Registration Dossier

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Toxicological information

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)
GLP compliance:
yes (incl. QA statement)
Remarks:
Charles River Laboratories Edinburgh Ltd Elphinstone Research Centre Tranent, East Lothian EH33 2NE, United Kingdom
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

On November 19th, 2018 ECHA informed the REACH lead registrant that the submitted testing proposal was accepted (Decision number: TPE-D-2114449867-30-01/F). Pursuant, the registrant was requested to carry out:
"Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation."

On November 21st, 2018 and February 13th, 2019 the Biocidal Geraniol Task Force (BGTF) was informed by Anses (French agency for food, environment and occupational health safety Regulated Products Assessment Directorate) that the following study for the active substance Geraniol is required under the Biocidal Products Regulation (BPR, Regulation (EU) 528/2012):
"The extended one-generation reproductive toxicity study - EOGRTS – (OECD TG 443) with ten weeks premating exposure duration for parental (P0) animals could be performed, if an equivalent level of information than for a two-generation is provided.
We advise you to include all cohorts, especially since the data set on the developmental neurotoxicity and the immunotoxicity is not sufficiently robust in your dossier. That is to say:
- extension of Cohort 1B by mating of Cohort 1B animals to produce F2 generation (necessary for investigations on reproductive toxicity in offspring),
- Cohorts 2A and 2B (developmental neurotoxicity) and
- Cohort 3 (developmental immunotoxicity).
The rat is the preferred species, oral route of administration is the preferred route.
The highest dose level should be based on toxicity and selected with the aim to induce reproductive and/or other systemic toxicity."

Neither the REACH registrants nor the BGTF has been made aware by any official body that two different decisions on OECD 443 data requirements for the substance Geraniol were available at the same time. However, the registrants involved have made every effort to avoid double testing in the interest of animal welfare. The REACH lead registrant and BGTF members found a scientific way forward to ensure that the study design will be set-up in such a way that it covers both regulatory needs (REACH and Biocidal Products Regulation). Therefore, the requested OECD 443 was carried out as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (developmental neurotoxicity) and
- Cohort 3 (developmental immunotoxicity).

The dose levels were selected based on a Reproduction/Developmental Toxicity Screening Study in Wistar Rats, Oral Administration (Gavage) with the registered substance Geraniol (BASF SE 2019; 80R0046/10R199) and the DECISION ON A TESTING PROPOSAL based on Article 40 of Regulation ((EC) No 1907/2006) for Geraniol (CAS 106-24-1).

Test material

Constituent 1
Chemical structure
Reference substance name:
Geraniol
EC Number:
203-377-1
EC Name:
Geraniol
Cas Number:
106-24-1
Molecular formula:
C10H18O
IUPAC Name:
3,7-dimethylocta-2,6-dien-1-ol
Test material form:
liquid
Specific details on test material used for the study:
Expiration Date: 08 April 2022 (Re-test Date: 15 Mar 2022)
Density: 0.8810 g/mL
Purity: 99.2% (Dose calculations were not corrected for purity)
Storage Conditions: Ambient, protected from light

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: F0 animals were approximately 6-7 weeks old (males) and approximately 5-6 weeks old (females)
- Weight at study initiation: F0 animals weighed between 160-261 g (males) and 106-157 g (females; target was minimum 125g but the lower starting weight was accepted at the time because the females were of the correct age and lower weight animals were present in all groups).
- Fasting period before study: no
- Housing: F0 animals were housed 2 or 3 per cage by treatment group and sex (unless reduced by mortality). A few days prior to mating, F0 males were transferred to individual cages. F0 females were transferred to these cages for mating. F0 females were transferred to individual solid bottomed cages after mating. F0 females with litters were retained in this type of cage until
termination. On a suitable day after completion of mating, the F0 males were re-housed with their original cage mates.
Cohort 1, 2 and 3 animals were housed 3 or 4 per cage by treatment group and sex (unless reduced by mortality). A few days prior to mating, Cohort 1B males were transferred to individual cages. F0 and Cohort 1B females were
transferred to these cages for mating.
Cohort 1B females were transferred to individual cages after mating. Cohort 1B females with litters were retained in this type of cage until termination. On a suitable day after completion of mating, the Cohort 1B males were re-housed with their original cage mates.
- Diet: SDS VRF-1 breeder diet (Special Diet Services, Essex, UK), ad libitum
- Water: water from the public supply, ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 26-66
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility (Formulation Process Document 998849-19-110) at appropriate concentrations to meet dose level requirements. The test item was mixed with the vehicle using a magnetic stirrer and/or a high shear homogeniser until formulations were visibly homogeneous. The dosing formulations were prepared at least weekly and either delivered to the animal unit immediately, or stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before, and then continuously during dosing.
Details on mating procedure:
F0 and Cohort 1B:
Pairing was on a 1 male to 1 female basis. Females were housed with their allocated co-group male partner during the evening (after 5 pm). For F0 animals, this commenced on Study Day 71 and for Cohort 1B animals this commenced between PND 100 and 116.
Vaginal lavages were taken early each morning from the day of pairing until mating had occurred and the stage of estrus observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated GD 0.
The pairing period for each pair of animals was a maximum of 14 nights.
If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for that female continued as if it had mated on the last night of pairing.
For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (estruses passed without a sign of mating) were evaluated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis on Day 1, Week 14 and Week 25 (all test groups for concentration analysis, Test Group 2-4 for homogeneity).

Concentration Analysis
Duplicate top, middle and bottom (duplicate middle only for control) samples for each sampling time point were sent to the analytical laboratory. Triplicate top, middle and bottom (triplicate middle only for control) samples were retained at the Test Facility as backup samples. Sample volumes (0.1 mL) were collected into appropriately sized volumetric flasks and stored in a refrigerator set to maintain 4°C, protected from light. Concentration results were considered acceptable if sample concentration results were within or equal to ± 15% of
theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of <= 10% for each group. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability analyses performed previously in conjunction with Lindsay, 2019, 423530 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Lindsay, 2019, 423530.

Analyses were performed by ultra-performance liquid chromatography (UPLC) with ultra-violet detection using a validated analytical procedure (Lindsay, 2019, 423530).
Duration of treatment / exposure:
F0 Males: 10 weeks prior to mating and continuing throughout and after mating, until termination
F0 Females: 10 weeks prior to mating and continuing throughout mating and gestation, until at least Lactation Day (LD) 21
Cohort 1A: From PND 21 until termination
Cohort 1B Males: From PND 21, through mating and after mating until termination after the majority of females reached LD 21 (a small number of animals from each group were not dosed on PND 21).
Cohort 1B Females: From PND 21, through mating, pregnancy and littering until F2 pups reached PND 22-24
Cohort 2A: From PND 21 until termination
Cohort 3 (Groups 1 to 4): From PND 21-59

F2 pups were not dosed directly.
Frequency of treatment:
once daily
Details on study schedule:
F0 animals were randomly assigned to groups. Males and females were randomised separately.
The F0 animals were allowed to acclimate to the Test Facility rodent toxicology accommodation for a period of at least 2 weeks before the commencement of dosing.

