the mean weight of each sex.
Selection of offspring to form F1 generation: Selection on Day 18-20 of age. Allocation at weaning on Day 21 of age. Formal start of F1 generation nominally on Day 28 of age (± 2 days of age) (direct dose administration commenced on Day 21 of age).
Where possible, two male and two female offspring were selected from each selected litter for F1 Cohorts 1A and 1B (up to the required number of offspring). For F1 Cohorts 2A, 2B and 3 at least 1 male or 1 female offspring was selected from each selected litter (up to the number required). Selected animals were microchipped on Day 18-21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (where possible 28±2 days of age for selected F1 animals). Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortalities. These spares had body weights and clinical signs monitored weekly and were terminated after commencement of the F1 generation; they did not receive direct treatment unless used as a replacement animal.

- Fasting period before blood sampling for clinical biochemistry (F0 and F1 Cohort 1A generations, 10 animals per sex per group): Overnight deprivation of food. Samples collected under light general anesthesia (isoflurane).
Blood sampling from sublingual vein of F0 and F1 Cohort 1A animals coincided with urine collection, and animals were therefore deprived of water overnight but had access to water for a minimum period of one hour prior to blood sampling.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS (F0 generation and F1 generation):
- Time schedule: At least twice daily for evidence of reaction to treatment or ill-health.
- Deviations from normal recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition.

PHYSICAL EXAMINATIONS:
- Time schedule: Once each week for all F0 and selected F1 generation animals. For F0 and F1 Cohort 1B females on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
- A detailed physical examination was performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals were assessed for physical condition and behavior during handling. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal were recorded with respect to nature, and, where appropriate, degree of severity.

DETAILED CLINICAL OBSERVATIONS (F0 and formal F1 generation animals):
- Time schedule: Week 1 – Daily; Weeks 2 to 4 – twice weekly (middle and end of the week); Week 5 onward – once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 and F1 Cohort 1B females).

BODY WEIGHT (F0 and F1 generation):
- Time schedule for examinations:
F0 Males: Day that treatment commenced; Each week; Before necropsy (at least 10 weeks dosing).
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; Before necropsy.
F1 selected animals: From nominal Week 4 of age, for Cohorts 1A, 1B (males only), 2A and 3: Twice during Week 1 of the F1 generation and weekly thereafter; On the day of attainment of sexual maturation; Before necropsy. For F1 Cohort 1B females: Twice during Week 1 of the F1 generation and weekly thereafter 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 Day 28 of lactation; Before necropsy.

FOOD CONSUMPTION (F0 and F1 generation):
- Time schedule:
F0 animals: Weekly. Food consumption was not recorded for males and females during the period when paired for mating, but for males it was resumed after maiting of all animals was completed. For females after mating, the food consumption schedule coincided with the body weight schedule: Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating; Days 1-4, 4-7, 7-14 and 14-21 of lactation.
F1 selected animals: For Cohorts 1A, 1B (males only), 2A and 3: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
Cohort 1B females: Twice during Week 1 of the F1 generation and weekly thereafter until mating detected; Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating; Days 1-4, 4-7, 7-14 and 14-21 of lactation.

Food consumption was not recorded for Cohorts 1B males and females during the period when paired for mating but was continued for Cohorts 1B males once pairing of all the animals was completed.

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
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.

HEMATOLOGY, PERIPHERAL BLOOD:
F0 and F1 Cohort 1A animals, 10 animals per sex per group. Blood sampling at termination.
Parameters examined: Hematocrit; Hemoglobin concentration; Erythrocyte count; Total leucocyte count; Differential leucocyte count; Platelet count; Mean cell hemoglobin; Mean cell volume; Mean cell hemoglobin concentration; Red cell distribution width; Reticulocytes (absolute and relative counts); Prothrombin time; Activated partial thromboplastin time.
A blood film was prepared for examination at the discretion of the analyst, for samples showing abnormalities in the automated analysis.

BLOOD CHEMISTRY:
F0 and F1 Cohort 1A animals, 10 animals per sex per group. Blood sampling at termination.
Parameters examined: Alkaline phosphatase; Alanine amino-transferase; Aspartate amino-transferase; Gamma glutamyl transpeptidase; Glucose; Bilirubin – total; Cholesterol – total; Creatinine; Urea; Total protein; Albumin - by chemical assay; Albumin/globulin ratio; Sodium; Potassium; Chloride; Calcium; Phosphorus; Bile Acids.

BIOMARKERS - TSH AND T4:
F0: 10 animals per sex per group at termination.
F1 offspring: 10 litters per group – pooled litter sample of pups culled on Day 4 of age (if analysis required, restricted to T4 only). 10 males and 10 females per group on Day 22 of age from as many litters as possible (1 male or 1 female per litter where possible).
F1 Cohort 1A: 10 animals per sex per group at termination.
Conditions: F1 Offspring on Day 4 and Day 22 of age: No overnight deprivation of food. F0 and F1 Cohort 1A animals: Following overnight deprivation of food. Blood sampling of F0 and F1 Cohort 1A animals coincided with urine collection and and animals were therefore deprived of water overnight but had access to water for a minimum period of one hour prior to blood sampling.
Following preparation, serum aliquots were placed on dry ice and stored frozen (-60˚C to -80˚C).

URINALYSIS:
F0 and F1 Cohort 1A animals, 10 animals per sex per group. Overnight urine collection at termination in individual metabolism cages with deprivation of food and water.
Parameters examined: Clarity/color (appearance); Volume; pH; Specific gravity; Glucose; Ketone; Bilirubin (bile pigments); Blood pigments; Protein (total and concentration); Sodium (total and concentration); Potassium (total and concentration); Chloride (total and concentration)
After centrifugation, the pellet was examined microscopically for: Epithelial cells; Leucocytes; Erythrocytes; Crystals; Casts; Spermatozoa and precursors; Other abnormal components
Oestrous cyclicity (parental animals):
F0:
Dry smears: Daily for 15 days before pairing.
Wet smears: Daily after pairing until proof of mating; For four days before scheduled termination (nominally Days 25 to 28 post partum).
Females showing no evidence of mating were separated from the males after completion of the pairing period and vaginal smearing continued for up to 5 days or until the first estrus smear was seen. If a female showed an estrus smear during this period, it was killed as soon as practically possible and subjected to macroscopic examination. If necropsy was not possible on the day of estrus, smears were continued until the morning of necropsy. If a female did not show an estrus smear, wet smears began again on Day 22 after separation from mating (with day of separation = Day 0) for a period of 4 days with the last smear on the morning of necropsy (Day 25 after mating).

F1 Cohort 1A:
Wet smears: Following onset of vaginal patency until the first cornified (estrus) smear was recorded. For at least three days prior to the start of the necropsy phase and on the day of termination.
Dry smears: For 14 days from nominal Day 75 of age (± 2 days of age).

F1 Cohort 1B:
Wet smears: After pairing until evidence of mating confirmed; For at least three days prior to the start of the necropsy phase and on the day of termination.
Females showing no evidence of mating were separated from the males after completion of the pairing period and vaginal smearing continued for up to 5 days or until the first estrus smear was seen. If a female showed an estrus smear during this period, it was killed as soon as practically possible and subject to macroscopic examination. If necropsy was not possible on the day of estrus, smears were continued until the morning of necropsy. If a female did not show an estrus smear, wet smears were continued on Day 22 after separation from pairing (with day of separation = Day 0) for a period of 4 days with the last smear on the morning of necropsy (Day 25 after mating).
Sperm parameters (parental animals):
Parameters examined in F0 males and F1 Cohort 1A males:
- Vas deferens (from left side) – each animal in each group: For all males in all groups, sperm sample (at least 200 spermatozoa, where possible) assessed for motility using a computer assisted sperm analyzer (CASA). A manual assessment of sperm morphology (at least 200 spermatozoa, where possible) was performed. For the F0 males and F1 Cohort 1A males in Groups 2 and 3 a manual assessment of sperm morphology was performed.
- Cauda epididymis (from left side): For all males in all groups, the cauda epididymis was weighed and homogenized and the number of sperm was counted using a computer assisted sperm analyzer (CASA).
- Testis (from left side): For all males in all groups, the testis was homogenized and the number of homogenization-resistant spermatids was counted using a computer assisted sperm analyzer (CASA).
Litter observations:
RECORDS MADE DURING LITTERING PHASE (F0 and F1 Cohort 1B generations):
- 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. 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.
- Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
- Individual offspring body weights: All offspring: Days 1, 4, 7, 14 and 21 of age. Unselected F1: Day 22 of age. Selected F1 (not including Cohort 2B): 23, 25, 27* and 29* of age. (*Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ± 2 days).
- Weaning of offspring: Day 21 of age.
- Ano-genital distance: Offspring on Day 1 of age, corrected for the body weight, using analysis of covariance.
- Nipple count: Male offspring Day 13 of age.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Litters culled to 10 (where possible 5 males and 5 females).
- All culled offspring were macroscopically examined (with thyroid hormone samples collected from 10 F1 litters per group).

