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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 November 2019 to 24 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
July 2018
Deviations:
no
Principles of method if other than guideline:
• OECD Guidance Document Supporting OECD Test Guideline 443 on the Extended One Generation Reproductive Toxicity Test, No. 151, July 2013.
• The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
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:
Study design according to decision number CCH-D-2114448635-42-01/F
- Premating exposure duration for parental (P0) animals: 10 weeks, as ten weeks exposure duration is supported by the lipophilicity of the substance (log Kow of 5.3) to ensure that the steady state in parental animals has been reached before mating.
- Basis for dose level selection: aim to induce systemic toxicity at the highest dose level. Dose levels were based on a 90-day repeated dose study, a OECD TG 414 (prenatal developmental toxicity) study (information provided by the Study Sponsor) and a DRF for this EOGRT study (CRL Study No. 20202555).
- Inclusion of extension of Cohort 1B to mate the Cohort 1B animals to produce the F2 generation. The use of the registered substance in the joint submission is leading to significant exposure of professionals and consumers. In addition, there are indications that the internal dose for the registered substance will reach a steady state in the test animals only after an extended period of exposure as the log Kow of the registered substance is 5.3, and there are weak indications of modes of action related to endocrine disruption from an available in vitro study (Schreurs et al., 2005).
- Termination time for F2: PND21 (no trigger for extension of the F2 generation).
- exclusion of developmental neurotoxicity Cohorts 2A and 2B: absence of triggers in dataset.
- exclusion of developmental immunotoxicity Cohort 3: absence of triggers in dataset.
- Route of administration: Since the substance to be tested is a liquid, the oral route of exposure was selected as this is a potential route of human exposure during manufacture, handling or use of the test substance as specified in the applicable guidelines.

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran
EC Number:
214-946-9
EC Name:
1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran
Cas Number:
1222-05-5
Molecular formula:
C18H26O
IUPAC Name:
4,6,6,7,8,8-hexamethyl-1,3,4,6,7,8-hexahydrocyclopenta[g]isochromene
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han (Crl:WI(Han) rat was chosen as the animal model for this study as it is an accepted rodent species for reproduction and developmental toxicity testing by regulatory agencies. Charles River Lyon has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Historical control data for the strain are available at the Test Facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6 wks; (F1)
- Weight at study initiation: (P) Males: 145.3 g to 204.3 g; Females: 104.4 g to 154.9 g
- Fasting period before study: no
- Housing: Pre-mating: 5 animals/sex/cage; Mating 1m + 1f housed together; Gestation: 5 males/cage and 1 female/cage; Lactation: 1 female with litter/cage; Post-weaning 5 animals/sex/cage. All animals were housed in plastic cages containing appropriate bedding (Cellulose bedding or dust-free sawdust made from spruce tree wood, analyzed once a batch for sawdust or twice a year for cellulose).
- Diet (e.g. ad libitum): Rat powdered complete diet ad libitum (Diet Reference No. A04C-10) sterilized by irradiation and analyzed by the supplier.
- Water (e.g. ad libitum): Softened and filtered (0.2 µm) mains drinking water was available ad libitum via an automatic watering system or bottles. Water is analyzed at least twice a year for bacterial and chemical contaminants.
- Acclimation period: 7 days between arrival and the start of dosing.
For psychological/environmental enrichment, animals were provided with items such as a wooden gnaw block and shredded paper, except when interrupted by study procedures/activities. No known contaminants were present in the diet, water, bedding and enrichment at levels which might have interfered with achieving the objective of the study.
ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 19 to 25
- Humidity (%): >35%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h (except during designated procedures)
IN-LIFE DATES: From: 18 November 2019 To: 24 July 2020

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a mixture in the control item at concentrations of 470, 825 and
1650 ppm for the pre-mating, mating and gestation periods of the F0 generation and after
weaning for the F1 generation. The concentrations were adjusted to 340, 600 and 1200 ppm
for LD1 to LD7, to 255, 450 and 900 ppm for LD8 to LD14, and to 205, 365 and 730 ppm for
LD15 to LD21-23 for the F0 and F1 (Cohort 1B) females.
The test item was stored in the container between 60-70°C for a minimum of 24 hours and then mixed well before use. The test item was heated gently again if necessary (due to solidification).
The test item was formulated in a form suitable for admixture in basic powdered diet. For each concentration, a weighed amount of test item was mixed into a small amount of diet in a blender to produce a pre-mixture of high test item concentration. The pre-mixture was then diluted to the final desired concentration with the necessary quantity of basic diet and mixed in a blender. The concentrations were fixed throughout the study with the exception of the lactation phases during which an adjustment of ppm was made based on the background data for body weight and food consumption.

DIET PREPARATION
- Rate of preparation of diet (frequency): Test item diet mixes were formulated weekly and divided into 2 aliquots, 1 stored refrigerated (2°C to 8°C) and the other at room temperature (15°C to 25°C) prior to use.
- Mixing appropriate amounts with (Type of food): Basic powdered diet (Diet Reference No. A04C P2.5, supplier SAFE)

- Storage temperature of food: Test item diet mixes were formulated weekly and divided into 2 aliquots, 1 stored refrigerated (2°C to 8°C) and the other at room temperature (15°C to 25°C) prior to use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): mixed in the diet
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 consecutive days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (GD0)
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol:-
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
concentration: day 1, week 11 and week 22 of dosing, all groups (samples collected from right, middle, left); week 16 of dosing 205 and 1650 ppm (sample collected from middle); week 33 of dosing, 0, 205 and 1650 ppm (sample collection from top, middle, bottom).
Homogeneity: day 1 of dosing, low and high dose (samples collected from right, middle, left); week 33 of dosing, 0, 205 and 1650 ppm (samples collected from top, middle, bottom).
Stability: week 16 of dosing, 4 and 8 days at room temperature, 4 days refrigerated (bag samples collected from middle).
Additional diet samples (collected at the top, middle and bottom position for evaluation of homogeneity) were prepared at 100 and 10000 ppm for evaluation of the homogeneity using a larger blender.
Analyses were performed by UPLC, using a validated analytical procedure (Test Facility Study No. 20202553).
Stability analyses performed previously in conjunction with Study No. 20202553 and demonstrated:
• A loss of 22% and 27% of the test item in the diets at 100 and 10000 ppm, respectively, when stored for 4 days at room temperature.
• A loss of 20% and 21% of the test item in the diets at 100 and 10000 ppm, respectively, when stored refrigerated (+2°C to +8°C) for 8 days.
• A stability of the test item in the diet of 25 days when stored frozen (-15°C to -25°C) at concentration range of 100 to 10000 ppm.
Formulations were analysed for achieved concentration of test item for Groups 2 to 4 and verification of the absence of test item in the control for Group 1. The homogeneity was confirmed between 100 to 10000 ppm (Charles River Study No. 20202553). Homogeneity was assessed for Groups 2 and 4 formulations by measuring the achieved concentration in the top or right, middle, and bottom or left samples.
Duration of treatment / exposure:
F0 animals (both sexes): 10 weeks before mating, throughout the mating period and up to the day before necropsy.
F0 animals (females): During gestation (the first day of gestation was designated as GD0) and at least 21 days after parturition, up to and including the day before scheduled necropsy (the first day of birth was designated as LD0).
Apparently unmated females for at least 23 days after the last day of the mating period.
F1 animals: During lactation (up to PND21), pups were not dosed directly but could potentially be exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, via spilled diet from the food hopper or when they started eating solid diet for themselves. After weaning (PND22), F1 animals were dosed up to and including the day before or the day of scheduled necropsy. The F1 surplus animals and F2 pups were not dosed.
Frequency of treatment:
Continuously (ad libitum)
Doses / concentrationsopen allclose all
Dose / conc.:
1 650 ppm (nominal)
Remarks:
group 4; F0 and F1 premating-mating-gestation-F1 after weaning, to give an approximate dose of 150 mg/kg bw/day
Dose / conc.:
825 ppm (nominal)
Remarks:
group 3; F0 and F1 premating-mating-gestation-F1 after weaning, to give an approximate dose of 75 mg/kg bw/day
Dose / conc.:
470 ppm (nominal)
Remarks:
group 2; F0 and F1 premating-mating-gestation-F1 after weaning, to give an approximate dose of 42.5 mg/kg bw/day
No. of animals per sex per dose:
F0: 25/sex/dose
F1 after allocation into cohort: 20 pups/sex/group/cohort
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on a 90-day repeated dose study, a OECD TG 414 (prenatal developmental toxicity) study and a DRF for this EOGRT study.
Considering systemic toxicity, a relative liver weight increase between 10%-20% is considered the maximum tolerable dose in an EOGRT study. A liver weight increase was anticipated for doses between 62.5 (DRF for EOGRT) and 150 mg/kg bw (90-day repeated dose). Also, relative thyroid weight was expected to increase to a similar extent as for liver weight.
The maximum tolerable decrease in pup weight should not exceed the 10% level. In the prenatal dev tox study no effects were seen on fetal weight at 150 mg/kg bw. In the DRF at 62.5 mg/kg bw, pup weights were within the historical control data, whilst at 225 mg/kg bw, a decrease was below the historical control data range on PND4 and PND14 but not at PND21. No effects on fertility were seen.
Based on systemic effects it was anticipated that 120 mg/kg bw presents the maximum tolerable dose. Transient developmental effects such as reduced pup weight may be seen at 120 and/or 60 mg/kg bw but not at 30 mg/kg bw.
Estimated to give approximate doses of 42.5, 75 and 150 mg/kg body weight/day, taking into account:
• Body weight of 220 g and food consumption of 20 g/animal/day for the premating, mating and gestation periods for the F0 animals or during the whole dosing period for the F1 animals.
• Body weight of 280 g and food consumption of 35 g/animal/day for the period from LD1 to LD7.
• Body weight of 300 g and food consumption of 50 g/animal/day for the period from LD7 to LD14.
• Body weight of 315 g and food consumption of 65 g/animal/day for the period from LD14 to LD21.
The dose levels were deliberately set at +25% when compared with the target doses to take into account a loss of test item of approximately 25% (see stability data) according to analytical results 4 days after test diet formulation (at room or refrigerated temperature).