Day 55, Animal 4516F – cannot be confirmed if animal was dosed

F0 and Cohort 1B females that were found to be in the process of littering, or had recently littered, at the time of scheduled dose administration, were not dosed on that day and dosing re-commenced the following day for these animals.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Test group 1
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Test group 2
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Test group 3
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
Test group 4
No. of animals per sex per dose:
F0 Generation: 30/sex/group
F1 Generation:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Cohort 2A: One male or one female/litter (10/sex/group)
Cohort 2B: One male or one female/litter (10/sex/group)
Cohort 3: One male or one female/litter (10/sex/group)
F2 Generation: max. 25 litters per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In an OECD 421 screening study in Wistar rats which used dose levels of 0, 100, 300 and 1000 mg/kg/day, there was no treatment-related mortality. However, body weight gain was reduced in males at 1000 mg/kg/day (by ~70%), with initial weight loss. Food consumption in females at 1000 mg/kg/day was reduced during the pre-mating period (by ~11%) and during lactation (~12%).
Clinical signs were reported in males at 100 mg/kg/day (noisy breathing [1/10] and ploughing [3/10]), at 300 mg/kg/day (ploughing [10/10]), and at 1000 mg/kg/day (noisy breathing [5/10], ploughing [10/10] and abdominal position [1/10]).
Clinical signs were reported during the pre-mating period in in females at 300 mg/kg/day (salivation [10/10]) and at 1000 mg/kg/day (salivation [10/10], unsteady gait [6/10], gasping [1/10], noisy breathing [6/10], piloerection [9/10], ploughing [9/10], reduced attention [6/10], apathy [1/10], abdominal position [5/10] and lateral position [1/10]). Clinical signs during gestation were seen in females at 100 mg/kg/day (salivation [9/10]), at 300 mg/kg/day (salivation [10/10] and ploughing [10/10]) and at 1000 mg/kg/day (salivation [9/9], ptosis [1/9], ploughing [9/9] and reduced attention [2/9]). Clinical signs during lactation were seen in females at 100 mg/kg/day (salivation [7/10] and ploughing [1/10]), at 300 mg/kg/day (salivation [8/8] and ploughing [7/8]) and at 1000 mg/kg/day (salivation [8/8] and ploughing [8/8]).
Decreased T4 values and increased serum TSH values in parental males.
Decreased T4 values in female PND13 pups.


- Fasting period before blood sampling for clinical biochemistry: yes
Positive control:
Developmental immunotoxicity examinations in cohort 3 animals.

For positive control animals (Cohort 3), cyclophosphamide was administered via oral gavage once daily on five consecutive days prior to necropsy (PND 51-55), at approximately the same time each day with a maximum of 6 hours difference between the earliest and the latest dose. Formulations were gently inverted prior to dosing. The dose volume for each animal was based on body weight measurement on the first day of treatment.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
F0 and F1 animals were observed twice daily, once at the start and once towards the end of the working day throughout the study for general health/mortality and moribundity.
F0 nimals were observed regularly throughout the day on each day of dosing (at least twice postdose) for signs of reaction to treatment, with particular attention being paid to the animals during the first hour after dosing. The onset, intensity and duration of any signs were recorded, as appropriate.
Cohort 1 and 2 animals were observed prior to dosing and when possible throughout the day of dosing for signs of reaction to treatment, with particular attention being paid to the animals during the first hour after dosing. Cohort 3 animals were also observed on the day of KLH administration, after injection. Cohort 3 positive control animals (Group 5) were observed during treatment with cyclophosphamide, at least once daily, up to the day prior to necropsy, for specific clinical signs after injection with cyclophosphamide. The time of onset, intensity and duration of any observed signs were recorded as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0 animals were subjected to detailed clinical observations at least weekly, beginning Week -1. Animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, palpebral closure, vocalisation, rearing, arousal, stains and autonomic activity (lacrimation, salivation, piloerection, unusual respiratory pattern). Changes in gait and posture, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were assessed.
F1 Cohorts animals were subjected to detailed clinical observations at least weekly from weaning, starting on a suitable day within one week of weaning (PND 21) of all litters. Animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, palpebral closure, vocalisation, rearing, arousal, stains and autonomic activity (lacrimation, salivation, piloerection, unusual respiratory pattern). Changes in gait and posture, as well as the presence of clonic or tonic
movements, stereotypy or bizarre behaviour were assessed.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Males were weighed weekly beginning Week -1. Females were weighed weekly beginning Week -1 until pairing for mating and then at least on GD 0, 7, 14 and 20 and on LD 1, 4, 7, 14 and 21. Body weights taken on LD 0 were for dosing purposes only and have not been reported but retained in the study records. F1 pups were weighed individually on PND 1, 4, 7, 14 and 21. A weight was also recorded on the day of scheduled necropsy.
Cohorts 1A, 2A, 3 (both sexes) and Cohort 1B males were weighed weekly from weaning, starting on a suitable day within one week of weaning of the majority of litters (beginning of Nominal Week 4). Cohort 1B females were weighed weekly from this time until pairing for mating and then on GD 0, 7, 14 and 20 and on LD 1, 7, 14 and 21. Body weights taken on LD 0 were for dosing purposes only and have not been reported but retained in the study records. F2 pups were weighed individually, by sex, on PND 1, 4, 7, 14 and 21. Cohort 3 positive control animals (Group 5) were weighed once on the day of dosing with cyclophosphamide.

FOOD CONSUMPTION: Yes
Food consumption for F0 animals was quantitatively measured for both sexes weekly, beginning Week -1 until pairing for mating, for mated females on GD periods 0 to 7, 7 to 14 and 14 to 20 and on LD periods 1 to 7, 7 to 14 and 14 to 21. Food consumption was resumed weekly for males from Study Day 78 after mating and re-housing.
Food consumption was quantitatively measured weekly for Cohort 1A, 1B (both sexes, until pairing for mating), 2A and 3 animals, starting on a suitable day within one week of weaning of all litters (nominal Week 4), and for mated females (Cohort 1B only) on GD periods 0 to 7, 7 to 14 and 14 to 20 and on LD periods 1 to 7, 7 to 14 and 14 to 21. Food consumption was resumed weekly for Cohort 1B males on a suitable day after mating and re-housing.

WATER CONSUMPTION: Yes
Water consumption for F0 animals was monitored on a regular basis throughout the study by visual inspection of the water bottles. No intergroup differences were noted.

URINALYSIS: Yes
- F0 animals and Cohort 1A animals
- last week of dosing (10 animals/sex/group)
- Colour, Appearance/Clarity, Specific gravity, pH, Volume, Protein, Glucose, Bilirubin, Ketones, Blood

HAEMATOLOGY, COAGULATION AND CLINCICAL CHEMISTRY: Yes
- F0 animals and Cohort 1A animals
- morning of necropsy (10 animals/sex/group)
- Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute)
- Activated partial thromboplastin time, Fibrinogen, Prothrombin time, Sample Quality
- Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine Kinase, Total bilirubina, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride, Sample quality, Total bile acids

BLOOD COLLECTION FOR TSH AND T4 MEASUREMENTS:
- F0 animals and Cohort 1A animals
- morning of necropsy (10 animals/sex/group)
- T4 was measured on Advia Centaur CP Immunoassay System by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a sold phase enzyme immunometric assay – ELISA kit manufactured by BioVendor, Cat. No. RTC007R.
Oestrous cyclicity (parental animals):
Estrus Cycle Monitoring (F0 females):
Vaginal lavages were taken early each morning and the stages of estrus observed were recorded from 2 weeks prior to pairing until the day of detection of a copulatory plug in situ and/or of sperm in the lavage.
Vaginal smears were examined on the morning of necropsy to determine the stage of the estrus cycle to allow correlation with histopathology of reproductive organs, where this was considered necessary.