SEXUAL MATURATION (All Selected F1 generation animals not including F1 Cohort 2B animals):
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 28 of age until vaginal opening occurs. Body weight recorded on day of vaginal opening.
Females in F1 Cohort 1A: A wet smear was taken daily from the day of vaginal opening until first estrus, and for at least three days prior to the start of necropsy and on the day of termination. A dry smear was taken for 14 days from approximately Day 75 of age.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
F1 Cohort 2A:
Sensory Function – Quantitative:
At approximately Day 24 of age, the animals were tested in an automated system for auditory startle habituation. Startle amplitudes measured over five consecutive blocks of 10 trials (total 50 trials).

Neurobehavioral Screening:
The functional observation battery recordings were performed at approx. the same time of day at approximately Day 63-75 of age (nominally Day 70±1). Not all animals were tested in one day, but the time of testing was balanced across the groups.
- In cage observations: Abnormal motor movements (e.g. fasciculation, convulsions, stereotypy, excessive sniffing, licking, grooming and twitches); Palpebral closure; Posture; Tremor
- In hand observations: Ease of removal from cage; Exophthalmos; Fur condition; Piloerection; Reactivity to handling; Salivation/lacrimation; Vocalization on handling
- Observation in the arena (2-min recording period): Activity count; Arousal; Abnormal motor movements; Fecal count; Gait; Palpebral closure; Posture; Rearing count; Urination; Tremor
- Reactivity investigations: Approach response; Body temperature; Body weight; Grip strength (fore and hindlimb); Landing footsplay; Pupil closure reflex; Righting reflex; Auditory startle reflex; Tail pinch response; Touch response (pinna reflex); Proprioception

Motor Activity:
Between Days 63-75 of age (nominally Day 65±1) motor activity was measured by automated infra-red equipment. High and low beams recorded rearing and cage floor activity, respectively. For testing, designated animals were placed singly into observation cages. The test session for each animal was one hour. Data were automatically collected and reported at regular intervals throughout the session.

F1 Cohort 2B:
No specific investigations in life, animals despatched to necropsy at weaning (Day 21 or Day 22 of age) for histopathological examinations.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
F1 Cohort 3:
T cell dependent antibody response (TDAR) using keyhole limpet hemocyanin (KLH):
- Administration of KLH: Intravenous (bolus) at 300 µg KLH/animal/occassion (0.1 mLvolume per animal) on Day 47 and Day 54 of age.
- Blood sampling for evaluation of IgM response to KLH administration: On Day 46 of age (pre 1st KLH administration), Day 53 and 60 of age (six days after each KLH immunization) from sublingual vein under isoflurane anaesthesis. Blood volume 0.3 mL. Number of blood samples per occasion: Day 46 of age - 80; Day 53 of age - 80; Day 60 of age - 80. Blood samples allowed to clot for 0.5 to 2 hours after collection at room temperature before preparation of single serum aliquots. Serum was placed on dry ice following collection and stored frozen (-60˚C to -80˚C).
- Analysis: Specific anti-KLH IgM antibodies were measured in the Department of Immunology and Immunotoxicology (I&I) using suitable validated methods (Method No. I&I/0090: Method for the Measurement of Anti-KLH IgM in Rat serum). Details are documented in a separate Methodology Validation report.
Postmortem examinations (parental animals):
NECROPSY:
- Time of necroppsy:
F0 adult animals: Females on Day 28 post-partum. Males after at least 10 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating required.
F0 females failing to mate: If an estrus smear was seen following completion of the pairing period animals were terminated as soon as logistically possible. If no estrus smear was seen, animals were terminated on Day 25 after the last day of pairing.
F0 females failing to produce a viable litter and those with total litter loss: Terminated with first cohort of females with live litters.
F1 Cohort 1B animals: Males at approx. 21-22 weeks of age. Females on Day 28 post-partum.
F1 Cohort 1B females failing to mate: If an estrus smear was seen following completion of the pairing period animals were terminated as soon as logistically possible. If no estrus smear was seen, animals were terminated on Day 25 after the last day of pairing.
F1 Cohort 1B females failing to produce a viable litter and those with total litter loss: Terminated with first cohort of females with live litters.
- Macroscopic Pathology:
Complete: All animals, including surplus F1 and F2 offspring culled on Day 4 of age, and unselected F1 and F2 offspring. Where possible, decedent offspring ≤21 days of age (found dead or welfare kill) were examined and carcass retained.
Implantation site count: F0 females and F1 Cohort 1B females.

HISTOPATHOLOGY / ORGAN WEIGHTS:
- Organ weights: For bilateral organs, left and right organs were weighed together unless otherwise specified. Organ weights were not routinely recorded for animals killed or that died prematurely; organ weights were recorded for groups terminated prematurely.
- Fixation (F0 animals, F1 Cohort 1B animals): Standard 10% neutral buffered formalin. Where possible, carcass retained for decedent offspring ≤21 days of age. Testes in modified Davidson’s fluid. Eyes in Davidson’s fluid.
- Histology: See tables.
Postmortem examinations (offspring):
NECROPSY:
- Time of necroppsy:
F1 unselected offspring: On Day 4 and Day 22 of age.
F1 Cohort 1A animals: At approx. 13 weeks of age.
F1 Cohort 2A animals: At approx. Day 75 of age.
F1 Cohort 2B animals: At Day 21/22 of age.
F1 Cohort 3 animals: At approx. Day 60 of age.
F2 offspring: On Day 4 and Day 22 of age.
- Macroscopic Pathology:
Complete: All animals, including surplus F1 and F2 offspring culled on Day 4 of age, and unselected F1 and F2 offspring. Where possible, decedent offspring ≤21 days of age (found dead or welfare kill) were examined and carcass retained.

HISTOPATHOLOGY / ORGAN WEIGHTS:
- Organ weights: For bilateral organs, left and right organs were weighed together unless otherwise specified. Organ weights were not routinely recorded for animals killed or that died prematurely; organ weights were recorded for groups terminated prematurely.
- Fixation:
Unselected F1 and F2 animals (including decedent offspring), F1 animals Cohort 1A and Cohort 3: Standard 10% neutral buffered formalin. Where possible, carcass retained for decedent offspring ≤21 days of age. Testes (F1 animals only) in modified Davidson’s fluid. Eyes in Davidson’s fluid.
F1 generation Cohort 2A and 2B: Standard glutaraldehyde:paraformaldehyde fixation by in situ perfusion followed by immersion. Peripheral nerves: Left nerve specimens were retained in situ in the carcass and could be teased and evaluated by light microscopy if any evidence of neurological change was detected in the resin sections or if neurobehavioral studies indicated evidence of peripheral neuropathy.
- Histology: See tables.

IMMUNOPHENOTYPING OF SPLEEN LEUCOCYTES:
F1 Cohort 1A animals: Ten males and ten females per group from F1 Cohort 1A animals, from as many litters as possible, were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter (i.e. all surviving litters should be represented by at least one offspring). The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological examination. The remaining portions of the spleen were weighed and then placed into a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held on wet ice until processing for analysis. Cell suspensions from each tissue section were prepared by mechanical dissociation, following method IAI/0304 and the samples were stained with a cocktail of directly conjugated monoclonal antibodies. Any contaminating red cells were removed using lysis buffer and the samples were fixed by re-suspension in phosphate buffered saline containing 1% formaldehyde. Samples were analyzed immediately or stored at 2-8°C until analysis. Each sample was analyzed for cell markers using a combination of antibody markers.
Statistics:
DATA TYPES:
The following data types will be analyzed at each timepoint separately, where required, in support of interpretation:
- body weight, using absolute weights and gains over appropriate study periods.
- food consumption, over appropriate study periods.
- estrous cycles, vaginal opening to first estrous and pre-coital interval.
- mating performance and fertility.
- gestation length.
- litter size and survival indices.
- pre-weaning examination (ano-genital distance, surface and righting reflexes)
- sexual maturation, age and body weight at completion.
- clinical pathology (hematology, blood chemistry, urinalysis).
- thyroid hormone analysis (TSH and T4).
- immunophenotyping.
- behavioral data (sensory function, arena rearing and activity counts, grip strength, landing footsplay, body weight, body temperature and motor activity).
- organ weights, both absolute and body weight relative.
- sperm analysis, motility, morphology and counts.

METHODS:
For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group.
For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Under the advice of the Associate Director, Global Statistics, or other qualified Statistician, alternative or additional methods may be carried out if deemed appropriate following data review.
Reproductive indices:
PRE-COITAL INTERVAL:
Individual intervals were tabulated for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 or 13-14 days of pairing.