- Fasting period before blood sampling for clinical biochemistry: o/n F0 and F1 cohort 1A animals surviving to scheduled necropsy
- Other:

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily. All animals were observed at least twice daily to detect any which were dead or moribund (except on the days of receipt and necropsy when frequency was at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least on each weighing day. Towards the end of the gestation, females were examined daily for signs of parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: Each male was weighed at least weekly.
Each female was weighed as follows:
• At least weekly during pre-mating and mating periods (only pre-mating data are reported).
• On GD0, 4, 7, 11, 14, 17 and 20.
• On LD1, 4, 7, 10, 14, 17 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption of males was recorded twice weekly during the pre-mating period.
Food consumption of females was recorded for the following periods:
• Twice weekly during the pre-mating period.
• Daily during gestation but reported as GD0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17, 17 to 20.
• Daily during lactation but reported as LD1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21.
Food consumption during mating was not recorded for practical reasons.

WATER CONSUMPTION: No

HEMATOLOGY: Yes
Time point: Day of necropsy
blood sampling from retro-orbital sinus under isoflurane after o/n fasting (about 16h), 10/sex/group
Parameters analysed: Red blood cell count, Hemoglobin, Pack cell volume (hematocrit), Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Reticulocyte count, Platelet count, Total white blood cell count, Neutrophil count, Lymphocyte count, Monocyte count, Eosinophil count, Basophil count, Large unstained cells.

COAGULATION: Yes
Time point: Day of necropsy
blood sampling from retro-orbital sinus under isoflurane after o/n fasting (about 16h), 10/sex/group
Parameters analysed: Activated partial thromboplastin time, Prothrombin time.

CLINICAL CHEMISTRY: Yes
Time point: Day of necropsy
blood sampling from retro-orbital sinus under isoflurane after o/n fasting (about 16h), 10/sex/group
Parameters analysed: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total bilirubin, Urea, Chloride, Creatinine, Calcium, Inorganic phosphorus, Total protein, Albumin, Globulin, Albumin/Globulin ratio, Glucose, Total cholesterol, Triglycerides, Sodium, Potassium, Bile acids.

URINALYSIS: Yes
Time point: Day of necropsy
Samples collected on Day of Necropsy (about 16h in metabolism cages; deprived of food and water but gavage with approximately 20 mL/kg of filtered water), 10/sex/group
Parameters analysed: Volume, Specific gravity, Appearance (color, turbidity), pH, Bilirubin, Urobilinogen, Ketones, Blood, Protein, Glucose, Sediment: White blood cells, Red blood cells, Casts, Epithelial cells, Crystals, Bacteria.

HORMONE ANALYSIS: Yes
Time point: Day of necropsy
Blood sampling from retro-orbital sinus under isoflurane after o/n fasting (about 16h), 10/sex/group, between 7.00 and 10.30 AM
Parameters analysed: T4 and/or TSH


Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal smears continued for those females with no evidence of copulation until termination of the mating period.
On the day of terminal necropsy or for any moribund animal, a vaginal lavage was also taken to determine the cycle stage.
Sperm parameters (parental animals):
Sperm analysis was performed for all surviving F0 males using automated equipment (Hamilton Thorne Research IVOS). The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility.
Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The numbers of sperm with each type of abnormality was recorded.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
detailed clinical observations at least once daily, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (on PND1, 4, 7, 14, 21), physical or behavioural abnormalities, anogenital distance (AGD, on PND1), pup weight on the day of AGD, presence of nipples/areolae (PND13) in male pups.

GROSS EXAMINATION OF DEAD PUPS:
For any pups found dead or euthanized moribund, the stomach was examined for the presence of milk and defects or cause of death were evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs. There were no external abnormalities collected.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

F1 GENERATION FROM WEANING ONWARD: Cohorts 1A, 1B and 1C (60 Animals/Sex/Group)

CAGE SIDE OBSERVATIONS: Yes, Cohorts 1A, 1B, 1C
- Time schedule: at least daily. Mortality/moribundity checks at least twice daily throughout the study (animals not removed from cage).

DETAILED CLINICAL OBSERVATIONS: Yes, Cohorts 1A, 1B, 1C
- Time schedule: at least on each weighing day. Animals were observed (full clinical observation) at least daily during the study.

BODY WEIGHT: Yes, Cohorts 1A, 1B, 1C
- Time schedule for examinations: At least weekly from weaning onwards. This started on a specific date on which all pups were at least at PND21.
In addition, the body weight was recorded of each female on the day of acquisition of vaginal opening and of each male on the day of acquisition of balanopreputial skinfold cleavage.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, Cohorts 1A, 1B, 1C
- Twice weekly from Day 1 and on the same day prior to necropsy for some batches of males (Cohorts 1A and 1C were necropsied over 4 and 2 days, respectively).

VAGINAL OPENING: Yes, Cohorts 1A, 1B, 1C
Daily for all females from PND28 onwards, continued until vaginal opening was detected.

BALANOPREPUTIAL SEPARATION: Yes, Cohorts 1A, 1B, 1C
Daily for all males from PND38 onwards, continued until balanopreputial skinfold cleavage was detected.

STAGE OF ESTROUS CYCLE DETERMINATION: Yes, Cohorts 1A and 1B
On the day of scheduled necropsy, a vaginal lavage was taken for examination of cytology.


COHORT 1A
ONSET OF ESTROUS CYCLING DETERMINATION: Yes
20 females/group: Daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was continued until the first estrus was determined, in order to determine the time interval between these 2 events. Daily vaginal lavage was also performed during at least
2 weeks before necropsy. Estrous stages and cycles were evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage.

SPERM ANALYSIS: Yes
All Cohort 1A surviving males. The left cauda epididymis was sampled and weighed and used for the assessment of caudal sperm reserves and sperm motility, including progressive motility. Sperm morphology was evaluated (at least 200 sperm per sample, where possible). The numbers of sperm with each type of abnormality was recorded.

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS: Yes
Cohort 1A animals


COHORT 1B (20 females/group)
BODY WEIGHT: Yes
Mated females were weighed at least on GD0, 4, 7, 11, 14, 17 and 20 and on LD1, 4, 7, 10, 14, 17 and 21.

FOOD CONSUMPTION: Yes
Food consumption was not determined for males after the start of the mating period and for females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured daily but reported during gestation as GD0 to 4, 4 to 7, 7 to 11, 11 to 14, 14 to 17, 17 to 20 and during lactation as LD1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21.

ESTROUS CYCLING DETERMINATION: Yes
Daily vaginal lavage was performed from the day after the start of the mating period until evidence of copulation was observed. Vaginal smears continued for those females with no evidence of copulation until termination of the mating period.

CLINICAL PATHOLOGY (F1 Animals Cohort 1A)

HEMATOLOGY: Yes
Time point: Day of necropsy
blood sampling from retro-orbital sinus under isoflurane after o/n fasting (about 16h), 10/sex/group
Parameters analysed: Red blood cell count, Hemoglobin, Pack cell volume (hematocrit), Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Reticulocyte count, Platelet count, Total white blood cell count, Neutrophil count, Lymphocyte count, Monocyte count, Eosinophil count, Basophil count, Large unstained cells.

COAGULATION: Yes
Time point: Day of necropsy
blood sampling from retro-orbital sinus under isoflurane after o/n fasting (about 16h), 10/sex/group
Parameters analysed: Activated partial thromboplastin time, Prothrombin time.

CLINICAL CHEMISTRY: Yes
Time point: Day of necropsy
blood sampling from retro-orbital sinus under isoflurane after o/n fasting (about 16h), 10/sex/group
Parameters analysed: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total bilirubin, Urea, Chloride, Creatinine, Calcium, Inorganic phosphorus, Total protein, Albumin, Globulin, Albumin/Globulin ratio, Glucose, Total cholesterol, Triglycerides, Sodium, Potassium, Bile acids.

URINALYSIS: Yes
Time point: Day of necropsy
Samples collected on Day of Necropsy (about 16h in metabolism cages; deprived of food and water but gavage with approximately 20 mL/kg of filtered water), 10/sex/group
Parameters analysed: Volume, Specific gravity, Appearance (color, turbidity), pH, Bilirubin, Urobilinogen, Ketones, Blood, Protein, Glucose, Sediment: White blood cells, Red blood cells, Casts, Epithelial cells, Crystals, Bacteria.