Estrus Cycle Monitoring (Cohort 1A and 1B):
Vaginal lavages were taken early each morning and the stages of estrus observed were recorded from the day after vaginal patency, continuing until the first confirmed estrus was determined (from approximately PND 75 for at least 14 consecutive days) for Cohort 1A females, and from 2 weeks prior to mating until detection of a copulatory plug in situ and/or of sperm in the lavage for Cohort 1B females. Vaginal smears were also examined on the morning of necropsy to determine the stage of the estrus cycle and to allow correlation with histopathology of reproductive organs, where considered necessary, for Cohort 1A females only.
Sperm parameters (parental animals):
F0 and Cohort 1A Males
- Computer Aided Sperm Assessment (CASA)
From all males, the right cauda epididymis was placed in 0.3% BSA in Medium 199 (as per Test Facility SOPs) and the sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was prepared and examined using a Hamilton Thorne sperm motility analyser.

- Sperm Count and Morphological Analysis
The cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From all samples of the sperm suspension described in the preceding paragraph, a sperm smear was prepared and stained with eosin Y solution. At least two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce (1975).

- Spermatid Count
The right testes were decapsulated and homogenised. The homogenate was sonicated to reduce tissue debris etc., if required. The number of homogenisation resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 10 pups/litter (5/sex/litter as nearly as possible) via random selection; excess pups were killed and discarded; when the total number of pups in a litter on PND 4 was ≤ 10 pups, no litter size adjustment occurred.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) on PND 1, pup weight on the day of AGD, presence of nipples/areolae in male pups on PND 13, gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS: YES
- Any pups that were found dead or were killed during lactation were sexed
and appropriately examined. Any externally abnormal decedent pup was preserved in 10% Neutral Buffered Formalin; externally normal pups were discarded.

WEANING AND SELECTION OF F1 ANIMALS FOR COHORTS 1A, 1B, 2A and 3:
From each group, 75 males and 75 females were selected at random on PND 21 and identified on that day for post-weaning assessments, nominally by selecting up to 5 males and 5 females from each litter. Where fewer than 60 litters were weaned, the necessary additional animals were obtained by selecting an additional pup from appropriate litters. These pups were removed from their mother on PND 21 and housed in their new cage.
Pups that were not selected for post-weaning assessments remained with their mother until termination.

ASSESSMENT OF SEXUAL MATURATION (COHORTS 1A, 1B, 2A and 3):
Commencing at PND 28, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day.
Commencing at PND 35, males were examined daily for balano-preputial separation.

BLOOD COLLECTION FOR TSH AND T4 MEASUREMENTS:
- F1 culled offspring (at least 2 pups/litter, where possible), PND 4 - at necropsy, T4 only
- F1 unselected pups (1 pup/sex/litter, where possible), at necropsy, TSH and T4
- T4 was measured on Advia Centaur CP Immunoassay System by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a sold phase enzyme immunometric assay – ELISA kit manufactured by BioVendor, Cat. No. RTC007R.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Assessment of Post-Natal Functional Development (Cohort 2A)
- Acoustic Startle: Between PND 23-25
- Motor Activity: Between PND 63-75
- Functional Observation Battery: Approximately PND 63-75
- Home Cage Observations: Posture/condition on first approach (animal undisturbed), checking for:, Prostration, Stereotypy / bizarre behaviour, Tremors (head, limbs, whole body), Convulsions, Ease of removal from the cage, Body temperature
- Handling Observations:
Pupillary function, Miosis / Mydriasis, Enophthalmos/ Exophthalmos, Lacrimation, Evaluation of diameter of the pupil, Body tone, Pinna response, Presence of salivation, Overall ease of handling, Respiration rate and pattern
- Air Righting
- Extensor Thrust
- Observations in a standardised arena (2 minute observation period): Rearing, Grooming, Urination and defecation, Arousal (level of alertness), Posture, Tremor (head, limbs, whole body), Convulsions, Piloerection, Palpebral closure, Gait abnormalities, Stereotypy (excessive repetition of behaviours) and/or unusual behaviours
- Functional Tests: Reaction to sudden sound (click above the head), Reaction to touch on the rump with a blunt probe, Grip strength, Pain perception, Landing Foot Splay, Other physical/functional abnormalities

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
KLH TDAR Assay – Cohort 3:
All animals were assessed for the ability to mount a T-cell dependent antibody response (TDAR) by measurement of circulating antibodies following administration of keyhole limpet haemocyanin (300 µg KLH on PND 51).
For Group 5, this immunisation was performed 2 to 4 hours after treatment of the positive control animals with cyclophosphamide on PND 51.
The animals were immunised by intravenous injection of KLH in 0.9% sodium chloride solution in the tail vein of each animal. Each immunisation was administered using sterile needles and disposable plastic syringes.
Blood samples (0.3 mL) were collected from the jugular vein using sterile hypodermic needles and plastic syringes before PND 51 and at PDN 55 or 56. Animals were not fasted prior to blood sampling.
Immediately after collection, the samples were transferred to glass tubes and allowed to stand at room temperature for at least 1 hour to allow complete coagulation. The samples were then centrifuged at approximately 1500 g at 4°C for 10 minutes and the serum separated into plain plastic tubes. The serum was then stored frozen at approximately -20°C until analysis for IgM antibodies to KLH was performed at the Test Facility. Samples were analysed by ELISA
Postmortem examinations (parental animals):
SACRIFICE
- Euthanised by exposure to carbon dioxide followed by exsanguination
- F0 Females: LD 22 or shortly thereafter
- F0 Males: After the majority of F0 females
- Cohort 2A and 2B animals underwent transcardial perfusion fixation

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
The reproductive tracts of all F0 and Cohort 1B females were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female. The uteri of non-pregnant females were fixed in buffered formalin and stained using 10% aq (v/v) ammonium sulphide solution and examined for implantation sites.

For further information on ORGAN WEIGHTS, HISTOPATHOLOGY please refer to the attached document "Material and Methods - Information on Organ Weights, Tissue Collection and Preservation".

Immunophenotyping Sample Collection, Processing, and Analysis (Cohort 1A):
Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group of the Cohort 1A animals at necropsy and were collected into tubes containing RPMI-1640 medium and placed immediately on wet ice until they were processed. The remainder of the spleen was preserved in 10% neutral buffered
formalin.

HISTOLOGY
Identified tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.

HISTOPATHOLOGY
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell- or stage-specificity of testicular findings was noted.
The kidneys of 5 control F0 generation males and 5 affected Group 4 F0 generation males
were stained with Mallory-Heidenhain stain.

OVARIAN FOLLICLE COUNTS (Cohort 1A)
From each ovary, 6 step serial sections at approximately 5 μm (a small amount into the formalin-fixed ovary e.g. 25 μm before the next section was taken) were taken. One section was stained with haematoxylin and eosin for routine evaluation and 5 sections stained for immunohistochemistry (IHC) staining using proliferating cell nuclear antigen (PCNA) marker for enumeration of primordial and primary follicles and for confirmation of the presence of or absence of the corpora lutea.

BONE MARROW SMEAR EVALUATION
Two bone marrow smears were taken from the femur of each euthanised F0 and Cohort 1A animal. Both bone marrow smears were air dried, fixed in methanol, stained with May-Grunwald-Giemsa stain and coverslipped. Bone marrow smears were not evaluated.
Postmortem examinations (offspring):
SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and euthanised by exposure to carbon dioxide followed by exsanguination.
- Animals less than 10 days old were killed by intraperitoneal injection of sodium pentobarbitone followed by exsanguination, by cervical dislocation. When possible, the animals were euthanised in a rotating order across dose groups such that similar numbers of animals from each group, including controls, were necropsied throughout the day.
- Culled PND 4 Pups from F1 and F2 Litters:
All culled pups at PND 4 of test group 1-4: Necropsy; Unscheduled Deaths: Necropsy
- Weanlings (unselected pups):
10 animals/sex/group, test group 1-4: Necropsy, Tissue Collection, Organ Weights

GROSS NECROPSY
Where practicable, offspring found dead or killed (unscheduled) were sexed internally, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate. Externally normal pups were discarded.