MATING PERFORMANCE & FERTILITY:
Individual data was tabulated. Group values calculated for males and females separately for the following:
Percentage mating (%) = (# of animals mating / Animals paired) x 100
Conception rate (%) = (# of animals achieving pregnancy / Animals mated) x 100
Fertility index (%) = (# of animals achieving pregnancy / Animals paired) x 100

GESTATION LENGTH & GESTATION INDEX:
Gestation length: Individual values tabulated for the number of days from mating to the start of parturition (inclusive), with half a day subtracted where parturition started overnight. Percentage of animals in appropriate categories tabulated for each group.
Gestation index was calculated for each group as:
Gestation index (%) = (# of live litters born / # pregnant) x 100
Offspring viability indices:
The following were calculated for each litter:
- Post implantation survival index (%) = (Total # of offspring born / Total # of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index (%) = (# of offspring on Day 1 after littering / Total # of offspring born) x 100
- Viability index (%) = (# of live offspring on Day 4 before culling / # of live offspring on Day 1 after littering) x 100
- Lactation index (%) = (# of live offspring on Day 21 after littering / # of live offspring on Day 4 (after culling)) x 100
Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test-item related clinical signs or post-dose observations related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Eight females from the F0 generation were euthanized for welfare reasons or died prematurely during the study. None of the deaths were associated with the administration of the test item:
On Day 14 of treatment, one female (3F No. 249) that received 300 mg/kg/day was euthanized for welfare reasons due to abnormal gait with hind limbs splayed; this animal had also previously shown hunched body posture and whole-body pallor. Macroscopic examination revealed dark and thickened meninges, thickened pericardium, enlargement of the spleen, pancreatic, lumbar and mediastinal lymph nodes and thick pale fluid in the thoracic cavity. The cause of death was considered lymphoma.
On Day 16 of treatment, one female (4F No. 275) that received 1000 mg/kg/day was found dead, this female had no previous clinical observations or observations relating to dose administration. Macroscopic examination revealed a perforated esophagus, clotted blood and pale caseous material in the pericardium, and thickened pericardium. These findings indicated that this female was mal-dosed.
One female (4F No. 281) that received 1000 mg/kg/day was underactive, slightly hunched with irregular breathing and pale eyes prior to dosing on Day 18 of treatment. This animal was not dosed, their condition deteriorated during the day and was therefore euthanized for welfare reasons. Macroscopic examination revealed a perforated esophagus, thickened pericardium and abnormal contents of the pericardium, dark areas of the jejunum and many organs were noted as pale. These findings indicated that this female was mal-dosed.
On Gestation Day 6, one Control female (1F No. 218) was found dead and had macroscopic findings that suggested the death was due to a dosing accident. The findings included a distended esophagus with firm and pale contents and there was abnormal fluid and clotted blood in the heart.
On Day 1 of lactation, one female that received 100 mg/kg/day (2F No. 235) was observed gasping and the offspring were observed as having little or no milk in their stomachs and therefore this female and the litter were killed for welfare reasons. Macroscopic findings included a pale and inactive mammary gland, and the jejunum was pale in colour; no specific cause of death was established at histopathology.
One female receiving 1000 mg/kg/day (4F No. 284) was found dead in the cage, partly cannibalised on Day 24 of lactation. The only macroscopic finding for this female was that all lobes of the lungs were described as firm all other findings were related to the damage caused by the cannibalisation. This female was in good clinical condition and has no signs observed related to the dosing procedure in days prior to the death, a cause of death was not established at histopathology.
One female receiving 100 mg/kg/day (2F No 236) was euthanized on Day 49 and one female receiving 1000 mg/kg/day (4F No. 276) was euthanized on Day 44 due to failure to litter, both females were found to be not pregnant.
All other F0 animals survived to their scheduled sacrifice.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Overall mean body weight gain for males at all dose levels was unaffected by treatment.
Body weight gains for females prior to pairing and during the gestation period were unaffected by test item administration.
Group mean body weight gain for females receiving 300 or 1000 mg/kg/day were statistically significantly higher during Days 7 to 14 of lactation, when compared to controls. As a result, overall weight gain during Days 1 to 21 of lactation was statistically significantly higher at 1000 mg/kg/day. Conversely, a statistically significantly greater mean body weight loss was seen in females receiving 1000 mg/kg/day during Days 21 to 28 of gestation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption for males receiving 1000 mg/kg/day prior to pairing was generally slightly higher compared to controls with differences attaining statistical significance during Weeks 5 and 7 to 9 of treatment, resulting in the overall mean being statistically significantly higher compared to controls (1.06X control). Mean food consumption for males receiving 100 or 300 mg/kg/day was similar to controls. Mean food intake for females at all dose levels before pairing and during gestation and lactation was unaffected by treatment.
Food efficiency:
no effects observed
Description (incidence and severity):
The efficiency of food utilization for both males and females during the two week treatment period before pairing was unaffected by administration of the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological investigations prior to the termination of the F0 generation revealed statistically significantly lower lymphocyte, eosinophil and basophil counts compared with controls (0.75X, 0.58X and 0.57X control, respectively) in males receiving 1000 mg/kg/day.
Males receiving 1000 mg/kg/day had statistically significantly low mean haematocrit (0.95X control), reticulocyte counts (0.72X control) and mean cell volume (0.95X control) compared with controls; mean cell volume was also significantly low among males at 300 mg/kg/day and females at 1000 mg/kg/day (0.96X and 0.97X control, respectively).
All other hematological differences from control observed during the treatment period were generally minor, confined to one sex or lacked dose relationship and were therefore attributed to normal biological variation. These included slight reductions in mean corpuscular haemoglobin concentration and red cell distribution width in females receiving 1000 mg/kg/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Biochemical analysis of the plasma at scheduled termination of the F0 animals revealed, when compared to controls, statistically significantly slightly higher mean urea concentrations in males receiving 1000 mg/kg/day (1.15X control).
All other biochemical differences from controls observed at scheduled termination were minor, limited to one sex and lacked a clear dose relationship and were therefore attributed to normal biological variation; this included a slight increase in sodium concentration in males at 300 mg/kg/day.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean T4 serum concentrations in F0 males given 1000 mg/kg/day was statistically significantly lower compared to controls although in absence of dose response. No other statistically significant difference in T4 concentrations was observed between the control and treatment groups.
Mean serum TSH concentrations were statistically significantly higher compared to controls in all groups of F0 males (2.19X, 2.18X and 2.65X control, at 100, 300 or 1000 mg/kg/day, respectively) although there was no dose response apparent; a similar difference was not apparent in F0 females.
Since these differences were slight and not accompanied by histological alterations in the thyroid, they were not considered adverse. Additionally, the majority of values were within the historical control data (HCD) range of the rat strain used, with only one F0 male TSH concentration (4M No. 82) outside the HCD range.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females given 1000 mg/kg/day had a statistically significantly higher total protein concentration compared with controls (approximately 1.45X control) and urinary pH was significantly lower; total chloride concentration was also significantly higher in females given 1000 mg/kg/day (1.42X control).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Findings related to the administration of the test item were present in the kidneys of F0 females and the thymus and epididymis of F0 males:
There was a higher incidence and severity of basophilic tubules in the kidneys of females given 1000 mg/kg/day, with more of multifocal distribution. This correlated with the higher organ weight at necropsy. The females given 100 or 300 mg/kg/day showed a slightly higher incidence of basophilic tubules when compared to contemporaneous controls. Whilst no increase in severity of the finding was present, in those given 300 mg/kg/day the distribution was multifocal similar to those given 1000 mg/kg/day. There was no true dose relationship established and the severity was minimal thus the significance of this change in females given 300 mg/kg/day is considered equivocal and in those given 100 mg/kg the severity and distribution were considered similar to controls and not histopathologically significant.
In males there was cellular debris present in the epididymis in those given 1000 mg/kg/day. Whilst there was no correlating change within the testes making interpretation of this change unclear, there were alterations in the sperm analysis indicating adverse changes in the male reproductive system. No change was present in males given 100 or 300 mg/kg/day.
There was a slightly higher incidence of decreased lymphocytes in the cortex of the thymus of males given 1000 mg/kg/day. This correlated with a slightly lower weight at necropsy but was minor, of low incidence and therefore of equivocal significance. No change was present in males given 100 or 300 mg/kg/day.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Mineralisation in the aorta was present only in females given 1000 mg/kg/day but this is seen as a background finding of variable incidence in Sprague Dawley rats in this laboratory and was considered to be incidental.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Five females receiving 1000 mg/kg/day exhibited irregular estrous cycles compared to one female exhibiting an irregular cycle in the controls. This incidence of females not showing regular estrous cycles had no effect on the pre-coital interval, with the majority of females at all dose levels showing positive evidence of mating within the four days of pairing. Estrous cycles of the F0 females at termination were unaffected by treatment.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, the percentage of motile and progressively motile sperm was lower (motile sperm: 81% versus 94% in controls), total testicular spermatid number was higher (221 millions/g versus 187 millions/g in controls), and all motion parameters assessed were statistically significantly lower with the exception of medium and static sperm, percentage of slow sperm which were higher when compared to controls. Sperm morphology was also affected for males given 1000 mg/kg/day with a higher percentage of abnormal sperm observed 20.1% compared to 2.2% in controls, included abnormalities such as decapitated sperm (13.3%), abnormal head (3.5%), abnormal tail (3.6%), abnormal midpiece (2.2%) and flat head (3.4%).
Sperm motility, counts and morphology were unaffected for males that received 300 mg/kg/day although numerous motion parameters (VAP, VCL, ALH, STR and LIN) were statistically significantly lower compared to controls.
At 100 mg/kg/day, as with 300 mg/kg/day but to a lesser extent, some motion parameters (VAP, VCL and ALH) were statistically significant lower compared to controls. Sperm morphology and counts were unaffected by test item administration.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital interval was unaffected by test item administration. All mating pairs showed positive evidence of mating within six days of pairing.
Mating performance and fertility were unaffected by the test item. The percentage mating was 100% in all groups, and conception rate and fertility index were 95-100 % in all groups; one F0 female given 100 mg/kg/day (2F No. 236) and one F0 female given 1000 mg/kg/day (4F No. 276) were not pregnant. In addition, for one control female found dead on Day 6 of gestation pregnancy status could not be determined.
A slight, but statistically significant, shift to gestation lengths was apparent for females given 1000 mg/kg/day compared to controls. The expected gestation length in CD rats ranges from 22 to 23 days. In the 1000 mg/kg/day group, more females had a 23-day gestation length, one female showed a 23.5-day gestation length and another female showed a 24-day gestation length. There was no effect of treatment on gestation index up to and including 1000 mg/kg/day, with all pregnant females successfully giving birth to live pups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive function (sperm measures)