HORMONE ANALYSIS: Yes
Time point: Day of necropsy, 10/sex/group
blood sampling under isoflurane from retro-orbital sinus after o/n fasting (about 16h), 10/sex/group, between 7.00 and 10.30 AM
Parameters analysed: T4 and/or TSH



F1 GENERATION PND4 PUPS
HORMONE ANALYSIS: Yes
Time point: PND4, 1 pup/sex/litter
blood sampling by decapitation, between 7.00 and 10.30 AM
Parameters analysed: T4 and/or TSH
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: After a minimum of 12 weeks of dosing (after successful mating).
- Maternal animals: Females which delivered on LD 22-24; Females that failed to deliver on GD 26 (with evidence of mating) or approximately 26 days after the last day of the mating period (without evidence of mating); Dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organs of the reproductive system.
The numbers of former implantation sites were recorded for all paired females. The uterus of all adult females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites. The number of corpora lutea was counted. Any foetuses were examined externally where possible and discarded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissue collection and preservation according to guideline
Postmortem examinations (offspring):
SACRIFICE
Unscheduled deaths F1 and F2 generation: Pups (extra pups on PND4 or any moribund pups) were euthanized by intraperitoneal injection of sodium pentobarbitone.
Pups (including any found dead or euthanized moribund) were necropsied. For any pups found dead or euthanized moribund, the stomach was examined for the presence of milk and defects or cause of death were evaluated, if possible.
Each pup was sexed and examined for external defects with special attention being paid to the external reproductive organs. There were no external abnormalities collected.

Scheduled euthanasia F2 generation: Scheduled necropsy of the F2 animals from Cohort 1B was conducted on PND21-23 (sodium pentobarbital, not fasted). Organ weight was determined of brain, liver, thyroid glands (+parathyroid glands), spleen and thymus from 10 animals/sex/group. Tissue was collected from these organs as well as from mammary gland and from macroscopic lesions. All animals were subjected to a limited macroscopic examination, with special attention being paid to the reproductive organs.

Scheduled necropsy of cohort surplus on PND21 (not fasted). Terminal body weight was recorded. On PND21, blood samples were collected between 7.00 and 10.30 AM from all animals by aorta puncture under anaesthesia using isoflurane as part of the necropsy for measurement of Thyroid-Stimulating Hormone (TSH) and Thyroxine (T4).
All animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Brain, liver, thyroid glands (together with parathyroid glands), spleen and thymus were weighed at scheduled necropsy for 10 animals/sex/group. Tissue from mammary gland was collected.

F1 generation from weaning onwards:
Cohort 1A (fasted): ≤ 20/sex/group, scheduled euthanasia on PND 85 to PND 93 (by means of carbondioxide inhalation and exsanguination), determination of terminal BW, organ weight (according to guideline); histology and histopathology according to guideline for groups 1 and 4, gross lesions and target tissues for groups 2 and 3.
Cohort 1B (non-fasted); ≤ 20/sex/group, scheduled euthanasia males after successful mating, females LD 21-23, determination of terminal BW, organ weight (according to guideline), histology and histopathology from reproductive organs of groups 1 and 4 processed to block stage.
Cohort 1C (non-fasted); ≤ 20/sex/group, scheduled euthanasia after sexual maturation, only necropsy
Cohort surplus (non-fasted); ≤ 20/ sex/group, scheduled euthanasia PND 22, determination terminal BW, organ weight and tissue collection (10/sex/group).
Spare animals: scheduled euthanasia PND 21; necropsy. Animals were examined externally, with particular attention to the external reproductive genitals, and sex was determined. Descriptions of
all external abnormalities were recorded. There were no external abnormalities collected.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues according to guideline] were prepared for microscopic examination and weighed, respectively.
Statistics:
The best transformation for the data (none, log or rank) was determined depending upon:
• The kurtosis of the data.
• The probability of the Bartlett's test for homogeneity of the variances.
• An assessment of whether the size of the groups are approximately equal or not.
Non- or log-transformed data were analyzed by parametric methods.
Rank transformed data were analyzed using non-parametric methods.
Data were then analyzed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by Analysis of Variances (ANOVA) for parametric data or Kruskal-Wallis test for non parametric data.
If no trend was found and means were not homogeneous, the data were analyzed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
Estrous cycles, anogenital distance, sperm analysis and pre-coital interval data were analyzed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogeneous variances and a normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test if the ANOVA was significant. Data showing non-homogeneous variances or a non-normal distribution in at least 1 group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test if the Kruskal-Wallis test was significant.
Reproductive indices:
Pre-coital interval (in days): Sum of days until successful insemination/Number of inseminated females

Male and female copulation index (in %): (Number of inseminated females/Number of paired females ) x 100

Male and female fertility index (in %): (Number of pregnant females/Number of inseminated females) x 100

Pre-birth loss (in %): ( Number of implantations - Number of offspring born/Number of implantations) x 100

Irregularity index: Mean standard deviation of length of the oestrous cycle/√ (length of the oestrous cycle)

Days in estrus: (Number of estrus days/Number of smears) x 100
Offspring viability indices:
Live birth index (in %): (Number of pups born alive/Number of pups born) x 100

Viability index (in %): (Number of pups alive on PND4/Number of pups alive at birth) x 100

Lactation index (in %): (Number of pups alive on PND 21/Number of pups alive on PND4 (after culling)) x 100

Sex ratio (proportion of male pups in %): (Number of males/Number of pups) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related clinical sign in any group for either sex.
Isolated clinical signs were noted including localised hairloss, hypersalivation, malocclusion, scabs, soft or no faeces, sores, swelling, piloerection, red stained fur, palor, or bent tail amongst treated and control groups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item-related death in any group.
One female in the 470 ppm group was euthanized with its litter for ethical reason on LD3. This female was thin and had piloerection and all pups were thin with no milk noted in the stomach at necropsy. Another female lost its entire litter and was therefore necropsied in the 825 ppm group. In the absence of a similar event in the high dose group, this was considered incidental.
There was macroscopic finding for either of these females.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related effect on body weight change in any group for either sex.
Despite some transient statistically significant changes noted for both sexes, overall mean body weight gain during the pre-mating period was comparable with that of the control group in all treated groups for both sexes.
There was a slightly but statistically significantly lower mean body weight gain at 1650 ppm (-9%) when compared with the control group during the gestation period (GD0 to GD20). Mean body weight was therefore slightly but statistically significantly lower at 1650 ppm on GD20 when compared with the control group (-5%). This was considered likely related to the lower mean litter weight in this group as noted on PND1.
Mean body weight gain was lower at 470 ppm during the lactation period (-17%) when compared with the concurrent control but higher at 825 and 1650 ppm. In the absence of dose-relationship, this was considered incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test-item related effect on mean food consumption for either sex during the premating period or for the females during the gestation and lactation periods in any group.
There was an apparent dose-related lower mean food consumption noted mainly towards the end of the lactation period that reached statistical significance at 825 and 1650 ppm (on average -5% and -8% from LD1 to LD21, respectively) when compared with the control group. Mean body weights were higher during the lactation period in both these groups so this finding was likely related to the spillage noted at a higher incidence in the control group that disproportionally influenced the mean control values and therefore considered incidental.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes amongst hematological parameters in any group for either sex.
Several statistically significant differences arising between control and treated animals, such as decreased haemoglobin concentration and haemetatocrit at 1650 ppm or decreased monocytes count in all treated groups for males, were considered not toxicologically relevant as they were of low magnitude and remained within the Historical Control Data (HCD) range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant serum clinical chemistry changes for either sex in any group.
There were dose-related decreases in serum protein and globulin concentrations for males in all treated groups when compared with the concurrent control. These changes were minor, remained within the HCD range, observed for males only and in the absence of similar findings for F1 animals (Cohort 1A) they were considered not toxicologically relevant.
Several other statistically significant differences arising between control and treated animals, such as decreased inorganic phosphorus concentration in all treated male groups, increased urea and alkaline phosphatase concentrations at 1650 ppm for males or decreased glucose concentration at 1650 ppm for females, were considered not toxicologically relevant as they were of low magnitude and/or they occurred in the absence of dose-relationship.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related lower mean total T4 serum concentration for males that reached statistical significance at 825 and 1650 ppm which was associated at 1650 ppm with a minor increase in mean thyroid weight when compared with the control group. However all mean values (5.4, 4.6 and 4.5 μg/dL) remained within the HCD range (4.1 to 7.8 μg/dL; p > 0.05) and therefore considered not toxicologically relevant.
There was no test item-related effect on serum TSH concentration for either sex or on total serum T4 concentration for females in any group.
See Table in 'any other information on results, incl tables.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no test item-related effect on urine analysis parameters in any group for either sex.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the thyroid gland, a bilateral diffuse follicular cell hypertrophy was observed in males and females at all doses. This finding was dose-related and was minimal or mild. It correlated with an increased organ weight and was considered related to HHCB administration.
See Table in 'any other information on results, incl tables.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related effect on the mean length and regularity of estrous cycles. The percentage of females acyclic or with acyclic period was statistically significantly higher at 1650 ppm (24%) but the value remained within the HCD range (0% to 33%) and was considered incidental.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There was no test item-related effect on mean epydidymal sperm count, sperm morphology or motility, including the percentage of progressively motile sperm, in any group.
Reproductive performance:
no effects observed
Description (incidence and severity):
The majority of females in all groups showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore comparable in all groups.
There was no test item-related effect on mating performance in any group. After 2 weeks of mating, all pairs of animals mated in all groups.
There was no test item-related effect on fertility in any group. All mated females became pregnant with the exception of 1 female in the 470 ppm group. This isolated case was incidental.
There was no test item-related effect on the gestation index and duration of gestation in any group.
There were 25, 24, 25 and 25 pregnant females in each of the control, 470, 825 and 1650 ppm groups, respectively, all of which successfully completed delivery of liveborn pups. The mean duration of gestation was approximately 22 (22.2-22.3) days in each group.