For further information on ORGAN WEIGHTS, HISTOPATHOLOGY please refer to the attached document "Material and Methods - Information on Organ Weights, Tissue Collection and Preservation".
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 1% and 5% levels.
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Any group with less than 3 observations was excluded from analysis.

Parametric/non-parametric statistical method:
- Levene’s test: The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test.
- Body Weight, Body Weight Gains, Food Consumption, Haematology Variables, Coagulation Variables, Clinical Chemistry Variables, Urinalysis Variables, Ovarian Scoring (total number of oocytes per animal), Organ Weights, Organ Weight Relative to Body Weight, Organ Weight Relative to Brain Weight, Sperm Morphology, Sperm Density and % Motility, Anogenital Distances (Litter Means), Motor Activity, FOB Quantitative Variables, Brain Morphometric Data, Litter Observations (Continuous) (e.g. Sex Ratio - Males, Mean Litter Body weights – total and sexes separate)

Non-parametric statistical method:
- Overall Kruskal-Wallis test: If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
- Pre-coital Interval, Day of Sexual Maturation, Natural Delivery and Litter Observations (Count) (e.g. Gestation Length, Live Pups, Implantation Sites), Developmental Landmark (Mean Day)

Incidence statistical method:
- Fisher’s exact test
- Pregnancy, Mating and Fertility Indices, Natural Delivery and Litter Observations
(Proportional) (e.g. Pregnant, Females with Liveborn, Gestation Index, Female with Liveborn)

Analysing Acoustic Startle:
SAS Software for Windows

Analysing TDAR data:
SAS v9.4, analysis was performed on the pooled (male and female) data
Reproductive indices:
Female Mating Index = Number of Females with Evidence of Mating (or no confirmed mating date and pregnant) / Number of Females Paired

Female Fertility Index = Number of Pregnant Females / Number of Females with Evidence of Mating (or no confirmed mating date and pregnant)

Female Pregnancy Index = Number of Pregnant Females / Number of Females Paired

Male Mating Index = Number of Males with Evidence of Mating (or female partner confirmed pregnant) / Number of Males Paired

Male Fertility Index = Number of Males Impregnating a Female / Number of Males with Evidence of Mating (or female partner confirmed pregnant)

Male Pregnancy Index = Number of Males Impregnating a Female / Number of Males Paired

Gestation Length = The gestation length was calculated from GD 0 to the day the first pup was observed.

Gestation Index = Percentage of pregnancies that result in birth of live litters:
Number of Animals with Live Offspring / Number of Animals Pregnant x 100
Offspring viability indices:
Birth Index (Percentage of pups born) = Total Number of Live and Dead Newborn Pups / Number of Implant Sites x 100

Live Birth Index (Percentage of pups born alive) = Number of Live Newborn Pups / Number of Live and Dead Newborn Pups x 100

Sex Ratio (% males) (Percentage of male pups per litter) = Number of Live Male Pup / Total Number of Live Pups x 100

Viability Index (Percentage of pups born that survive 4 days postpartum) = Number of Live Pups on Day 4 Postpartum / Number of Live Newborn Pups x 100

Lactation Index (Percentage of pups that survive 21 days postpartum) = Number of Live Pups on Day 21 Postpartum / Number of Live Pups on Day 4 (postculling) Postpartum x 100

Post-Implantation Loss/Litter = (Number of Implants – Total Newborn Pups) / Number of Implants x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A dose-related higher incidence and duration of transient salivation and ploughing behaviour in response to dosing was seen:
- in all males and females at 800 mg/kg/day, with a slightly lower incidence in females during gestation;
- in most females premating and males at 200 mg/kg/day and fewer females during gestation;
- in some females premating and males at 50 mg/kg/day – for a shorter duration - and no females during gestation. One or two control males showed these signs on a single day.

Red paws were noted in all males and females for several hours after dosing at 800 mg/kg/day on Day 8. Red ears were similarly noted for these animals and for all males and several females at 200 mg/kg/day between Days 8 and 10.

Treatment at 800 mg/kg/day was also associated with very low incidences/frequencies of (i) transient post dose abnormal (uncoordinated) gait, decreased activity/subdued behaviour, low carriage, erect fur and chewing action sporadically throughout the study; and (ii) increased respiration rate and hunched posture in females during the pre-mating period.

Other clinical observations were considered incidental due to their nature and/or distribution across the groups.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two animals were euthanised for welfare reasons.
- F4509 (receiving 800 mg/kg/day) on Day 17: on removal from the cage for dosing, the animal showed increased respiration rate, decreased activity and was cold to touch with erected fur. It was not dosed and, half an hour later also had partly closed eyes, hunched posture and was subdued. Euthanasia was performed for welfare reasons.
At necropsy, cloudy red liquid was noted in the thoracic cavity; all other tissues were without
visible lesions. Histologically, a diffuse, mild infiltration by mixed cells in the lung was considered the cause of the animal’s terminal condition. It was possibly due to locally-deposited test item as a result of misdosing or reflux from previous days. The infiltrate included fibrinous exudate and macrophages with vacuolated cytoplasm. There was a mild mononuclear cell infiltrate in the pleura around the left lobe of the lung and adjacent to the sternum, and minimal inflammation in the thymic capsule. In the axillary lymph node there was a moderate accumulation of macrophages, some vacuolated, in the subcapsular sinus. This was possibly due to earlier misdosing. There were reactive changes in the mesenteric lymph node and vacuolation in the central nervous system, renal pelvis and splenic red pulp.
- F2527 (receiving 50 mg/kg/day) on Day 89: a toe injury sustained during gestation was successfully remedied under surgical anaesthesia. However, the animal was soon seen chewing her injured foot and with blood in the cage. She was therefore euthanised for welfare reasons. The only noteworthy finding was mild extramedullary haematopoiesis in the spleen.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on adult body weight or weight gain during the
pre-mating period, gestation or lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: no test item-related effects, despite occasional statistically significant differences from the control group.

Females: At 800 mg/kg/day, statistically significant slightly higher food consumption values than
controls were noted intermittently during the pre-mating period (up to 15%) and for GD 14-20 (small at 9% but consistent with values up to 13% higher during GD 0-14). During lactation, intake was consistently slightly lower than controls (by 10 to 16%), attaining statistical significance for this group and for 200 mg/kg/day during Lactation Days (LD) 1-7 (-10%).
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Variation in haematology parameters, including some statistically significant differences between test item group and control group means, was considered normal. In all cases where statistical significance occurred (males: monocyte and reticulocyte counts), values were within the historical control range and not associated with changes in any other white or red blood cell parameters.

Coagulation: Despite some statistically significant differences from control in group mean values, these were generally small in magnitude and considered not to represent a test item effect.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
800 mg/kg/day: group mean values for the following parameters were higher in males and females than for the respective controls: bile acids, by 1.5- to 1.8-fold; ALP by approximately
1.4-fold and GGT by 1.5- to 1.7-fold. Intergroup differences for these parameters were not always statistically significant and the standard deviations for bile acids reflected wider ranges of values at 800 mg/kg/day than in the control group.