Results: P1 (second parental generation)

General toxicity (P1)

Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the F1 generation males lower T4 serum concentrations were observed in males that received 300 or 1000 mg/kg/day, with the difference from control attaining statistical significance. No other statistically significant difference in T4 concentrations was observed between the control and treatment groups.
There was no effect of test item administration on mean serum TSH concentration in F1 offspring on Day 22 of age or in Cohort 1A animals when compared to controls.
Since these differences were slight and not accompanied by histological alterations in the thyroid, they were not considered adverse. Additionally, the majority of values were within the historical control data (HCD) range of the rat strain used, with only one F1 male T4 concentration (4M No. 476) outside the HCD range.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
F1 generation (Cohort 1B):
Limited tissues were examined from all Cohort 1B males and females from Group 1, 3 and 4 due to a significant reduction in the number of females producing and maintaining litters in animals given 1000 mg/kg/day. In those females given 1000 mg/kg/day eleven animals failed to litter and a further animal did not mate. Of those females given 1000 mg/kg/day littering as expected, four suffered total litter losses resulting in only eight with litters surviving to completion of the study. Microscopically there were no consistent changes present to account for the differences. Of those animals failing to litter one had involuting implantation site(s) (Animal 956), another had a large necrotic fetus present in the uterus (Animal 961), one had inflammation in the uterus and cervix which may have affected its ability to conceive or maintain pregnancy (Animal 960) and another had no corpora lutea present in the ovaries (Animal 971) suggesting abnormal cyclical activity. No histological changes were present in the remaining seven. Of the eight females with viable litters at study completion there was a higher weight of the uterus/cervix/oviducts which correlated with a higher proportion with estrus morphology in the vagina. It is not clear, due to the comparatively small pool of animals given 1000 mg/kg/day remaining, if there is any disruption of resumption of normal cyclical activity.
In males, findings related to the administration of the test item were present in the testes and epididymis of males. Cellular debris in the epididymis was present in most males given 1000 mg/kg/day and occasional males given 300 mg/kg/day. There was minimal degeneration of occasional cells in the seminiferous tubules of males given 1000 mg/kg/day indicating progression of the findings seen in the F0 males and F1A males. The F1A males received the test item for a shorter duration of time which may account for the difference.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous Cycles - Cohort 1A:
A slightly higher number of females showing irregular cycles was observed in F1 Cohort 1A females compared to controls, with two females showing irregular cycles in females given 300 or 1000 mg/kg/day and three females exhibited irregular cycles at 100 mg/kg/day compared to no control females showing irregular cycles. Furthermore, one female in both the 300 and 1000 mg/kg/day groups were acyclic. As the majority of females receiving the test item showed 4-, 4/5- or 5-day cycles, and as no dose relationship was apparent, it is considered that estrous cycle regularity of the Cohort 1A females during the last two weeks of treatment was unaffected by test item administration. In addition, estrous cycle at termination for these females were unaffected at all dose levels investigated.

Stage of Estrous Cycle at Termination - Cohort 1B:
The majority of females that received 100, 300 or 1000 mg/kg/day were showing estrous morphology at termination.

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A:
At scheduled termination of the Cohort 1A females, ovarian follicle counts were slightly lower in females given 1000 mg/kg/day when compared to Controls. There was no similar decrease in the number of corpora lutea present.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Sperm Assessment - Cohort 1A:
At 1000 mg/kg/day, the percentage of motile and progressively motile sperm was lower (90% versus 95% in controls for motile sperm), total epididymal sperm count was lower (103 millions/g versus 140 millions/g in controls) and VAP, VSL, VCL, ALH and Rapid motion parameters assessed were statistically significantly lower and medium, slow and static sperm were significantly higher than in controls. Sperm morphology was also affected for males given 1000 mg/kg/day with a higher percentage of abnormal sperm observed 15.8 % compared to 2.8% in controls, included abnormalities such as decapitated sperm (6.4%), abnormal head (7.1%), abnormal tail (3.3%), and flat head (6.7%).
Sperm motility and morphology were unaffected for males that received 300 or 100 mg/kg/day although epididymal sperm counts were slightly lower for both groups but were within the historical control data (HCD) range.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Pre-Coital Interval - Cohort 1B:
The pre-coital interval for all F1 Cohort 1B females receiving 100 mg/kg/day including controls was within 1 to 4 days. Among females receiving 300 mg/kg/day, two females had pre-coital interval of 5 to 8 days and one female had a pre-coital interval of 9 to 12 days, the remaining 21 females had pre-coital interval of 1 to 4 days. A similar trend was observed in F1 Cohort 1B females that received 1000 mg/kg/day with the majority of females (21) having a pre-coital interval of 1 to 4 days, two females had longer pre-coital intervals with one female having a 5 to 8 days pre-coital interval and one female having a 13 to 14 days pre-coital interval. As the majority of females receiving the test item showed a positive evidence of mating within a 4-day period it is considered that these low incidences of extended pre-coital intervals are due to normal biological variation and therefore pre-coital interval is unaffected by test item administration at all dose levels investigated.

Mating Performance and Fertility - Cohort 1B:
The conception rate and fertility index were statistically significantly lower compared to controls, at 88% or 57% for conception rate and 88% or 54% for fertility index, for F1 Cohort 1B females that received 300 or 1000 mg/kg/day, respectively. At 300 and 1000 mg/kg/day, 24 and 23 females were confirmed to have mated with 21 and 13 females achieving pregnancy, respectively. Mating performance and fertility of the Cohort 1B animals given 100 mg/kg/day were unaffected by test item administration.

Gestation Length and Gestation Index - Cohort 1B:
A slight, but statistically significant, shift in gestation length was apparent for females given 300 and 1000 mg/kg/day compared to controls. The expected gestation length in CD rats ranges from 22 to 23 days. In the 300 mg/kg/day group, three females showed a 23.5-day gestation length compared to two females given 100 mg/kg/day and one control female. Since this difference was very slight, it was considered non-adverse. In the 1000 mg/kg/day group, three females showed a 23.5-day gestation length, four females showed a 24-day gestation length and two females showed a 24.5-day gestation length. Gestation index was unaffected by test item administration.

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed among the F1 offspring that were considered to be related to parental treatment with the test item.
There were no signs associated to dose administration and there were no test item-related changes in general clinical condition among F1 males and females of all cohorts throughout the study, at any dose level investigated.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
F1 Litter Responses:
Mean number of uterine implantations and Day 1 total or live litter sizes were essentially similar to the control group for females that received 1000 mg/kg/day; however, Day 4 viability index (%) was lower, as offspring deaths during the first four days of lactation occurred in 9/21 litters in females given 1000 mg/kg/day compared to offspring deaths in 2/23 litters in controls during the first four days of lactation and the difference attained statistical significance. Sex ratio for the offspring did not indicate any selective effect on survival for either sex. Offspring survival after culling of litters on Day 4 to weaning was similar to control.
There was no effect of parental treatment with the test item at 100 or 300 mg/kg/day on the mean number of uterine implantations, Day 1 total and live litter size, post-birth offspring survival, or sex ratio.