There was no test item-related effect on pre-birth loss in any group.
Although lower than in the concurrent control group, the mean number of implantations was above the mean HCD value (12.2) in all treated groups (12.8, 12.6 and 12.3 at 470, 825 and 1650 ppm, respectively). The mean numbers of pups delivered in the treated groups (11.7, 11.0 and 11.8 at 470, 825 and 1650 ppm, respectively) were comparable with that of the control group (12.0) and/or the historical control data mean (11.1).
The mean percentage of pre-birth loss in the treated groups (8.6%, 12.4% and 5.5% at 470, 825 and 1650 ppm, respectively) was consequently lower when compared with the control group (10.1%) and/or within the HCD range (2.0% to 20.7%).

Details on results (P0)

There was no evidence for HHCB-related reproductive toxicity in the F0 generation.
Systemic effects of the F0 generation were limited to bilateral diffuse follicular cell hypertrophy of the thyroid gland observed for males and females at all doses. This finding was dose-related and was minimal or mild, correlated with an increased organ weight and a minor dose-related decrease in mean total T4 serum concentration for males (remained within the HCD range). These findings were considered not adverse in view of the low magnitude of the increased thyroid weight (maximum +24% of absolute weight for females at 1650 ppm versus control group). There were no macroscopic or other microscopic findings and no effects on serum thyroid hormones (T4 and TSH), clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) or other organ weights for F0 animals at any dosage level.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 650 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
Cohorts 1A, 1B and 1C - Post-Weaning
There was no test item-related clinical sign in any group for either sex.
Isolated clinical signs noted amongst treated and control groups including vocalisation, bent tail, scabs, broken tooth, chromodacryorrhea, localised or extensive hairloss, sores, piloerection lacrimation or partly closed eyes, were considered incidental or related to the pregnancy status of the females.
Mortality:
no mortality observed
Description (incidence):
Cohorts 1A, 1B and 1C - Post-Weaning
There was no unscheduled death in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Cohorts 1A, 1B and 1C - Post-Weaning
There was a dose-related reduction in mean body weight gain in all groups prior to mating (males and females) and during the gestation period (Cohort 1B), considered not toxicologically significant in view of the low magnitude of the changes and considering that the terminal mean body weight for males at 1650 ppm on Day 120 was comparable with the HCD.
Males:
There was an overall statistically significant dose-related lower mean body weight gain in all treated groups from Day 1 to Day 120 (-8%, -12% and -18% at 470, 825 and 1650 ppm, respectively - Cohort 1B) and from Day 1 to Day 64 (-7%, -10% and -16% at 470, 825 and 1650 ppm, respectively - Cohorts 1A and 1B) and a lower mean body weight gain in all groups from Day 1 to Day 57 (-8%, -8% and -14% at 470, 825 and 1650 ppm, respectively - Cohorts 1A, 1B and 1C) when compared with the control group.
Mean absolute body weights were consequently lower with a maximum change on Day 120 (-8%, -11% and -17% at 470, 825 and 1650 ppm, respectively - Cohort 1B) when compared with the concurrent control group but the mean value at 1650 ppm (396.3 g) was comparable with the HCD (432.6 ± 37.76).
Females:
Prior to mating, there was an overall dose-related lower mean body weight gain (Day 1 to Day 72) for Cohort 1B females (-5%, -5% and -9%, at 470, 825 and 1650 ppm, respectively) that reached statistical significance at 1650 ppm when compared with the control group. There was no clear differences in mean body weight gain for females from Day 1 to Day 57 (Cohorts 1A, 1B and 1C) and from Day 1 to Day 64 (Cohorts 1A and 1B) in any group when compared with the concurrent control.
There was an overall dose-related lower mean body weight gain during the gestation period at 825 and 1650 ppm (-8% and -16%, respectively - Cohort 1B) that reached statistical significance at 1650 ppm when compared with the control group considered not toxicologically significant in view of the low magnitude of the change.
There was an overall non-dose-related higher mean body weight gain in all treated groups during the lactation period when compared with the control group (+21%, +21% and +5% at 470, 825 and 1650 ppm, respectively). However, mean body weight gain on LD21 remained statistically significantly lower at 1650 ppm when compared with the control group (-7%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Cohorts 1A, 1B and 1C) - Post-Weaning
Males:
There was an overall statistically significant dose-related lower mean food consumption in all treated group when compared with the control group from Day 1 to Day 57 (Cohorts 1A, B and C), Day 1 to Day 64 (Cohorts 1A and B) and Days 1 to 72 (Cohort 1B) with a maximum change from Days 1 to 72 (-5%, -7% and -10% at 470, 825 and 1650 ppm, respectively).
Females:
Prior to mating, there was no clear test item-related effect on mean food consumption in any group from any cohort (difference < 5% when compared with the control group).
There was an overall statistically significant dose-related lower mean food consumption during the gestation period (-6%, -7% and -12% at 470, 825 and 1650 ppm, respectively - Cohort 1B) when compared with the control group.
Mean food consumption was comparable between treated and control groups during the lactation period.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A - PND85-93
There were no test item-related changes amongst the hematological parameters in any group for either sex.
Dose-related statistically significant higher lymphocytes count and total white blood cells count were noted at 825 and 1650 ppm for females. This was not considered toxicologically relevant in view of the low magnitude of the change and given that mean values remained within the HCD.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A - PND85-93
There was a dose-related increase of albumin concentration in all treated groups that reached statistical significance at 1650 ppm and an increase of albumin/globulin ratio in all treated male groups when compared with the control group. All values were within the HCD and/or no similar findings were observed for F0 animals and thus considered incidental.
Other statistically significant differences arising between control and treated animals, such as increased urea concentration for both sexes at 1650 ppm and females at 825 ppm and increased creatine concentration for females at 1650 ppm, were considered not toxicologically relevant as they were of low magnitude and within the historical control data.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A - PND85-93
For males, there was a dose-related lower mean total serum T4 level (6.2, 5.6 and 4.4 µg/dL) that reached statistical significance at 1650 ppm when compared with the control group (6.7 µg/dL) and a non statistically significant slightly increased serum TSH level at 1650 ppm (0.28 μIU/mL) when compared with the control group (0.15 μIU/mL). These findings were potentially related to increased thyroid weight but the values remained within the HCD range (0.04 to 0.54 μIU/mL for TSH level and 4.2 to 8.2 µg/dL for total T4 level; p < 0.05), so there was considered to be no toxicological significance. There was no clear difference in TSH levels in any group for males.
For females, there was a statistically significantly lower serum TSH level for females at 1650 ppm when compared with the concurrent control. Mean TSH levels for females was disproportionately influenced by 1 female with a high value of 6.08 μIU/mL. After excluding this female, the mean value (0.22 μIU/mL) was slightly above the HCD range (0.02 to 0.20 μIU/m; p < 0.05) and therefore likely related to the increased thyroid weight noted in this group. There was no difference in serum TSH levels for females at 470 and 825 ppm or in serum T4 levels in any group.
See table in 'any other information on results incl tables'.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Cohort 1A PND85-93
There was no test item-related effect on urine analysis parameters in any group for either sex .
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
Cohort 1A
There was no test item-related effect on the immunophenotyping of splenocytes in any group for either sex from Cohort 1A.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohorts 1A and 1B and Surplus
In the thyroid gland, higher mean absolute and relative organ weights were noted in male and female groups from Cohorts 1A and 1B. A relationship to HHCB was considered likely at the high dose in males (1650 ppm), where it correlated with follicular cell hypertrophy. In females, there was no histopathologic correlate in the thyroid gland, and a relationship to HHCB administration was therefore considered equivocal.
In the liver, higher mean relative organ weights were noted in male given 1650 ppm from both Cohorts 1A and 1B. The absolute values did not show relevant differences, due to the lower terminal body weights in these groups. These higher liver weights were considered related to an adaptive response to the administration of HHCB at the high dose in males (1650 ppm). Of note, slightly higher liver weights (relative to body) were present in high dose males and females from the cohort surplus (euthanized on PND21).
The lower mean adrenal gland weight noted in all treated female groups from Cohort 1A was statistically significant but did not have any histopathologic correlation, and was not present in females from Cohort 1B. These organ weight differences were considered incidental and unrelated to administration of HHCB.
Of note, slightly higher mean relative liver weights were noted in high dose males and females from the cohort surplus (animals euthanized on PND21). There were no relevant differences to be noted in the thyroid gland weights.
See table in 'any other information on results incl tables'.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Cohorts 1A, 1B, 1C and Surplus
There was no test item-related macroscopic changes in any cohort.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
In the thyroid gland, minimal bilateral diffuse follicular cell hypertrophy was observed in high dose males (1650 ppm). This was correlated with an increased organ weight and was considered related to HHCB administration. In females, the incidence of follicular cell hypertrophy did not show relevant differences between controls and treated groups.
In the liver, minimal diffuse centrilobular hypertrophy was observed in high dose males (1650 ppm). This finding correlated with an increased organ weight and was considered related to HHCB administration.
Of note, the histopathologic evaluation of the testes and the quantitative evaluation of ovarian follicles and corpora lutea did not show any difference between high dose and control groups.
See table in 'any other information on results incl tables'.
Histopathological findings: neoplastic:
no effects observed
Details on results:
There was no evidence for HHCB-related reproductive toxicity in the F1 generation (Cohort 1B).
For F1 animals, there was a dose-related reduction in mean body weight gain prior to mating in all groups (males and females) and during the gestation period (Cohort 1B) together with a slight reduction in mean food consumption, considered not toxicologically significant in view of the low magnitude of the changes and considering that the terminal mean body weight for males at 1650 ppm on Day 120 was comparable with the HCD.
At necropsy, higher mean absolute and relative thyroid weights were noted for male and female groups from Cohorts 1A and 1B and correlated with a minor increase in TSH level for females at 1650 ppm and a slight dose-related decrease in all groups in T4 levels for males when compared with the concurrent control. A relationship with HHCB was likely at 1650 ppm for males but considered not adverse in view of the low magnitude of the thyroid weight increase, where it correlated with follicular cell hypertrophy. For females, there was no histopathologic correlate in the thyroid gland, and a relationship with HHCB administration was therefore considered equivocal. In the liver, higher mean relative organ weights were noted for males given 1650 ppm from Cohorts 1A and 1B and were considered not adverse in view of the low magnitude of the change and in the absence of any histology findings. There were no test item-related macroscopic or other microscopic findings and no other differences in organ weights for F1 animals at any dosage level. See table in 'any other information on results incl tables'.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Cohort 1A. Mean estrous cycles lengths for the F1 females in the treated groups (4.0 , 4.0 and 3.9 at 470, 825 and 1650 ppm, respectively) during the last 2 weeks of dosing were comparable to the control group value (4.0). In addition, the percentage of females cycling in the treated groups (31.6%, 30.3% and 30.0% at 470, 825 and 1650 ppm, respectively) were comparable with that of the concurrent control group (31.3%).
The duration from vaginal opening to first estrus in the treated groups (4.0, 2.2 and 3.2 days at 470, 825 and 1650 ppm, respectively) was comparable with that of the control group (3.4 days).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Cohort 1A. There was no test item-related effect on mean sperm counts, sperm morphology or motility, including the percentage of progressively motile sperm, in any group. All values were comparable with the concurrent control and/or within the historical control data range. Similarly, mean cauda epididymis weights were comparable in all groups.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1B. There was no test item-related effect on the gestation index and duration of gestation in any group. There were 19, 20, 19 and 19 pregnant females in each of the control, 470, 825 and 1650 ppm groups, respectively, all of which successfully completed delivery of liveborn pups.
There was no test item-related effect on pre-birth loss in any group.
The mean percentage of pre-birth loss in the treated groups (8.2%, 4.7% and 4.8% at 470, 825 and 1650 ppm, respectively) was lower when compared with the control group (6.5%) and/or within the HCD range (2.0% to 20.7%).
There was a slightly lower mean number of implantations in the 1650 ppm (11.3) group when compared with the concurrent control (12.5) but the value remained within the HCD range (11.0 to 12.8 for main studies) and therefore considered incidental. The mean numbers of pups delivered was consequently slightly lower at 1650 ppm (10.7) when compared with the concurrent control (11.6) but remained within the HCD range (9.9 to 11.8 for main studies) and thus considered incidental.
Mean number of implantations/pups delivered at 470 and 825 ppm (12.0/11.0 and 12.1/11.5, respectively) were comparable with that of the HCD mean (12.2/11.1).