Variation in the remaining clinical chemistry parameters, including a statistically significant
difference from control for female globulin levels that was within the historical control range and not associated with any other protein changes, was considered normal. Creatinine kinase (CK) was 1.8-fold higher for females at this dose level but the standard deviation was large and there was no similar magnitude change in males.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Stimulating Hormone (TSH) levels were slightly higher than the concurrent control for F0 males and females at all dose levels (up to 1.5-fold at 50 mg/kg/day and approximately 2-fold at 200 and 800 mg/kg/day ). Group mean values were influenced by individual results that were below the reportable range and all listed as 0.63 ng/mL. Such values were likely due to a limit in assay sensitivity. However, the test item effect described above remained if these values were removed from the mean calculations. Individual values for all males and most females were within the historical control ranges, with only 1, 2 and 3 females at 50, 200 and 800 mg/kg/day respectively having values above these ranges compared with none for control group females.

There was no effect on circulating TSH values at PND 21. It was noted that all unselected (circa PND 21) female TSH values were below the reportable range, due to the limit in assay sensitivity.

Values for T4 were generally variable and did not suggest any effect of test item administration.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Despite some statistically significant differences from control in group mean values, these were considered to be small in magnitude and not representative of a test item effect.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related microscopic findings at 800 mg/kg/day in the nasal cavity in males (primarily degeneration in the olfactory epithelium and nasopharynx), thyroid in females (diffuse follicular cell hypertrophy), kidney in males (hyaline droplet accumulation) and spleen in males (increased haematopoiesis).
For further details please refer to "Any other information on results incl. tables".

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Females were cycling normally and pre-coital intervals were similar in all groups, with one female at 800 mg/kg/day having a pre-coital interval of 11 days – similar to that seen in an F1B control group female.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no clear dose-related trends in spermatid counts and differences from control were small, despite values attaining statistical significance at 50 and 800 mg/kg/day. It was therefore considered that there was no test item-related effect on these parameters and no effects on sperm counts, motility or morphology.
Reproductive performance:
no effects observed
Description (incidence and severity):
MATING FERTILITY, PREGNANCY INDICE:
similar in all groups.

GESTATION, LITTER PERFORMANCE AND SURVIVAL
- Gestation: no test item-related effects on the duration or the Gestation Index.
- Birth Index: The mean percentage of male pups per litter on LD 1 was almost identical in all groups.
- Live Birth Index: marginally lower at 800 mg/kg/day than in the control group (94.1% compared to 99.6%), with 3 litters at the highest dose level losing more than 1 pup – including two total litter losses by LD 1 –compared with none in the other groups. However, the values and incidences obtained in this high dose level group are not uncommon for control groups and were considered not to be an effect of the test item.
Pup survival between LD 0 and 4 was slightly lower at 800 mg/kg/day than in the control group, as shown by the Viability Index (85.9%, with several litters losing 2 or 3 pups, compared to 99.7% in the control group and no less than 97% for either generation in historical control data. Pup mortality occurred in 11 litters at 800 mg/kg/day compared with single litters at each of 0 (Control), 50 and 200 mg/kg/day.
Pup survival after the Day 4 cull was similar in all groups.
Pup and litter observations were also similar in all groups, with multiple nests and ‘scattering’ of pups noted for control and test item group litters.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: olfactory epithelium degeneration
Dose descriptor:
NOAEL
Remarks:
fertility/reproductive performance
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed at highest dose tested

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Note: the high dose level of 800 mg/kg/day was reduced to 600, then 400 mg/kg/day due to intolerance – see Mortality section - and titrated back via 600 to 800 mg/kg/day within the first two weeks of dosing. This dose level is simply referred to as 800 mg/kg/day.

A dose-related increase in incidence and duration of salivation and ploughing in response to dosing was seen in all cohorts: in most males and females at 800 mg/kg/day and in some males and females at 200 mg/kg/day. A small number of Cohort 2A control females and one Cohort 1B female at 50 mg/kg/day had salivation on isolated occasions.
Treatment at 800 mg/kg/day was associated with very low incidences/frequencies of transient post dose abnormal (uncoordinated) gait, decreased activity, low carriage, erected fur, chewing action, partially closed eyes, irregular respiration/breathing and pallor.
A small number of Cohort 1B animals at all dose levels were noted to have thinning fur/fur loss later on in the treatment period, compared with single control animals.
Other clinical observations were considered incidental due to their nature and/or distribution across the groups.
Mortality:
mortality observed, treatment-related
Description (incidence):
In Cohort 1B, F4641 was euthanised on Day 1 of dosing (Post Natal Day (PND) 21), M4131 was euthanised on Day 2 (PND 22) and F2635 was found dead on Day 18 (PND 38). Both F4641 and M4131 were euthanised because they did not tolerate the high dose level. For M4131, Respiratory Rate Abnormal, Decreased; Fur, Erected; Eyes Closed, Partly; Low Carriage, and Activity Decreased were recorded 1-2 hours after dosing at 800 mg/kg/day. When the animal was also noted as being subdued, the decision was made to euthanise. For F4641, clinical signs were similar to those of other animals dosed at 800 mg/kg/day initially but worsened by 1 hour postdose and the animal was effectively moribund by approximately 2 hours post dose. The observations were Respiratory Rate Abnormal, Decreased/Irregular; Skin, Discoloured, Generalized, Pale; Activity Decreased/Abnormal Gait; Hunched Posture; Fur, Erected; Eyes Closed, Partly/Completely; Lying on Side/Prostrate, and Muscle Tone, Generalized, Decreased.
For F2635 receiving 50 mg/kg/day, this animal was found dead only 1 minute after dosing. A gavage dosing incident was suspected although there was no physical evidence of relevant trauma found at necropsy. In fact, none of these animals had any abnormalities at necropsy and they were not evaluated histologically.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
All Cohorts receiving 800 mg/kg/day (with the exception of Cohort 2B that was last weighed on PND 21) had slightly lower mean body weight values than concurrent control groups during Weeks 1 and 2 of dosing, which corresponded with the time period during which intolerance and mortality occurred. Differences from control were up to 18% for males and 13% for females, sometimes attaining statistical significance. Week 2 body weight for Cohort 3 males at 200 mg/kg/day was 11% lower than the concurrent control but, in isolation, this was considered not to be an effect of the test item.
Maternal body weight values during gestation and lactation (Cohort 1B) were essentially similar in all groups, even though this included statistically significant 6% higher weights on Gestation Day (GD) 20 at 200 and 800 mg/kg/day. Maternal weight gain from LD 1-21 was lower than control in a dose-related manner: -12 and -24% at 200 and 800 mg/kg/day respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Consistently slightly higher food consumption values than controls were recorded for Cohort 1B males at 800 mg/kg/day from Week 16 (10-18%, statistically significant).
Gradually increasing slightly higher intake than controls was noted at 800 mg/kg/day for Cohort 1A females (9-15% between Weeks 7 and 10, statistically significant in the last week) and 1B females (10-20% between Weeks 6 and 13, statistically significant from Week 7).
These differences were not seen in Cohorts 2A and 3.
Slightly higher food consumption than controls was also seen throughout gestation (Cohort 1B): up to 16% at 200 and 800 mg/kg/day (statistically significant). Intake during lactation was similar in all groups.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Some white blood cell counts were higher in Cohort F1A test item groups than in controls, sometimes attaining statistical significance, in particular:
Basophils: 1.6-fold for females at 200 mg/kg/day and 1.7- to 2.1-fold for males and females at 800 mg/kg/day;
Monocytes: 1.6-fold for females at 800 mg/kg/day;
Large unstained cells: 1.6- to 1.7-fold for males and females at 800 mg/kg/day.
Variation in the remaining haematology parameters, including some statistically significant differences between test item group and control group means, was considered normal. In cases where statistical significance occurred (females: lymphocyte count, haematocrit), differences from control were small, values were within the historical control range and there were no associated red blood cell parameter changes.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Stimulating Hormone (TSH) levels were slightly higher than the concurrent control for Cohort F1A males at all dose levels. The summary table lists these differences as approximately 2- to 3-fold. However, group mean values were influenced by individual results that were below the reportable range and all listed as 0.63 ng/mL. The test item effect described above remained if these values were removed from the mean calculations, giving differences of 1.5- to 1.7-fold. Most of the values below the reportable range were in the control group. Most of the individual measurable values in the test item groups and both of those in the control group were within the historical control ranges (combining F0 and F1 historical control data), with only 1 and 3 males at 200 and 800 mg/kg/day respectively having values above these ranges. It was noted that including F0 generation and similar age range historical control data resulted in comparing with animals older than those in the F1 generation, with a potential for inherently slightly higher TSH values in the historical control data.
There were few TSH values above the reportable range for females and a trend for higher values was therefore not suspected.
Values for T4 were generally variable and did not suggest any effect of test item administration.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Cohort F1A females in all groups reached their first estrus within similar day ranges. Most Cohort F1B females were cycling normally, except for F3630 at 200 mg/kg/day which appeared to enter a pseudopregnancy and one control group female (F1637) which did not cycle. These were considered typical anomalies to see within a set of females. Pre-coital intervals were similar in all groups, with one control female having a pre-coital interval of 13 days.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
In Cohort 1A at 800 mg/kg/day, total and per g testis spermatid counts were statistically
significantly 13-24% lower than the control value, also being just below the historical control data range. However, sperm counts were superior to control in the test item groups. The percentage of abnormal sperm was also higher at 800 mg/kg/day – the values still being very low, however: 0.43 and 1.18% compared with 0.30 and 0.78% in the control group). However, even this control group mean for abnormal sperm excluding tail defects was above the historical control data range (up to 0.2%). Motility was unaffected.
Reproductive performance:
no effects observed
Description (incidence and severity):
FERTILITY, PREGNANCY INDICES:
Male and female fertility and pregnancy indices were marginally lower at 200 and 800 mg/kg/day (between 83 and 88%) than the control group (96%). Considering F0 and F1 generation historical control data together – because the number of studies was limited - fertility indices were within this range; there being no historical control data for pregnancy indices. With the additional observation that the concurrent control group fertility indices were high compared to the historical control data, an effect of the test item was therefore not suspected.