F1 Generation:
Increased premature euthanasia related to the test item administration was seen in females, due to reduced fertility in the F1 generation in Cohort 1B. At 1000 mg/kg/day, twelve animals were euthanized early, eleven due to failure to litter and one which failed to mate. A further four were euthanized due to total litter loss. One control female (1F No. 887) was euthanized for welfare reasons on Day 42 after the formal commencement of F1 treatment and this was due to a liver lobe torsion. One female given 300 mg/kg/day (3F No. 947) was euthanized on Day 13 of gestation due to a mammary tumor. In addition, three females given 300 mg/kg/day failed to litter where one female (3F No. 938) was paired with a male (3M No. 542) with notable tubular atrophy in the testes which would have resulted in infertility. The remaining two females had no obvious cause for the lack of litter but at this low incidence it was considered incidental and not related to treatment. One female that received 100 mg/kg/day (1F No. 921) was euthanized for welfare reasons on Day 14 of lactation. From Day 10 of lactation onwards the animal had decreased activity, thin build, rapid breathing, piloerection, hunched posture and its perigenital area was dark with a firm superficial mass present that ruptured on Day 14 of lactation. Macroscopic examination of the animal found that the adrenal glands had dark areas, the lungs and bronchi had pale areas, there were depressions on the tongue and ruptured masses present on the mammary tissue in the genital region.
Two further premature deaths occurred in the F1 generation; however these deaths were not attributable to test item administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring Body Weight:
The group mean body weight of male and female F1 offspring on Day 1 of age, and subsequent body weight gain of the offspring to weaning on Day 21 of age, was unaffected by parental treatment with the test item at all dose levels investigated.

F1 Generation Body Weight:
A slight reduction in body weight and body weight gain was recorded from weaning until Day 25 of age for males and females given 1000 mg/kg/day. From the formal start of the F1 generation, body weight gains in males only receiving 1000 mg/kg/day continued to be reduced during the first 4 weeks of the F1 generation (0.89X control from Day 1 to 29 of the formal start of F1 generation); thereafter there was no conclusive or consistent effect of treatment. Body weight gains of males and of females before pairing were unaffected by treatment at 100 or 300 mg/kg/day.
Mean body weight gain for the F1 Cohort 1B females that received 1000 mg/kg/day was statistically significantly lower compared to control during the gestation period (0.57X control). Group mean body weight gain for the F1 Cohort 1B females given 100 or 300 mg/kg/day during gestation and all dose levels investigated during lactation was essentially similar to controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The overall mean food consumption value for males that received 1000 mg/kg/day was slightly lower (0.95X control) compared to controls, this difference attained statistical significance. Mean food intake values for individual periods for males given 1000 mg/kg/day were low at the start of the treatment and towards the end of the treatment period showed slightly higher mean intake values compared to controls. As these differences were minor, lacked consistency and showed no dose response relationship, they were attributed to normal biological variation. Food consumption for males at 100 or 300 mg/kg/day was unaffected by treatment.
Food consumption was statistically significantly lower (0.92X control during Days 1 to 4) than control for females at 1000 mg/kg/day for the first week of treatment (Days 1-8), thereafter food consumption was comparable with control. Overall food consumption was unaffected at any dose level for F1 females or for F1 Cohort 1B females before pairing and during gestation.
Mean food consumption for females that received 1000 mg/kg/day was statistically significantly lower compared to control for the lactation period (0.75X control). Mean food consumption for females at 100 or 300 mg/kg/day was unaffected by treatment during lactation.
Food efficiency:
no effects observed
Description (incidence and severity):
Food conversion efficiency for F1 Cohort 1B animals prior to pairing was unaffected by test item administration.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematology - Cohort 1A:
Haematological investigations prior to the termination of the F1 Cohort 1A animals revealed slightly lower mean cell hemoglobin, mean cell volume and red cell distribution width in males and females that received 1000 mg/kg/day when compared with controls, with all differences attaining statistical significance. Mean cell hemoglobin was lower in males and females receiving 300 mg/kg/day, and mean cell volume was lower in females given 300 mg/kg/day, with all differences from control attaining statistical significance. Red blood cell concentration was slightly higher (1.05X control) for females that received 1000 mg/kg/day when compared to controls.
All other differences from control were minor, limited to one sex or lacked a dose response relationship and were therefore attributed to normal biological variation, such as the low eosinophil counts among males at 1000 mg/kg/day and high leukocytes and lymphocytes in females that received 300 mg/kg/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood Chemistry - Cohort 1A:
Biochemical analysis of the plasma at scheduled termination of the F1 Cohort 1A animals revealed, when compared to controls, slightly higher mean urea concentrations in males and females receiving 1000 mg/kg/day (1.22X and 1.14X control, for males and females, respectively). These differences attained statistical significance, but no dose relationship was apparent in both sexes.
Mean plasma bile acids were moderately higher and achieved statistical significance in all groups of treated females although in the absence of a dose response relationship.
All other biochemical differences from controls observed at scheduled termination were minor or lacked clear dose relationship or were confined to one sex and therefore attributed to normal biological variation. These differences included slightly high gamma-glutamyl transferase activity in males at 100 mg/kg/day and statistically significant differences in glucose concentration were conflicting for males and females at 1000 mg/kg/day, mean glucose concentration were also low for males receiving 100 or 300 mg/kg/day, but a dose relationship was not apparent.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinalysis - Cohort 1A:
Analysis of the clarity/color and composition of the urine of F1 Cohort 1A animals prior to scheduled termination revealed, when compared to controls, statistically significantly lower pH in males and females in the 1000 mg/kg/day group. Males in this dose group also showed slightly lower urinary protein, sodium, potassium, and chloride concentrations. Microscopy of the urine sediment did not reveal any test item-related changes.
Sexual maturation:
no effects observed
Description (incidence and severity):
The age and body weight at which F1 females attained vaginal opening in all treatment groups, and at which F1 males at 100 or 300 mg/kg/day attained balano preputial separation was unaffected by test item administration.
The body weight at completion of balano preputial separation was statistically significantly lower for F1 males that received 1000 mg/kg/day when compared to controls, however the age of completion was similar to controls.

Vaginal Opening - Cohort 1A: There was no effect of test item administration on the duration between vaginal opening and the first estrus occurring in the Cohort 1A females.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance of F1 offspring at dose levels up to 1000 mg/kg/day on Day 1 of age was unaffected by 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):
Unselected Offspring:
There was no clear effect of parental test item administration on the weights of the brain, liver, spleen or thymus of the unselected F1 offspring killed on Day 22 of age.
It was noted that at 1000 mg/kg/day there was a statistically significantly lower absolute brain, spleen and thymus weight in males and lower absolute brain weight in females. At 300 mg/kg/day there was a statistically significantly lower absolute brain weight in females. After adjustment for body weight the thymus weight of males was lower at 1000 mg/kg/day but the variation was not statistically significant. After adjustment for body weight the spleen weight was lower for males but higher for females, neither change reaching statistical significance. The brain weight showed no variation from contemporaneous control animals after adjustment for body weight.
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

F1 generation (Cohorts 1A and 1B):
In Cohort 1A there were several statistically significant lower absolute organ weights, particularly in males, which were considered likely to be related to the lower body weight in animals given 1000 mg/kg/day. In males, lower weights were present in the brain, epididymis, pituitary, spleen, thymus, thyroids and parathyroids and the prostate. After adjustment for body weight there was a statistically significant higher weight in the heart, kidneys and seminal vesicles with coagulating gland. There was a statistically significant lower weight in the pituitary and thymus. In Cohort 1B there was a statistically significant lower absolute pituitary and epididymides weight in males given 1000 mg/kg/day. After adjustment for body weight there was still a marginal, although not statistically significant, lower pituitary weight. After adjustment for body weight there was a statistically significant higher seminal vesicles with coagulating gland weight. No changes were noted at pathology in the seminal vesicles and the variation occurred only in this cohort, thus this is considered to be incidental.
In F1A females given 1000 mg/kg/day lower absolute weights were present in the adrenal glands, brain, ovaries and pituitary. After adjustment for body weight there was a statistically significant higher weight in the kidneys and lower weight in the pituitary. In the F1B females given 300 and 1000 mg/kg/day there was a statistically significant lower pituitary weight and higher uterus, cervix and oviduct weight, both absolute and body weight adjusted. The higher weight of the uterus/cervix/ovaries is likely to be related to cyclical activity with an increased proportion of females in estrus compared to control animals, however the low number of surviving females given 1000 mg/kg/day makes interpretation complex thus relationship to treatment is equivocal.
Whilst there were no histopathology correlates for any of the organ weight changes in the F1 Cohort 1A animals, the lower weight of the thymus and pituitary and higher weight of the kidneys were consistent with the F0 animals and thus were considered to be related to the administration of the test item.