Details on results (P1)

In the F1 generation, there was no test item-related effect on sexual maturation or on reproductive development (Cohorts 1A and 1B), as indicated by estrous cyclicity, sperm parameters, microscopic examination or reproductive performance.

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 650 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects at the highest dose tested

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
F1 pups during lactation. There was no test item-related clinical signs amongst the offspring in any group.
Isolated clinical signs were noted including occasional pale/cyanotic/cold pups, hematoma, complete or partial absence of the tail, weakness, thinness, incomplete hair growth, scabs or sores. In view of their nature and/or sporadic occurrence amongst treated and control groups, these findings were considered incidental.

Cohorts 1A, 1B, 1C - post weaning.
There was no test item-related clinical sign in any group for either sex.
Isolated clinical signs noted amongst treated and control groups including vocalisation, bent tail, scabs, broken tooth, chromodacryorrhea, localised or extensive hairloss, sores, piloerection lacrimation or partly closed eyes, were considered incidental or related to the pregnancy status of the females.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 pups. There was no test item-related effect in any group on the number of live offspring at birth compared with the total number of offspring born (live birth index).
There were 0, 1, 0 and 0 stillborn pups in the control, 470, 825 and 1650 ppm groups, respectively.
There was no test item-related effect on the number of live offspring on PND4 before culling compared to the number of offspring on PND0 (viability index).
For females that raised offspring to weaning, there were 6, 5, 5 and 2 dead, missing or cannibalized pups from 5, 4, 4 and 2 litters, respectively, in the control, 470, 825 and 1650 ppm groups, respectively, between PND0 and PND4.
There was no test item-related effect on the number of live offspring at weaning (PND21) compared to the number of live offspring on PND4 after culling.
There were 0, 0, 1 and 0 dead, missing or cannibalized pups in the control, 470, 825 and 1650 ppm groups, respectively. The lactation index was therefore comparable in all groups (100%, 100%, 99.5% and 100% at 0, 470, 825 and 1650 ppm, respectively).
Cohorts 1A, 1B, 1C - post weaning.
There was no unscheduled death in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 pups during lactation. There was a slight dose-related lower mean pup body weight in the 825 and 1650 ppm groups when compared with the control group and the HCD mean on PND1, maximally -6.1%/-8.6% for males at 825 ppm and -8.9%/-11.3% for males at 1650 ppm.
The difference was persistent throughout the lactation period in the 1650 ppm group with a maximum difference of -11.2%/-8.0% for males on PND21.
However this finding was not considered adverse given that the mean pup values at 1650 ppm (48.2 g for females and 49.9 g for males) remained within or very close to the HCD range on PND21 (49.7 g to 57.6 g for males and 48.8 g to 56.0 g for females).
The difference noted at 470 and 825 ppm on PND21 when compared with the control group and HCD mean was considered not adverse in view of the recovery noted from PND1 to PND21.

Cohorts 1A, 1B, 1C - post weaning.
There was a dose-related reduction in mean body weight gain in all groups prior to mating (males and females) and during the gestation period (Cohort 1B), considered not toxicologically significant in view of the low magnitude of the changes and considering that the terminal mean body weight for males at 1650 ppm on Day 120 was comparable with the HCD.
Males:
There was an overall statistically significant dose-related lower mean body weight gain in all treated groups from Day 1 to Day 120 (-8%, -12% and -18% at 470, 825 and 1650 ppm, respectively - Cohort 1B) and from Day 1 to Day 64 (-7%, -10% and -16% at 470, 825 and 1650 ppm, respectively - Cohorts 1A and 1B) and a lower mean body weight gain in all groups from Day 1 to Day 57 (-8%, -8% and -14% at 470, 825 and 1650 ppm, respectively - Cohorts 1A, 1B and 1C) when compared with the control group.
Mean absolute body weights were consequently lower with a maximum change on Day 120 (-8%, -11% and -17% at 470, 825 and 1650 ppm, respectively - Cohort 1B) when compared with the concurrent control group but the mean value at 1650 ppm (396.3 g) was comparable with the HCD (432.6 ± 37.76).
Females:
Prior to mating, there was an overall dose-related lower mean body weight gain (Day 1 to Day 72) for Cohort 1B females (-5%, -5% and -9%, at 470, 825 and 1650 ppm, respectively) that reached statistical significance at 1650 ppm when compared with the control group. There was no clear differences in mean body weight gain for females from Day 1 to Day 57 (Cohorts 1A, 1B and 1C) and from Day 1 to Day 64 (Cohorts 1A and 1B) in any group when compared with the concurrent control.
There was an overall dose-related lower mean body weight gain during the gestation period at 825 and 1650 ppm (-8% and -16%, respectively - Cohort 1B) that reached statistical significance at 1650 ppm when compared with the control group considered not toxicologically significant in view of the low magnitude of the change.
There was an overall non-dose-related higher mean body weight gain in all treated groups during the lactation period when compared with the control group (+21%, +21% and +5% at 470, 825 and 1650 ppm, respectively). However, mean body weight gain on LD21 remained statistically significantly lower at 1650 ppm when compared with the control group (-7%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Cohorts 1A, 1B, 1C - post weaning.
Males:
There was an overall statistically significant dose-related lower mean food consumption in all treated group when compared with the control group from Day 1 to Day 57 (Cohorts 1A, B and C), Day 1 to Day 64 (Cohorts 1A and B) and Days 1 to 72 (Cohort 1B) with a maximum change from Days 1 to 72 (-5%, -7% and -10% at 470, 825 and 1650 ppm, respectively).
Females:
Prior to mating, there was no clear test item-related effect on mean food consumption in any group from any cohort (difference < 5% when compared with the control group).
There was an overall statistically significant dose-related lower mean food consumption during the gestation period (-6%, -7% and -12% at 470, 825 and 1650 ppm, respectively - Cohort 1B) when compared with the control group.
Mean food consumption was comparable between treated and control groups during the lactation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A - PND85-93.
There were no test item-related changes amongst the hematological parameters in any group for either sex.
Dose-related statistically significant higher lymphocytes count and total white blood cells count were noted at 825 and 1650 ppm for females. This was not considered toxicologically relevant in view of the low magnitude of the change and given that mean values remained within the HCD.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
PND4 and PND21 T4 and TSH levels. There was no test item-related effect on the total T4 levels in F1-PND4 pups and F1 PND21 pups.