DURATION OF GESTATION, LITTER PERFORMANCE AND SURVIVAL
There were no test item-related effects on the duration of gestation, Gestation Index or pup survival indices during lactation.
The mean percentage of male pups per litter on LD 1 was also unaffected, with no trends evident.
Pup and litter observations were generally similar in all groups, with multiple nests noted for control and test item group litters. While ‘scattering’ of pups was noted for some litters at all dose levels and not in the control group, it was recorded for control litters in the F0 generation. In the absence of other effects on F1 litters, this was considered to be an unfortunate distribution and not an effect of the test item.

OVARIAN FOLLICLE COUNTS
In Cohort 1A, total ovarian follicle counts at 800 mg/kg/day were lower than the control group, reflecting slightly lower primordial and primary follicle counts. However, these data are known to be quite variable, as can be seen from the historical control data range and a test item effect was therefore not suspected in this case. Corpora lutea.
were present in all females.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
other: olfactory epithelium degeneration
Dose descriptor:
NOAEL
Remarks:
fertility/reproductive toxicity
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed at highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
In Cohort 1B, F4641 was euthanised on Day 1 of dosing (Post Natal Day (PND) 21), M4131 was euthanised on Day 2 (PND 22) and F2635 was found dead on Day 18 (PND 38).
Both F4641 and M4131 were euthanised because they did not tolerate the high dose level. For M4131, Respiratory Rate Abnormal, Decreased; Fur, Erected; Eyes Closed, Partly; Low Carriage, and Activity Decreased were recorded 1-2 hours after dosing at 800 mg/kg/day. When the animal was also noted as being subdued, the decision was made to euthanise. For F4641, clinical signs were similar to those of other animals dosed at 800 mg/kg/day initially but worsened by 1 hour postdose and the animal was effectively moribund by approximately 2 hours post dose. The observations were Respiratory Rate Abnormal, Decreased/Irregular; Skin, Discoloured, Generalized, Pale; Activity Decreased/Abnormal Gait; Hunched Posture; Fur, Erected; Eyes Closed, Partly/Completely; Lying on Side/Prostrate, and Muscle Tone, Generalized, Decreased.

For F2635 receiving 50 mg/kg/day, this animal was found dead only 1 minute after dosing. A
gavage dosing incident was suspected although there was no physical evidence of relevant trauma found at necropsy. In fact, none of these animals had any abnormalities at necropsy and they were not evaluated histologically.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean values for litter and pup weights were not significantly different, test item group values
being within 10% of control group values. The smallest litter and pup weights were recorded
at 800 mg/kg/day but the magnitude of difference from control was considered insufficient to
support a test item-related effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Consistently slightly higher food consumption values than controls were recorded for Cohort 1B males at 800 mg/kg/day from Week 16 (10-18%, statistically significant).
Gradually increasing slightly higher intake than controls was noted at 800 mg/kg/day for Cohort 1A females (9-15% between Weeks 7 and 10, statistically significant in the last week) and 1B females (10-20% between Weeks 6 and 13, statistically significant from Week 7).
These differences were not seen in Cohorts 2A and 3.
Slightly higher food consumption than controls was also seen throughout gestation (Cohort 1B): up to 16% at 200 and 800 mg/kg/day (statistically significant). Intake during lactation was similar in all groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some white blood cell counts were higher in Cohort F1A test item groups than in controls,
sometimes attaining statistical significance, in particular:
- Basophils: 1.6-fold for females at 200 mg/kg/day and 1.7- to 2.1-fold for males and females
at 800 mg/kg/day;
- Monocytes: 1.6-fold for females at 800 mg/kg/day;
- Large unstained cells: 1.6- to 1.7-fold for males and females at 800 mg/kg/day.

Variation in the remaining haematology parameters, including some statistically significant differences between test item group and control group means, was considered normal. In cases where statistical significance occurred (females: lymphocyte count, haematocrit), differences from control were small, values were within the historical control range and there were no associated red blood cell parameter changes.

COAGULATION:
Despite some statistically significant differences from control in group mean values, these were generally small in magnitude and considered not to represent a test item effect.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Group mean values for bile acids were higher than controls by 1.9-fold for Cohort F1A males at 800 mg/kg/day and by 5.3-fold for Cohort F1A females at 200 and 800 mg/kg/day.
Differences from control attained statistical significance in all cases.
At 800 mg/kg/day, group mean values for ALT and ALP were also higher in males and females than for the controls: by 1.4- to 1.5-fold.
For females only, in comparison with control means, triglycerides were approximately 1.5-fold higher at 800 mg/kg/day and cholesterol was 1.3- to 1.4-fold higher at 200 and 800 mg/kg/day.