F1 generation (Cohorts 2A and 2B):
There was a marginally lower brain weight, both absolute and relative to body weight in animals given 1000 mg/kg/day in both Cohorts 2A and 2B.

F1 generation (Cohort 3):
No changes were apparent in the weight of the spleen from Cohort 3 animals which could be related to the administration of the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The predominant macroscopic observation detected in the F1 offspring that died prior to scheduled termination was the absence of milk in the stomach.
There were no macroscopic abnormalities detected among the unselected F1 offspring killed at scheduled termination on Day 22 of age.

The macroscopic examination performed at scheduled termination of F1 Cohort 1A, 1B, 2A, 2B and 3 animals revealed no test item-related lesions. The incidence and distribution of all findings were considered to be unrelated to treatment.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 generation (Cohort 1A, 2A and 2B):
In the F1 Cohort 1A findings related to the administration of the test item were present in the epididymis of males. There was minimal cellular debris present in the epididymis in those given 1000 mg/kg/day. Whilst there was no correlating change within the testes making interpretation of this change unclear, there were alterations in the sperm analysis indicating adverse changes in the male reproductive system. No change was present in males given 100 or 300 mg/kg/day. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
No changes related to the administration of the test item were seen in the brain and nerves, including both paraffin and resin sections from Cohort 2A and the brain sections from Cohort 2B including brain morphometry from both cohorts.
All other 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 this strain. Therefore, they were considered not test article related.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Auditory Startle Response Habituation - Cohort 2A:
Auditory startle response habituation for F1 Cohort 2A animals at Day 24±1 of age, both in terms of latency to peak amplitude values and peak amplitude values, was unaffected by treatment with the test item at all dose levels investigated. The group mean percent habituation values in both sexes at all dose levels was considered to be unaffected by test item administration. There was no effect on the latency to the peak response or on the amplitude of the peak response during the initial 10 trials, only one individual block of 10 trials for mean latency to peak attained statistical significance in males given 1000 mg/kg/day during trials 21-30 which was marginally higher compared to controls.

Motor Activity - Cohort 2A:
The locomotor activity of Cohort 2A F1 animals on nominal Day 65±1 of age was unaffected by test item administration. One difference from the control group attained statistical significance in treated females, with a lower number of low beam breaks recorded during the 36-minute test for females given 300 mg/kg/day. The overall total number of high and low beam breaks during the 1-hour test was unaffected, and therefore this difference was considered incidental and unrelated to test item administration.

In-Cage Observations - Cohort 2A:
A detailed functional observational battery was performed for all Cohort 2A animals on nominal Day 70±1 of age. There were no test item-related changes evident in the behavior of the Cohort 2A animals during the in-cage observations.

In-Hand Observations - Cohort 2A:
During the in-hand observations there were no findings observed which represented neurological changes related to administration of the test item.

Arena Observations - Cohort 2A:
The 2-minute arena observation of the Cohort 2A animals did not reveal any neurological changes in the Cohort 2A animals at any dose level investigated.

Reactivity Investigations - Cohort 2A:
Reactivity investigations of the Cohort 2A animals revealed, when compared to controls, statistically significantly lower mean body weight in males given 1000 mg/kg/day and a statistically significantly lower hind limb grip strength in males in the 1000 mg/kg/day, although in the absence of a dose relationship.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
T-Cell Dependent Antibody Response (F1 Cohort 3):
No treatment-related differences in IgM responses were observed in F1 Cohort 3 male and female rats when the test item was administered at 100, 300 and 1000 mg/kg/day when compared to the control. Apparent differences, observed when comparing mean IgM levels in some of the treated groups to the control group, were considered to represent normal variation based on the high variability in the individual results and the lack of a clear dose-related response.

Spleen Cell Immunophenotyping (F1 Cohort 1A):
Statistically significantly lower male NK cell cells/spleen were observed at doses 300 mg/kg/day and 1000 mg/kg/day when compared to the control group. Statistically significantly lower female WBC, lymphocyte, T cell (including CD4+ and CD8+ T cells), NK cell and monocyte cells/spleen were observed at dose 1000 mg/kg/day when compared to control group, as well as a statistically significantly lower female NK cell and monocyte cells/spleen at dose 300 mg/kg/day when compared to the control group. A statistically significantly lower female monocyte percentage was observed at dose 1000 mg/kg/day when compared to the control group.
Whilst the aforementioned statistically significant differences were observed in Sprague-Dawley rat spleen leukocytes, these differences were not related to the oral gavage administration of the test item as the differences were slight and the variation observed in the immunophenotyping cell percentage and cells/spleen parameters at doses 300 mg/kg/day and 1000 mg/kg/day were within the ranges observed in the control animals.
There were no further observable or statistically significant effects on the immunophenotyping parameters measured in Sprague-Dawley rat spleen leukocytes as a result of the oral gavage administration of the test item.

Spleen Weight (F1 Cohort 3):
No changes were apparent in the weight of the spleen from Cohort 3 animals which could be related to the administration of the test item.

Details on results (F1)

As two F0 females (F0 female No. 236 given 100 mg/kg/day and F0 female No. 276 given 1000 mg/kg/day) were not pregnant, three F0 females died prematurely and all deaths were related to mis-dosing (1F No. 218, 4F No’s. 275 and 281) and two additional F0 females were killed for welfare reasons (2F No. 235 and 3F 249), the assessment of F1 litter responses is based on 23, 22, 23 and 21 litters at 0, 100, 300 and 1000 mg/kg/day, respectively.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed among the F2 offspring that were considered to be related to parental treatment with the test item.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Litter Size, Survival Indices and Sex Ratio: The mean number of implantation sites, litter size, post-implantation survival and live birth index were all statistically significantly lower than controls in the 1000 mg/kg/day group (implantation sites: 7.9 versus 15.1 in controls; litter size: 7.2 versus 14.3 in controls; post-implantation survival: 84.8% versus 94.1% in controls). Mean litter size on Day 1 to Day 4 of lactation before the cull was lower compared to controls, litter size remained stable following the Day 4 cull to the Day 21 of age. The lower Viability index reflected one total litter loss and pup mortality in two other litters.
There was no effect on post-implantation survival, litter size or offspring survival from Day 1 of lactation at 100 or 300 mg/kg/day. Offspring sex ratio was unaffected by test item administration at all dose levels investigated.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The group mean body weight of male and female F2 offspring on Day 1 of age, and subsequent body weight gain of the offspring to weaning on Day 21 of age, was unaffected by parental treatment with the test item for males at all dose levels investigated and for females at 100 or 300 mg/kg/day. For female offspring at 1000 mg/kg/day, overall weight gain was slightly lower at 90% of controls; in the absence of any effect on male sibling weight gain an effect of treatment is considered highly unlikely.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, mean ano-genital distance was slightly longer in male and female F2 offspring, both differences attained statistical significance. There was no effect of treatment on the ano-genital distance of F2 offspring in the 100 or 300 mg/kg/day groups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
All F2 male offspring were assessed on Day 13 of age for the presence of nipples; no nipples were observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The predominant macroscopic observation detected in the F2 offspring that died prior to scheduled termination was the absence of milk in the stomach. There were no macroscopic abnormalities detected among the F2 offspring killed at scheduled termination on Day 21 of age.

Details on results (F2)

Eleven Cohort 1B females in the 1000 mg/kg/day group (No’s. 953, 955, 956, 960, 961, 965, 969, 971, 973, 975 and 976) and three Cohort 1B females in the 300 mg/kg/day group (No’s. 931, 938 and 943) failed to litter, one female given 1000 mg/kg/day (No. 964) failed to mate, one Control female (No. 887) was killed for welfare reasons prior to pairing and one female given 300 mg/kg/day (No. 947) was killed for welfare reasons on Day 13 of gestation. In addition, three females given 1000 mg/kg/day (No’s. 958, 959, 966 and 970) had a total litter loss on Days 4, 1, 1 and 6 of lactation, respectively. Therefore, the assessment of F2 litter responses is based on 23, 24, 20 and 8 litters at 0, 100, 300 and 1000 mg/kg/day, respectively.