Cohort 1A - PND85-93.
There was a dose-related increase of albumin concentration in all treated groups that reached statistical significance at 1650 ppm and an increase of albumin/globulin ratio in all treated male groups when compared with the control group. All values were within the HCD and/or no similar findings were observed for F0 animals and thus considered incidental.
Other statistically significant differences arising between control and treated animals, such as increased urea concentration for both sexes at 1650 ppm and females at 825 ppm and increased creatine concentration for females at 1650 ppm, were considered not toxicologically relevant as they were of low magnitude and within the historical control data.
Thyroid Hormone Analysis:
For males, there was a dose-related lower mean total serum T4 level (6.2, 5.6 and 4.4 µg/dL) that reached statistical significance at 1650 ppm when compared with the control group (6.7 µg/dL) and a non statistically significant slightly increased serum TSH level at 1650 ppm (0.28 μIU/mL) when compared with the control group (0.15 μIU/mL). These findings were potentially related to increased thyroid weight but the values remained within the HCD range (0.04 to 0.54 μIU/mL for TSH level and 4.2 to 8.2 µg/dL for total T4 level; p < 0.05), so there was considered to be no toxicological significance. There was no clear difference in TSH levels in any group for males.
For females, there was a statistically significantly lower serum TSH level for females at 1650 ppm when compared with the concurrent control. Mean TSH levels for females was disproportionately influenced by 1 female with a high value of 6.08 μIU/mL. After excluding this female, the mean value (0.22 μIU/mL) was slightly above the HCD range (0.02 to 0.20 μIU/m; p < 0.05) and therefore likely related to the increased thyroid weight noted in this group. There was no difference in serum TSH levels for females at 470 and 825 ppm or in serum T4 levels in any group.
See table in 'any other information on results incl tables'.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Cohort 1A - PND85-93.
There was no test item-related effect on urine analysis parameters in any group for either sex.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related effect on sexual maturation for either sex in any group.
The mean occurence of balanopreputial cleavage for treated males was statistically significantly longer in a non-dose-related trend in all treated groups (52.5, 52.4, and 51.4 days in the 470, 825 and 1650 ppm groups, respectively) when compared with the control group (50.0 days). Mean body weights at the age of attainment were comparable at 470, 825 ppm when compared with the control group but lower at 1650 ppm (-8%). The mean value were slightly above the HCD range (41.7 to 47.5 days) but in the absence of a dose relationship and in view of the low magnitude of the change when compared with the control group, this was considered incidental.
The mean occurence of vaginal opening for treated females (32.6, 32.8 and 32.9 days at 470, 825 and 1650 ppm, respectively) was comparable with the concurrent control group (32.6 days). Mean body weights at the age of attainment was statistically significantly lower at 1650 ppm when compared with the concurrent control group (-6%).
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 pups. There was no test item-related effect on the anogenital distance in any group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 pups. Male pups did not present any areola nor nipple in any group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohorts 1A, 1B and Surplus. In the thyroid gland, higher mean absolute and relative organ weights were noted in male and female groups from Cohorts 1A and 1B. A relationship to HHCB was considered likely at the high dose in males (1650 ppm), where it correlated with follicular cell hypertrophy. In females, there was no histopathologic correlate in the thyroid gland, and a relationship to HHCB administration was therefore considered equivocal.
In the liver, higher mean relative organ weights were noted in male given 1650 ppm from both Cohorts 1A and 1B. The absolute values did not show relevant differences, due to the lower terminal body weights in these groups. These higher liver weights were considered related to an adaptive response to the administration of HHCB at the high dose in males (1650 ppm).
The lower mean adrenal gland weight noted in all treated female groups from Cohort 1A was statistically significant but did not have any histopathologic correlation, and was not present in females from Cohort 1B. These organ weight differences were considered incidental and unrelated to administration of HHCB.
Of note, slightly higher mean relative liver weights were noted in high dose males and females from the cohort surplus (animals euthanized on PND21). There were no relevant differences to be noted in the thyroid gland weights. See table in 'any other information on results incl tables'.

Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A. In the thyroid gland, minimal bilateral diffuse follicular cell hypertrophy was observed in high dose males (1650 ppm). This was correlated with an increased organ weight and was considered related to HHCB administration. In females, the incidence of follicular cell hypertrophy did not show relevant differences between controls and treated groups.
In the liver, minimal diffuse centrilobular hypertrophy was observed in high dose males (1650 ppm). This finding correlated with an increased organ weight and was considered related to HHCB administration.
Of note, the histopathologic evaluation of the testes and the quantitative evaluation of ovarian follicles and corpora lutea did not show any difference between high dose and control groups. See table in 'any other information on results incl tables'.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Test item-related preweaning developmental effects for the F1 offspring consisted of a dose related lower mean pup body weight in all groups when compared with the control group and the HCD mean on PND1 and persisted throughout the lactation period at 1650 ppm. This finding was considered not adverse since the mean pup values remained within or very close to the HCD range on PND21. There were no other test item-related effects on F1 litter parameters, preweaning developmental endpoints (anogenital distance and areolae/nipple retention), neonatal and thyroid hormone levels at weaning, organ weights or any macroscopic findings in F1-pups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 650 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no test item-related clinical signs for pups.
Isolated clinical signs including hematoma, weakness, thinness, cold body, incomplete hair growth, teeth marks, abnormal behaviour or sores were considered incidental in view of their nature and/or sporadic occurrence amongst treated and control groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item-related effect on the number of live offspring at birth compared with the total number of offspring born (live birth index).
There were 8, 3, 2 and 1 stillborn pups in the control, 470, 825 and 1650 ppm groups, respectively.
There was no test item-related effect on the number of live offspring on PND4 before culling compared with the number of offspring on PND0 (viability index).
There were 5, 2, 4 and 0 dead, missing or cannibalized pups in the control, 470, 825 and 1650 ppm groups, respectively, between PND0 and PND4. The viability index was consequently higher in all treated groups when compared with the control group.
There was no test item-related effect on the number of live offspring at weaning (PND21) compared with the number of live offspring on PND4 (after culling).
There was only 1 dead, missing or cannibalized pup at 825 ppm. The lactation index was therefore comparable in all groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no clear differences in mean pup body weight on PND1 when compared with the control group. However, a dose-related lower mean pup body weight was noted at 825 and 1650 ppm for both sexes when compared with the control group and the HCD mean. This finding was considered not adverse given that there was no differences in mean pup body weight on PND1 and that mean pup values on PND21 at 825 (52.4 g for males and 51.3 g for females) and at 1650 ppm (49.6 g and 49.5 g for males and females, respectively) remained within or very close to the HCD range (48.8 g to 56.0 g for females and 49.7 g to 57.6 g for males). See table in 'any other information on results incl tables'.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related effect on anogenital distance for either sex in any group.
There was a statistically significant shorter anogenital distance in all groups for males (2.3, 2.0 and 1.9 at 470, 825 and 1650 ppm, respectively) and at 470 and 825 ppm for females (1.0 and 0.9, respectively) when compared with the control groups (2.4 and 1.2 for males and females, respectively) that remained comparable with the HCD mean (2.0 and 1.0 for males and females, respectively, N = 636 pups for males and 686 pups for females) and in the absence of similar findings for F1-pups, this was considered due to incidental high values in the control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Male pups did not present any areola nor nipple in any group.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the liver, higher mean relative organ weights were noted in males and females of the high dose group (1650 ppm). The absolute values did not show relevant differences, due to the lower terminal body weights in these groups. These higher liver weights were considered related to the administration of HHCB at the high dose in males and females (1650 ppm). See table in 'any other information on results incl tables'.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross findings were noted at necropsy of F2 generation animals (PND21).
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Pre-birth loss: There was no test item-related effect on pre-birth loss in any group.
The mean percentage of pre-birth loss in the treated groups (8.2%, 4.7% and 4.8% at 470, 825 and 1650 ppm, respectively) was lower when compared with the control group (6.5%) and/or within the HCD range (2.0% to 20.7%).
There was a slightly lower mean number of implantations in the 1650 ppm (11.3) group when compared with the concurrent control (12.5) but the value remained within the HCD range (11.0 to 12.8 for main studies) and therefore considered incidental. The mean numbers of pups delivered was consequently slightly lower at 1650 ppm (10.7) when compared with the concurrent control (11.6) but remained within the HCD range (9.9 to 11.8 for main studies) and thus considered incidental.
Mean number of implantations/pups delivered at 470 and 825 ppm (12.0/11.0 and 12.1/11.5, respectively) were comparable with that of the HCD mean (12.2/11.1).

sex ratio: There was no test item-related effect on sex ratio in any group.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

Test item-related preweaning developmental effects for the F2 offspring consisted of a dose related lower mean pup body weight at 825 and 1650 ppm for both sexes when compared with the control group considered not adverse given that there was no differences in mean pup body weight on PND1 and that mean pup values on PND21 at 825 and at 1650 ppm remained within or very close to the HCD range. There were no other test item related effects on F2 litter parameters, preweaning developmental endpoints (anogenital distance and areolae/nipple retention).
At necropsy of F2 animals, findings were limited to a test item-related increased liver weight for males and females at 1650 ppm considered adaptive in view of the low magnitude of the change (< 20% versus control group). There was no macroscopic finding in any group.