Variation in the remaining clinical chemistry parameters, including some statistically significant differences between test item and control group means, was considered normal. At 800 mg/kg/day, slightly but statistically significantly lower Na concentrations: by 0.99-fold, were on the limit or just outside the historical control range (see Appendix 54). However, the minor magnitude of this difference and the absence of any other test item-related changes in
electrolytes did not support this as a test item-related result. In other cases where statistical
significance occurred (males: urea and K concentrations; females: cholesterol concentration),
values were within the historical control range and there were no other associated clinical chemistry parameter changes. The significant result for female Na concentration at 50 mg/kg/day was not part of a dose-related trend and was within the historical control range.
Creatinine kinase was 1.3- to 1.9-fold higher for males at all dose levels but there was no dose-relationship and no similar magnitude change in females.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Despite some statistically significant differences from control in group mean values, these were considered to be small in magnitude and not representative of a test item effect.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Vaginal opening and balano-preputial separation were considered to be unaffected by the test item.
The age for preputial separation at 800 mg/kg/day was statistically significantly higher than the control group, at 42.2 days; however all groups were slightly above the historical control range (see Appendix 54) and the difference between all was less than 1 day.
Body weights at sexual maturation were essentially similar in all groups for males and for females separately. Despite a statistically significant difference in mean weight for females at 800 mg/kg/day compared with control, that was also below the historical control range at 99 g, the difference was small and the age for vaginal opening was within the historical control range.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distances were similar in all groups. Nipples were not retained in males
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The only general organ weight differences were noted in Cohort 1A animals at 800 mg/kg/day, where relative thyroid weight was higher than controls in females (all absolute thyroid weights were within the historical control range), absolute and relative kidney weight was higher than controls in males (2 of the 20 males at this dose level had absolute kidney weights greater than the historical control range), and absolute and relative liver weight was higher than controls in both sexes (1 of the 20 males and 8 of the 20 females at this dose level had absolute liver weights greater than the historical control range).
For further details please refer to "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross pathology findings in any cohort.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
In Cohort 1A animals, in the nasal cavity, 1 female at 800 mg/kg/day had degeneration/regeneration of the nasopharyngeal epithelium. In the thyroid, at all dose levels
there was minimal, diffuse follicular cell hypertrophy (affecting both sexes). In the kidney of males at 800 mg/kg/day, and in 1 male at 200 mg/kg/day, there was hyaline droplet accumulation. In the liver at 800 and 200 mg/kg/day there was centrilobular hepatocyte
hypertrophy (affecting both sexes). In the spleen of males at 800 and 200 mg/kg/day there was minimal increased haematopoiesis.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
ACOUSTIC STARTLE: effects not treatment-related
Within Cohort 2A, for males, there were no statistically significant intergroup differences and, although average and maximum response amplitudes were lowest at 800 mg/kg/day, at approximately 13-14% below control values, this is quite a variable parameter by nature and the differences observed were slight. For females, statistical significance was not attained for these parameters and differences were generally less than 10% and not consistent across the 5 trials (sometimes lower, sometimes higher than control). Overall, it was considered that there was no effect of the test item on the acoustic startle response.
Latency to maximum response was not affected by the test item for males or females. Despite
a statistically significant difference in mean latency time for females at 800 mg/kg/day compared with control, the difference was small (4%) and all group means were below the historical control range.

MOTOR ACTIVITY: effects treatment-related
Within Cohort 2A, there were some lower levels of motor activity assessed at PND 63-75 for males at 800 mg/kg/day and for females at all dose levels, in a dose-related manner. The effect occurred in both sexes at 800 mg/kg/day, being more marked and attaining statistical significance for females. Statistical significance was also recorded for females at 200 mg/kg/day but not at 50 mg/kg/day where the magnitude of the effect was similar to that seen for high dose level males. These overall values also reflected changes seen in increasing numbers of individual sessions, from at least half at 50 mg/kg/day to most/all at 200 and 800 mg/kg/day.
For further details please refer to "Any other information on results incl. tables".

FOB: effects not treatment-related
In the Cohort 2A FOB assessed at PND 63-75, the low incidences of salivation and uncoordinated landing on air righting at 800 mg/kg/day were considered to be related to the clinical observations at this dose level. One male at this dose level had no pupil response when tested but, in isolation, this was considered of unlikely relationship to the test item.
There were no other notable qualitative results.
A small difference from control in mean forelimb grip strength was noted for males: -17% at 50 and 200 mg/kg/day, -14% accompanied by -11% for hindlimb grip strength at 800 mg/kg/day. Statistical significance was not attained. No other notable differences in quantitative parameters were seen.

NEUROPATHOLOGY: effects not treatment-related
At 800 mg/kg/day, Cohort 2A males and Cohort 2B and unselected pups of both sexes had lower brain weights; this was considered to be due to the lower body weight of the animals. There were no test item-related microscopic findings in Cohort 2A animals and no microscopic abnormalities were noted in Cohort 2B animals.

BRAIN MORPHOMETRY 2A/2B: effects not treatment-related
In Cohort 2A males, the brain morphometry measurements were less than controls at 800 mg/kg/day, by 2% to 8%. No effect was seen in females. A similar response was seen in the Cohort 2B males and females (2-9%). This was considered due to the lower body weight of the animals.
The mean length and width of the brain, as measured grossly, were similar to controls at all dose levels in Cohort 2A and were considered not to show a test item-relationship. In Cohort 2B males and females, the mean length and width of the brain at 200 and 800 mg/kg/day were lower than the control values by 6 to 16%. At 800 mg/kg/day, this was considered due to the lower body weight of the animals. Although at 200 mg/kg/day, body weight was similar to controls, brain weight at this dose level was only 3% less than controls in females, and was greater than controls in males. In view of the lack of effect on brain weight and the lack of statistical significance, the lower length and width of the brain at 200 mg/kg/day was considered not test item-related.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
For all Cohort 1A splenic cell populations measured, there were no dose-dependent effects observed. Male animals in the control and 50 mg/kg/day dose groups had a higher percentage of B cells and lower percentage of T cells when compared with the 200 and 800 mg/kg/day dose groups. The 200 mg/kg/day and 800 mg/kg/day dose groups were comparable to all female dose groups, including the control, and showed no differences in percentage of B cells and T cells. Therefore, the difference noted in between the male groups could not be attributed to dosing with the test substance. The geomean of T helper cells percentage in male animals from the 800 mg/kg/day group was higher than that of the control group. However, this difference was marginal and the geomean was comparable to that of the female control group. There were no differences observed for the male Cytotoxic T cell or Natural Killer cell populations.

KLH TDAR Assay:
A clear humoral response to immunisation with KLH was observed in all animals; with KLH-specific IgM levels being greater on PND 56 than baselines values from before PND 51.
Mean levels of IgM were higher in female animals than in the male animals. There were no significant differences or dose-related changes in the concentration of KLH specific IgM antibodies in test-substance treated groups compared to the control groups. The test-susbtance-treated groups (Groups 2-4) and the 0 mg/kg/day negative control treated group (Group 1) showed a higher response than that of the 6 mg/kg/day Cyclophosphamide treated group, which was not unexpected as cyclophosphamide is a known immunosuppressant (significantly (p≤0.05) lower IgM levels at PND 56 in the positive control group compared to the negative control group).
The results indicated that the test-substance did not suppress the IgM response following administration when compared with the results obtained from the control animals.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity (physical development until adulthood)
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: offspring viability, neonatal health, developmental status at birth
Dose descriptor:
NOAEL
Remarks:
developmental neurotoxicity
Generation:
F1
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed at highest dose tested
Dose descriptor:
NOAEL
Remarks:
developmental immunotoxicity
Generation:
F1
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed at highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
Organ:
other: olfactory epithelium
Treatment related:
yes

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pup and litter observations were generally similar in all groups, with multiple nests noted for control and test item group litters. While ‘scattering’ of pups was noted for some litters at all dose levels and not in the control group, it was recorded for control litters in the F0 generation. In the absence of other effects on F1 litters, this was considered to be an unfortunate distribution and not an effect of the test item.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test item-related effects on pup survival indices during lactation.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean values for litter and pup weights were not significantly different, test item group values
being within 10% of control group values. The smallest litter and pup weights were recorded
at 800 mg/kg/day but the magnitude of difference from control was considered insufficient to
support a test item-related effect.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance data were similar in all groups. Nipples were not retained in males.
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related effects on the duration of gestation or Gestation Index.
The mean percentage of male pups per litter on LD 1 was also unaffected, with no trends evident.