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Any other information on results incl. tables

Summary of Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F0)
































































































































































 

Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



300



1000



0



100



300



1000



Kidneys



 



 



 



 



 



 



 



 



Absolute Weight (g)



3.76



102



97



103



2.15



97



102



109**



Body Weight Relative (%)



0.67



100



100



106*



0.74



96



101



106**



Testes



 



 



 



 



 



 



 



 



Absolute Weight (g)



3.70



107



103



107**



 



 



 



 



Body Weight Relative (%)



0.66



104



105



110*



 



 



 



 



Thymus



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.25



109



95



80*



0.26



98



109



97



Body Weight Relative (%)



0.044



107



97



83*



0.090



97



109



95



Pituitary



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.014



100



100



100



0.017



94



100



94*



Body Weight Relative (%)



0.0026



92



96



100



0.0059



93



97



90*



* = Statistically significant difference (absolute or relative) compared with respective control mean value
*p 0.05; **p 0.001.


Note: Values for absolute weight and body weight adjusted weight for dosed groups expressed as percentage control mean value.



 


Summary of Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice (F0)
























































































































































































































































 

Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



300



1000



0



100



300



1000



Kidneys



 



 



 



 



 



 



 



 



Number Examined



24



0



2



24



23



22



23



20



Basophilia, Tubular, Focal



 



 



 



 



 



 



 



 



Minimal



7



0



1



5



2



5



1



3



Slight



0



0



0



0



0



0



0



1



Basophilia, Tubular, Multifocal



 



 



 



 



 



 



 



 



Minimal



1



0



1



2



1



0



5



1



Slight



0



0



0



0



0



1



0



3



Moderate



0



0



0



0



0



0



0



1



Total Incidence of Basophilic  Tubules



8



0



2



7



3



6



6



9



Epididymides



 



 



 



 



 



 



 



 



Number Examined



24



24



24



24



NA



NA



NA



NA



Debris, Cellular



 



 



 



 



 



 



 



 



Minimal



0



0



0



10



 



 



 



 



Total



0



0



0



10



 



 



 



 



Thymus



 



 



 



 



 



 



 



 



Number Examined



24



24



24



24



23



0



0



20



Lymphocytes Decreased, Cortical



 



 



 



 



 



 



 



 



Minimal



1



2



1



0



0



0



0



2



Slight



1



0



0



7



0



0



0



0



NA = Not applicable



 


Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 1A)































































































































 

Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



300



1000



0



100



300



1000



Kidneys



 



 



 



 



 



 



 



 



Absolute Weight (g)



3.23



104



107



104



1.86



100



102



103



Body Weight Relative (%)



0.67



103



104



113**



0.70



101



99



105*



Thymus



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.40



96



99



78**



0.32



112



112



94



Body Weight Relative (%)



0.083



96



96



85**



0.12



113



110



96



Pituitary



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.013



100



100



85**



0.015



107



100



80**



Body Weight Relative (%)



0.0028



96



96



86**



0.0058



103



93



81**



NA = Not applicable.


* = Statistically significant difference (absolute or relative) compared with respective control mean value
*p 0.05; **p0.001.


Note: Values for absolute weight and body weight adjusted weight for dosed groups expressed as percentage control mean value.



 


Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 1B)
































































































































































 

Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



300



1000



0



100



300



1000



Pituitary



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.015



100



93



80**



0.018



100



89*



72**



Body Weight Relative (%)



0.0025



100



100



92



0.0058



100



90*



76**



Seminal Vesicles with Coagulating Gland



 



 



 



 



 



 



 



 



Absolute Weight (g)



2.31



105



95



107



NA



NA



NA



NA



Body Weight Relative (%)



0.40



105



96



117**



NA



NA



NA



NA



Epididymis



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.49



103



99



90**



NA



NA



NA



NA



Body Weight Relative (%)



0.25



103



101



98



NA



NA



NA



NA



Uterus, Cervix and Oviducts



 



 



 



 



 



 



 



 



Absolute Weight (g)



NA



NA



NA



NA



0.64



102



126*



135**



Body Weight Relative (%)



NA



NA



NA



NA



0.21



102



127*



140*



NA = Not applicable.


* = Statistically significant difference (absolute or relative) compared with respective control mean value
*p 0.05; **p0.001.


Note: Values for absolute weight and body weight adjusted weight for dosed groups expressed as percentage control mean value.



 


Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 2A)





























































 

Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



300



1000



0



100



300



1000



Brain



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.99



98



99



95*



1.85



100



99



93*



Body Weight Relative (%)



0.456



99



100



105



0.744



104



99



93



* = Statistically significant difference (absolute or relative) compared with respective control mean value
*p 0.05; **p0.001.


Note: Values for absolute weight and ratio of organ weights (relative to body) for dosed groups expressed as percentage control mean value.



 


Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 2B)






























































 



Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



300



1000



0



100



300



1000



Brain



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.47



103



98



94



1.43



101



105



96



Body Weight Relative (%)



2.86



98



97



93



2.75



96



103



99



Note: Values for absolute weight and ratio of organ weights (relative to body) for dosed groups expressed as percentage control mean value.



 


Summary Table: Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice (F1 Cohort 1A)
























































 



Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate



Sex



Males



Dose Level (mg/kg/day)



0



100



300



1000



Epididymides



 



 



 



 



Number Examined



19



20



19



20



Debris, Cellular



 



 



 



 



Minimal


Moderate



0


1



0


0



0


0



6


0



Total



1



0



0



6



 


Summary Table: Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice (F1 Cohort 1B)


































































































 



Tris[2-[2-(2-methoxyethoxy)


ethoxy]ethyl] orthoborate



Sex



Males



Dose Level (mg/kg/day)



0



100



300



1000



Epididymides



 



 



 



 



Number Examined



24



0



24



24



 Debris, Cellular



 



 



 



 



Minimal



0



0



2



9



Slight



0



0



0



4



Total



1



0



2



13



Testes



 



 



 



 



Number Examined



24



0



24



24



 Degeneration, Tubular



 



 



 



 



Minimal



0



-



0



8



Total



0



-



0



8



 


 

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 300 mg/kg/day due to the high incidences of reduced fertility in females of F1 Cohort 1B receiving 1000 mg/kg/day and the increased incidences of minimal epididymal cellular debris coupled with sperm motility and morphology changes in both F0 and F1 Cohort 1B males given 1000 mg/kg/day, accompanied with degeneration in the testes for F1 Cohort 1B males at 1000 mg/kg/day only.
Aside from the above-mentioned instances of reduced fertility and male reproductive system changes at 1000 mg/kg/day, increased incidences of basophilic tubules in the kidneys of F0 females and increased incidence of decreased lymphocytes in the cortex of the thymus in the F0 generation males were observed at 1000 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 300 mg/kg/day.
The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 300 mg/kg/day due to reduced early post-partum survival at 1000 mg/kg/day in both generations and low litter size in F2 litters.
There was no evidence of developmental neurotoxicity or developmental immunotoxicity in this study, therefore the NOAEL for these endpoints was concluded to be 1000 mg/kg/day.
Executive summary:

The purpose of this study was to assess the influence of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate on reproductive performance when administered by oral gavage to Sprague-Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation, estrous cycles, and reproductive performance. In addition, cohorts of F1 animals were used to assess developmental neurotoxicity and developmental immunotoxicity.


In the F0 generation, three groups of 24 male and 24 female rats received Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate at dose levels of 100, 300 or 1000 mg/kg/day at a volume dose of 4 mL/kg/day. Males were treated for two weeks before pairing, up to necropsy after litters were weaned. Females were treated for two weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In the F1 generation, 74 males and 74 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. Similarly constituted Control groups received the vehicle, Corn oil, at the same volume dose and throughout the same relevant period.


For the F0 generation data were recorded on clinical observations, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid related hormones, sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.


For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age. Serum samples that were collected from selected offspring on Day 22 of age were analyzed for thyroid-related hormones.


At weaning the F1 generation was split into five cohorts:


For F1 Cohort 1A, data were recorded on clinical signs and condition, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid‑related hormones, sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed.


For F1 Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles, mating performance, fertility, gestation length and gestation index. Organ weight, macroscopic pathology and microscopic pathology investigations were performed. For F2 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age.


For F1 Cohort 2A, data was recorded on clinical condition, body weight, food consumption and sexual maturation. Sensory function, neurobehavioural screening, brain weight, macroscopic pathology and microscopic pathology investigations were performed.


For F1 Cohort 2B, animals received no direct treatment and no specific in-life investigations were performed. Animals were dispatched to necropsy at weaning for microscopic pathology investigations of the brain.


For F1 Cohort 3, data was recorded on clinical signs, body weight, food consumption and sexual maturation. Spleen weight, macroscopic pathology and T-cell dependent antibody response investigations were performed.


Results:


The mean concentrations of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate in test formulations analyzed during the study were within +10/-15% of the nominal concentration, confirming the accuracy of formulation.


There were slightly lower circulating levels of thyroxine (T4) in F0 adult males at 1000 mg/kg/day and the F1 generation males at 300 or 1000 mg/kg/day. 


F0 responses:


Administration of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption or food conversion efficiency.


Estrous cycles, mating performance, pre-coital interval, fertility and gestation index were unaffected by treatment. Females in the 1000 mg/kg/day group showed a trend towards slightly extended gestation lengths.


The hematological examinations for F0 animals revealed, when compared with controls, lower values in some white blood cell parameters (lymphocyte, eosinophil and basophil counts), haematocrit, reticulocyte counts and mean cell volume in males given 1000 mg/kg/day. Mean cell volume were slightly lower than that of controls for males receiving 300 mg/kg/day and females receiving 1000 mg/kg/day. Blood chemistry investigations revealed higher mean urea concentrations in males at 1000 mg/kg/day. The analysis of urine prior to the termination of F0 animals, revealed higher total urinary protein and lower urinary pH in both sexes at 1000 mg/kg/day, total chloride concentration was also higher for females given 1000 mg/kg/day.


Sperm motility was reduced, and abnormal sperm morphology was increased, and total testicular spermatid counts were higher in males given 1000 mg/kg/day. Only slight changes in sperm motility were observed in males at 100 or 300 mg/kg/day.


Changes in organ weights consisted of slightly higher body weight relative kidney weights in males and females at 1000 mg/kg/day. In males at 1000 mg/kg/day, slightly higher absolute and body weight relative testes weights and lower absolute and body weight relative thymus weights were seen. In addition, absolute and body weight relative pituitary weight were slightly lower in females given 1000 mg/kg/day when compared to controls.


There were no test item related macroscopic findings observed at the scheduled sacrifice.


Histopathological evaluation of a full list of retained tissues revealed treatment related changes in the kidneys of the high dose females and the thymus and epididymis of the high dose males. In the kidneys, a higher incidence and severity of basophilic tubules with more multifocal distribution was observed in females at 1000 mg/kg/day. Minimal cellular debris was observed in the epididymis and a slight reduction in lymphocytes in the cortex of the thymus was observed in males that received 1000 mg/kg/day.


F1 litter responses:


The general condition of offspring, litter size on Day 1 of age, sex ratio, offspring body weight or development and macropathology were unaffected by treatment. Offspring survival was lower during the first four days of lactation in the litters of females given 1000 mg/kg/day.


Organ weights for unselected F1 offspring on Day 22 of age were unaffected by parental treatment and there were no macroscopic findings for offspring that either died prematurely or at scheduled termination that could be attributed to Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate.


F1 selected animals:


Administration of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate was generally well tolerated with no treatment-related mortality in F1 generation cohorts 1A or 1B, 2A, 2B and 3. Early euthanasia related to the test item was required in females, related to reduced fertility in the F1 generation in Cohort 1B. At 1000 mg/kg/day, there were twelve animals euthanized early, eleven due to failure to litter and one which failed to mate. A further four females that received 1000 mg/kg/day were euthanized due to total litter loss.


There were no adverse effects on general condition or food conversion efficiency. Body weight gain was generally unaffected during all phases of the study with the exception of Cohort 1B females during the gestation period, where body weight gain for females given 1000 mg/kg/day was statistically lower compared to controls. The food intake for animals during all phases in the study was generally similar to controls, the only difference was observed for F1 Cohort 1B females at 1000 mg/kg/day during the lactation period. There was also no effect of treatment on the attainment of sexual maturation of the F1 animals, or 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 females or the F1 Cohort 1A and 1B females


Estrous cycle regularity and pre-coital interval were unaffected by test item administration. A slight shift towards longer gestation lengths was apparent for females receiving 300 mg/kg/day and a stronger shift to longer gestation length was seen in females at 1000 mg/kg/day in comparison with the concurrent control. The number of females with live litters born were lower due to treatment at 1000 mg/kg/day. The rate of conception and fertility index for females receiving 1000 mg/kg/day was low compared to controls. The majority of females that received 100, 300 or 1000 mg/kg/day were showing estrous morphology at termination.


The hematological examinations at schedule termination revealed, when compared with controls, a lower mean cell hemoglobin, mean cell volume and red cell distribution width amongst males and females receiving 1000 mg/kg/day. Mean cell haemoglobin were slightly lower than that of controls in both sexes receiving 300 mg/kg/day and mean cell volume was low in females at 300 mg/kg/day. 


Blood chemistry investigations at scheduled termination for F1 animals revealed statistically significant higher urea concentration for males and females at 1000 mg/kg/day and higher bile acid concentrations at all dose levels in females. Urinary assessment revealed lower urinary pH in males and females given 1000 mg/kg/day and slightly lower urinary protein, sodium, potassium, and chloride concentrations in males at 1000 mg/kg/day.


Assessment of the F1 Cohort 2A and 2B animals did not reveal any evidence of developmental neurobehavioural or neuropathology changes.  Auditory startle response habituation, locomotor activity and the performance of the F1 Cohort 2A animals in the Functional Observational Battery (FOB) tests were unaffected by treatment.  During the reactivity investigations in the FOB, F1 Cohort 2A males at 1000 mg/kg/day showed a slightly lower hind limb grip strength; this difference was considered related to the lower mean body weight for this group. Brain weights were slightly lower in F1 Cohort 2A and 2B animals given 1000 mg/kg/day although the brain morphometry of the F1 Cohort 2A and 2B animals and detailed histopathological evaluation of the neurological tissues of the F1
Cohort 2A animals were unaffected by test item administration. 


The qualitative evaluation of the ovaries of F1 Cohort 1A females given 1000 mg/kg/day revealed a slightly lower number of follicles. In the absence of an effect on the number of corpora lutea or any histopathological changes in the ovary of the F1 Cohort 1B females, this finding was considered to be of no toxicological significance.


Sperm motility was reduced, epididymal sperm counts were lower compared to controls and normal sperm morphology was reduced in males receiving 1000 mg/kg/day. Sperm motility, counts and morphology of the F1 Cohort 1A males receiving 100 or 300 mg/kg/day were unaffected by test item administration.


Several changes in organ weights for the Cohorts 1A and 1B animals were observed, only the low thymus weights in males given 1000 mg/kg/day, the low pituitary weights and high kidney weight in both sexes given 1000 mg/kg/day were considered test item-related as these changes were consistent with the F0 generation.


There were no test item related macroscopic findings observed at the scheduled termination of F1 Cohort 1A, 1B, 2A, 2B and 3 animals.


Histopathological evaluation of full list retained tissues revealed treatment related changes in the epididymis of the high dose males in F1 Cohort 1A and 1B. Minimal cellular debris was observed in the epididymis of the F1 Cohort 1A males, minimal to slight cellular debris in the epididymis as well as minimal tubular degeneration of the testes was observed in F1 Cohort 1B males given 1000 mg/kg/day.


Assessment of the F1 Cohort 3 animals did not reveal any evidence of developmental immunotoxicity. Spleen weights at scheduled termination were unaffected, and there was no impact on the T-Cell Dependent Antibody Response (TDAR) following immunization with 300 µg of Keyhole Limpet Hemocyanin (KLH) on Days 47 and 54 of age.


F2 litter responses:


The general condition of offspring, sex ratio, offspring body weight or development and macropathology were unaffected by treatment. Ano-genital distances were similar to controls and there were no nipples observed in male offspring assessed on Day 13 of age.


Mean number of implantation sites, litter size, post-implantation survival and live birth index were all statistically significantly lower than controls in the 1000 mg/kg/day group. Mean litter size during the first four days of lactation prior to scheduled cull on Day 4 was lower compared to controls and Day 4 Viability index was low, litter size remained stable following the Day 4 cull to Day 21 of age. There was no effect on post-implantation survival, litter size or offspring survival from Day 1 of lactation at 100 or 300 mg/kg/day.


Conclusion:


Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 300 mg/kg/day due to the high incidences of reduced fertility in females of F1 Cohort 1B receiving 1000 mg/kg/day and the increased incidences of minimal epididymal cellular debris coupled with sperm motility and morphology changes in both F0 and F1 Cohort 1B males given 1000 mg/kg/day, accompanied with degeneration in the testes for F1 Cohort 1B males at 1000 mg/kg/day only.


Aside from the above-mentioned instances of reduced fertility and male reproductive system changes at 1000 mg/kg/day, increased incidences of basophilic tubules in the kidneys of F0 females and increased incidence of decreased lymphocytes in the cortex of the thymus in the F0 generation males were observed at 1000 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 300 mg/kg/day.


The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 300 mg/kg/day due to reduced early post-partum survival at 1000 mg/kg/day in both generations and low litter size in F2 litters.


There was no evidence of developmental neurotoxicity or developmental immunotoxicity in this study, therefore the NOAEL for these endpoints was concluded to be 1000 mg/kg/day.

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