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed at highest dose tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

F0 generation: Organ weights

Table 1: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F0 males)




























































































































































































Group



1



2



3



4



Dose (mg/kg/day)



0



50



200



800



No. animals examined



30



30



30



30



Terminal body weight (g)



486.1



501.1



474.5



459.4



% difference from control



NA



+3.1



-2.4



-5.5



Thyroid Gland



(30)



(30)



(30)



(30)



Absolute value (g)



0.02148



0.02482



0.02312



0.02637++



% difference from control



NA



+15.53142



+7.61831



+22.76183



% of body weight



0.00446



0.00494



0.00491



0.00576**



% difference from control



NA



+10.79192



+10.10130



+29.17267



% of brain weight



1.01944



1.15392



1.09583



1.25666++



% difference from control



NA



+13.19164



+7.49390



+23.27061



Kidney



(30)



(30)



(30)



(30)



Absolute value (g)



2.6299



2.7613



2.7863



3.3248 ++



% difference from control



NA



+4.9964



+5.9470



+26.4218



% of body weight



0.54436



0.55327



0.58890**



0.72606**



% difference from control



NA



+1.63632



+8.18195



+33.37799



% of brain weight



124.67748



128.26854



131.98862



158.48324**



% difference from control



NA



+2.88028



+5.86404



+27.11457



Liver



(30)



(30)



(30)



(30)



Absolute value (g)



14.6346



15.6058



15.3122



18.4433**



% difference from control



NA



+6.6359



+4.6299



+26.0250



% of body weight



3.01378



3.12248



3.23292**



4.01001**



% difference from control



NA



+3.60675



+7.27136



+33.05586



% of brain weight



693.16598



725.00322



725.57913



878.84113**



% difference from control



NA



+4.59302



+4.67610



+26.78654



Kruskal-Wallis & Dunn: + = p ≤ 0.05, ++ = p ≤ 0.01 Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable


 


Table 2: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F0 females)



















































































Group



1



2



3



4



Dose (mg/kg/day)



0



50



200



800



No. animals examined



30



30



30



30



Terminal body weight (g)



258.0



263.9



263.6



262.7



Liver



(30)



(29)



(30)



(29)



Absolute value (g)



13.6516



13.8466



14.4242



15.8284*



% difference from control



NA



+1.4288



+5.6597



+15.9455



% of body weight



5.27498



5.19145



5.45229



6.00124+



% difference from control



NA



-1.58338



+3.36140



+13.76810



% of brain weight



720.94328



712.09368



753.89009



829.49782*



% difference from control



NA



-1.22750



+4.56996



+15.5729



Kruskal-Wallis & Dunn: + = p ≤ 0.05, ++ = p ≤ 0.01 Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable


 


The only as adverse considered histopathological effect was the degeneration of the olfactory epithelium. The referring table is listed below.


Table 3: Microscopic Pathology (F0 Animals)































































Removal Reason(s): TERMINAL EUTHANASIA


Summary: Incidence


MaleFemale
050200800050200800
mg/kg/
day
mg/kg/
day
mg/kg/
day
mg/kg/
day
mg/kg/
day
mg/kg/
day
mg/kg/
day
mg/kg/
day
Group1Group2Group3Group4Group1Group2Group3Group4
Number of Animals3030303030293029

BODY CAVITY, NASAL


Degeneration; olfactory epithelium


...mild


...moderate



 


 


0


0


0



 


 


0


0


0



 


 


0


0


0



 


 


2


1


1



 


 


0


0


0



 


 


0


0


0



 


 


0


0


0



 


 


0


0


0



 


F1 generation: Organ weights


Table 4: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F1 Cohort 1A males)











































































































































Group



1



2



3



4



Dose (mg/kg/day)



0



50



200



800



No. animals examined



20



20



20



20



Terminal body weight (g)



355.1



368.9



366.5



345.2



% difference from control



NA



+3.9



+3.2



-2.8



Kidney (No. weighed)



(20)



(20)



(20)



(20)



Absolute value (g)



2.3487



2.5471



2.5450



2.6141*



% difference from control



NA



+8.4474



+8.3601



+11.3022



% of body weight



0.66327



0.69344



0.69637



0.75965**



% difference from control



NA



+4.54796



+4.98892



+14.53094



% of brain weight



117.27415



125.03116



127.14442



133.07809**



% difference from control



NA



+6.61442



+8.41640



+13.47606



Liver (No. weighed)



(20)



(20)



(20)



(20)



Absolute value (g)



13.0374



13.7026



14.0452



15.7124**



% difference from control



NA



+5.1026



+7.7305



+20.5184



% of body weight



3.65644



3.69577



3.82116



4.53905**



% difference from control



NA



+1.07561



+4.50481



+24.13826



% of brain weight



651.22680



671.34847



702.84981



799.75331**



% difference from control



NA



+3.08981



+7.92704



+22.80719



Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable


 


Table 5: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F1 Cohort 1A females)











































































































































Group



1



2



3



4



Dose (mg/kg/day)



0



50



200



800



No. animals examined



20



20



20



20



Terminal body weight (g)



212.2



212.0



216.0



209.6



% difference from control



NA



-0.1



+1.8



-1.2



Thyroid Gland



(20)



(20)



(20)



(20)



Absolute value (g)



0.01553



0.01677



0.01627



0.01800



% difference from control



NA



+8.01017



+4.78983



+15.90000



% of body weight



0.00732



0.00793



0.00758



0.00870*



% difference from control



NA



+8.44613



+3.64023



+18.84588



% of brain weight



0.82952



0.89298



0.86836



0.98988**



% difference from control



NA



+7.65002



+4.68199



+19.33080



Liver (No. weighed)



(20)



(20)



(20)



(20)



Absolute value (g)



7.9172



8.0360



8.6843



10.4670**



% difference from control



NA



+1.5012



+9.6891



+32.2060



% of body weight



3.71811



3.78564



4.01365



4.98153**



% difference from control



NA



+1.81621



+7.94848



+33.98002



% of brain weight



423.60244



428.49490



463.96970



575.46120**



% difference from control



NA



+1.15496



+9.52952



+35.84936



Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable


Other noteworthy organ weight differences are detailed in the Pathology phase report and were considered not test item-related.


 


F1 generation: Motor activity


Table 6: Motor activity















































 


Dose level (mg/kg/day)



Female



Female



Male



Female



50



200



800



800



Basic Movement



-



-14



-15



-24



Fine Movement



-



-



-16



-17



X Ambulation



-12



-26



-



-39



Y Ambulation



-19



-33



-16



-43



A dash (-) indicates absence of a test item-related change. Numerical values indicate the percentage difference of group mean values from the Overall control group.


Bold values were statistically significant from the control group (P ≤ 0.05/0.01).

Applicant's summary and conclusion