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2 (cohort 1B)
Effect level:
>= 1 650 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Formulation analysis.


Accuracy and Homogeneity:


Samples collected from formulations at nominal concentrations of 470, 825 and 1650 ppm for Day 1, Week 11 and Week 22 of treatment and from nominal concentrations of 205 and 1650 ppm for Week 16 and on Week 32.


All results were in agreement with acceptance criteria (± 20%).


The deviations from the nominal concentrations ranged from -18.4% to 0.3% and the formulations were homogenous as the RSD on the 6 aliquots (right, middle, left) was ≤ 3.2%.


No test item was present in the vehicle sample.


Stability:


Stability evaluation of samples collected from the rodent unit at concentration of 205 and 1650 ppm showed:



  • A loss of 14.8% and 16.9% of the test item in the diet when formulations were stored for 4 days refrigerated (+2°C to +8°C).

  • A loss of 16.9% and 18.6% of the test item in the diet when formulations were stored for 4 days at room temperature (+15°C to +25°C).

  • A loss of 19.9% and 25.1% of the test item in the diet when formulations were stored for 8 days at room temperature (+15°C to +25°C).


This was consistent with the overestimation of the dose levels (+25%) when compared with the target doses.


 


Achieved doses-F0 generation.


The following number of animals had no access to the diet and thus were not exposed to the test item for a maximum period of 24 hours. This was likely due to spillage since the same quantity of feed was given to all animals and considered sufficient based on the experience of the Test Facility. This was considered without impact on the overall exposure of the animals in view of the study duration.


0 ppm: 4 males, 16 females (only once, except for 6 females: max of 2 occasions and 1 female max of 3 occations)


470 ppm: 0 males, 7 females


825 ppm: 0 males, 5 females


1650 ppm: 0 males, 7 females


During lactation period only and for a maximum of 3 occasions per animal, food spillage was confirmed by the presence of powdered diet in the cage. These isolated events had no impact on the food consumption interpretation.


Test item intake:


Mean Achieved Dose Levels (mg/kg bw/day) for F0 Males and Females









































Dietary Concentration (ppm)



HHCB Intake (mg/kg bw/day)*



Males



Females



Prior to Mating



Prior to Mating



Gestation



Lactation(1)



470



25.8



29.0



26.8



34.4



825



45.9



50.6



45.6



57.5



1650



94.1



99.8



91.7



116.3



*: Taken into account a test item loss of approximately 25% in the diet mix (see above).


(1): The concentrations in ppm were adapted during the lactation period based on historical control food consumption and body weight data for females due to the higher ratio food consumption/body weight.


 


Achieved doses-F1 generation (cohorts 1A, 1B, 1C).


The following number of animals had no access to the diet and thus were not exposed to the test item for a maximum period of 24 hours (except for two low dose females: 2 occurences). This was likely due to spillage since the same quantity of feed was given to all animals and considered sufficient based on the experience of the Test Facility. This was considered without impact on the overall exposure of the animals in view of the study duration.


0 ppm: 5 males, 14 females  (for 1 of these: 2 occasions)


470 ppm: 0 males, 15 females (for 2 of these: 2 occasions)


825 ppm: 0 males, 0 females


1650 ppm: 0 males, 5 females


During lactation period food spillage was noted only and for a maximum of 3 occasions per animal, at comparable incidence amongst treated and control groups (5, 5, 3 and 9 females at 0, 470, 825 and 1650 ppm, respectively) during lactation only and maximum of 2 occasions per animal which suggest that there were not particular concern with respect to the taste/palatability of the test item and therefore no potential impact of the spillage on the food consumption interpretation.


Test item intake:


Mean Achieved Dose Levels (mg/kg bw/day) for F0 Males and Females.









































Dietary Concentration (ppm)



HHCB intake (mg/kg bw/day)*



Males



Females



Prior to Mating



Prior to Mating



Gestation



Lactation(1)



470



33.9



35.0



27.4



34.4



825



59.6



60



47.6



59.9



1650



123.2



122.1



95.2



121.0



*: Taken into account a test item loss of approximately 25% in the diet mix (see above).


(1): The concentrations in ppm were adaptated during the lactation period based on historical control food consumption and body weight data for females due to the higher ratio food consumption/body weight.


 


F0 Generation - Summary of thyroid weight data






































































 



Males



Females



Group



2



3



4



2



3



4



Dose (ppm)



470



825



1650



470



825



1650



Number of. Animals per Group



25



25



25



23



24



25



Gland, Thyroid
(No. Weighed)a



(25)



(25)



(25)



(23)



(24)



(25)



Absolute Value



+1



+4



+14



+15



+16



+24



% of Body Weight



+1



+6



+18



+21



+16



+28



a: All values expressed as percent difference of control group means.
Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - P £ 0.05; refer to data tables for actual significance levels and tests used.



 


F0 Generation - Summary of microscopic findings





























































































 



Males



Females



Group



1



2



3



4



1



2



3



4



Dose (ppm)



0



470



825



1650



0



470



825



1650



Number of Animals per Group



25



25



25



25



25



24



24



25



Gland, Thyroid (No. Examined)



(25)



(25)



(25)



(25)



(24)



(24)



(24)



(25)



Hypertrophy, Follicular



(0)a



(1)



(5)



(5)



(0)



(1)



(2)



(5)



Minimal



0



1



5



5



0



1



2



3



Mild



0



0



0



0



0



0



0



2



a: Numbers in parentheses represent the number of animals with the finding.



 


F0 Generation -  Mean thyroid hormone values




















































 



 



Group 1


0 ppm



Group 2


470 ppm



Group 3


825 ppm



Group 4


1650 ppm



F0-Males



Total T4 (μg/dL)



Mean


St.dev


N



6.05


1.07


10



5.38


0.81


10



4.55**


0.88


10



4.45**


1.10


10



TSH (μIU/mL)



Mean


St.dev


N



0.123


0.098


10



0.140


0.119


10



0.105


0.065


10



0.143


0.079


10



F0-Females



Total T4 (μg/dL)



Mean


St.dev


N



6.84


1.85


10



5.93


1.12


10



5.94


1.14


10



6.11


0.62


10



TSH (μIU/mL)



Mean


St.dev


N



0.175


0.083


10



0.306


0.156


10



0.232


0.143


10



0.367


0.260


10



**: p ≤ 0.01


 


 


F1 Generation - Mean pup body weight during lactation (versus control and versus historical control data (%)

































































 



Group 2



Group 3



Group 4



Group 2



Group 3



Group 4



Doses (ppm)



470



825



1650



470



825



1650



 



Versus HCD Mean



Versus Control Mean



Males D1



-5.6



-8.6



-11.3



-3.1



-6.1



-8.9



Males D21



+3.7



-2.7



-8.0



-2.8



-6.1



-11.2



Females D1



-7.2



-8.1



-10.7



-3.9



-4.9



-7.6



Females D21



+3.0



-1.7



-8.1



-2.8



-4.6



-10.8



D: Day; HCD: Historical Control Data.


 


F1 Generation -  Mean thyroid hormone values F1 pups PND 4

























 



 



Group 1


0 ppm



Group 2


470 ppm



Group 3


825 ppm



Group 4


1650 ppm



F1-PND 4 Pups



Total T4 (μg/dL)



Mean


St.dev


N



1.73


0.29


23



1.68


0.42


21



1.77


0.54


20



1.67


0.30


22



 


F1 Generation Post weaning - Organ weight cohort 1A


































































































 



Males



Females



Group



2



3



4



2



3



4



Dose (ppm)



470



825



1650



470



825



1650



Number of Animals per Group



20



20



20



20



19



19



Gland, Thyroid (No. Weighed)a



(20)



(20)



(20)



(20)



(19)



(19)



Absolute Value



+15



+16



+21



0



-4



+8



% of Body Weight



+25



+30



+44



+5



-2



+15



Liver (No. Weighed)a



(20)



(20)



(20)



(20)



(19)



(19)



Absolute Value



-2



-6



+2



-4



-2



-6



% of Body Weight



+5



+5



+20



+1



0



0



a: All values expressed as percent difference of control group means.


Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - P ≤ 0.05; refer to data tables for actual significance levels and tests used.



 



 


F1 Generation Post weaning - Organ weight cohort 1B


































































































 



Males



Females



Group



2



3



4



2



3



4



Dose (ppm)



470



825



1650



470



825



1650



Number of Animals per Group



20



20



20



20



20



20



Gland, Thyroid (No. Weighed)a



(20)



(20)



(20)



(20)



(20)



(20)



Absolute Value



-5



+1



+4



+1



+11



+13



% of Body Weight



+2



+13



+25



+4



+14



+24



Liver (No. Weighed)a



(20)



(20)



(20)



(20)



(20)



(20)



Absolute Value



-2



-5



-8



+3



+1



0



% of Body Weight



+6



+7



+11



+5



+4



+9



a: All values expressed as percent difference of control group means.


Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - P ≤ 0.05; refer to data tables for actual significance levels and tests used.



 



 


F1 Generation Post weaning - Organ weight cohort surplus







































































 



Males



Females



Group



2



3



4



2



3



4



Dose (ppm)



470



825



1650



470



825



1650



Number of Animals per Group



10



10



10



10



10



10



Liver (No. Weighed)a



(10)



(10)



(10)



(10)



(10)



(10)



Absolute Value



-3



-4



-4



+6



+5



+5



% of Body Weight



+2



+1



+7



+3



+8



+9



a: All values expressed as percent difference of control group means.


Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - P ≤ 0.05; refer to data tables for actual significance levels and tests used.



 



 


F1 Generation Post weaning - Microscopic findings cohort 1A



















































































































 



Males



Females



Group



1



2



3



4



1



2



3



4



Dose (ppm)



0



470



825



1650



0



470



825



1650



Number of Animals per Group



20



20



20



20



20



20



20



20



Gland, Thyroid (No. Examined)



(20)



(20)



(20)



(20)



(20)



(0)



(0)



(20)



Hypertrophy, Follicular



(0)



(0)



(0)



(3)



(1)



(0)



(0)



(2)



Minimal



0



0



0



3



1



0



0



2



Liver (No. Examined)



(20)



(20)



(20)



(20)



(20)



(0)



(0)



(20)



Hypertrophy, Centrilobular



(0)



(0)



(0)



(6)



(0)



(0)



(0)



(0)



Minimal



0



0



0



6



0



0



0



0



Numbers in parentheses represent the number of animals with the finding.



 


F1 Generation post weaning -  Mean thyroid hormone values




















































 



 



Group 1


0 ppm



Group 2


470 ppm



Group 3


825 ppm



Group 4


1650 ppm



F1-PND 21 Male Pups



Total T4 (μg/dL)



Mean


St.dev


N



4.62


0.88


10



5.45


0.75


10



5.04


0.49


10



5.65


1.08


10



TSH (μIU/mL)



Mean


St.dev


N



0.062


0.032


10



0.064


0.024


10



0.073


0.055


10



0.102*


0.038


10



F1-PND 21 Female Pups



Total T4 (μg/dL)



Mean


St.dev


N



5.08


1.11


10



4.71


0.72


10



5.44


0.92


10



5.78


0.63


10



TSH (μIU/mL)



Mean


St.dev


N



0.053


0.016


10



0.068


0.025


10



0.089*


0.046


10



0.099*


0.050


10



 


 




















































 



 



Group 1


0 ppm



Group 2


470 ppm



Group 3


825 ppm



Group 4


1650 ppm



F1-Cohort 1A Males



Total T4 (μg/dL)



Mean


St.dev


N



6.65


1.21


10



6.17


0.95


10



5.60


1.86


10



4.38***


0.68


10



TSH (μIU/mL)



Mean


St.dev


N



0.152


0.246


10



0.171


0.153


10



0.158


0.143


10



0.276


0.191


10



F1-Cohort 1A Females



Total T4 (μg/dL)



Mean


St.dev


N



3.81


1.20


10



3.77


0.90


10



3.67


1.43


10



3.61


0.59


10



TSH (μIU/mL)



Mean


St.dev


N



0.059


0.079


10



0.057


0.032


10



0.105*


0.088


10



0.808*


1.885


10



*: p ≤ 0.05; ***: p ≤0.001


 


F2 Generation - Body weight during lactation (versus control and versus historical control data (%)

































































 



Group 2



Group 3



Group 4



Group 2



Group 3



Group 4



Doses (ppm)



470



825



1650



470



825



1650



 



Versus HCD Mean



Versus Control Mean



Males D1



-3.3



-7.0



-4.7



-1.1



-5.0



-2.7



Males D21



+4.4



-3.4



-8.6



+0.2



-7.2



-12.2



Females D1



-3.7



-6.0



-3.3



+1.6



-0.8



+2.1



Females D21



+2.1



-2.4



-6.0



-1.7



-6.0



-9.4



 


F2 generation - liver weight







































































 



Males



Females



Group



2



3



4



2



3



4



Dose (ppm)



470



825



1650



205 to 470



365 to 825



730 to 1650



Number of Animals per Group



10



10



10



10



10



10



Liver (No. Weighed)a



(10)



(10)



(10)



(10)



(10)



(10)



Absolute Value



+6



+2



+2



+5



+2



+9



% of Body Weight



+6



+8



+14



+6



+9



+18



a: All values expressed as percent difference of control group means.


Based upon statistical analysis of group means, values highlighted in bold are significantly different from control group - P ≤0.05; refer to data tables for actual significance levels and tests used.



 



 


 


 


 


 


 


 


 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, HCCB administered continuously in the diet to male and female Wistar Han rats at dose levels of 470, 825 and 1650 ppm induced no parental or reproductive toxicity for F0 males exposed for 10 weeks prior to mating, during mating, and up to termination or for F0 females exposed for 10 weeks prior to mating, during mating, gestation and lactation through to weaning. There was no sign of reproductive toxicity for F1 males and F1 females exposed at the same dose levels during approximately 19/20 weeks for Cohort 1B.
A parental and reproduction No Observed Adverse Effect Level (NOAEL) of at least 1650 ppm was derived, corresponding to a minimum achieved intake of 94.1 and 91.7 mg/kg bw/day in males and females, respectively (taken into account the lowest HHCB intake prior to mating or during gestation and lactation).
Test item-related preweaning developmental effects consisted in a dose-related lower mean pup body weight in all groups from PND1 for the F1 generation or at 825 and 1650 ppm on PND 21 for both sexes for the F2 generation. This finding was considered not adverse as all mean values on PND21 for both generations remained within or very close to the HCD range. Postweaning developmental effects included a dose-related effect in all groups on mean body weight gain prior to mating (males and females) and during the gestation period (Cohort 1B) associated with a slight reduction in mean food consumption considered not toxicologically significant in view of the low magnitude of the changes and given that terminal mean body weight for males was comparable with the HCD. In addition, the minor differences in mean pup body weight or postweaning mean body weight gains were not associated with any other test item-related developmental effect. A developmental No Observed Adverse Effect Level (NOAEL) of at least 1650 ppm was derived, corresponding to a minimum achieved intake of 123.2 and 95.2 mg/kg bw/day in males and females, respectively (taken into account the lowest HHCB intake during development of the pups).
Executive summary:

Method: F0-generation: The test item was administered by the oral route (diet admixture) continuously to groups of 25 male and 25 female Wistar rats of the F0 generation at nominal doses of 470, 825 and 1650 ppm (adapted during the lactation period to the higher body weight/food consumption ratio). F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. A fourth group received the control item (basic powdered diet) under the same conditions. The inseminated females were allowed to litter and development of the offspring was observed up to weaning on Postnatal Day (PND) 21. The following parameters and endpoints were evaluated in all F0 animals: Mortality, clinical signs, body weights and body weight gains, food consumption, clinical pathology, thyroid hormone levels, estrous cycles, mating performance and fertility, delivery and litter data, sperm quality, macroscopic observations, organ weights and histology findings. F0 males were euthanised after at least 18 weeks of exposure and F0 females after at least 17 weeks of exposure.


Method: F1 generation: From weaning, the selected F1-pups were allocated to 3 cohorts (1A, 1B and 1C) of 20 males and 20 females each. F1 animals were exposed to the test item by the oral route (diet admixture) continuously from PND21 up to the day before or the day of scheduled necropsy at the same nominal doses of 470, 825 and 1650 ppm (adapted during the lactation period to the higher body weight/food consumption ratio). A fourth group received the control item (basic powdered diet) under the same conditions. F1 animals were observed for mortality, clinical signs, body weights and body weight gains, food consumption, clinical pathology (Cohort 1A), thyroid hormone levels (PND4 pups and Cohort 1A), pup anogenital distance, pup number of aerola/nipples, estrous cycles (Cohort 1A), sexual maturation, sperm quality (Cohort 1A), macroscopic observations, organ weights and histology findings. Additionally, spleen samples were taken for splenic lymphocyte subpopulation analysis from 10 rats/sex/group from F1 Cohort 1A animals. F1 animals were sacrificed after sexual maturation for Cohort 1C or between PND85 and PND93 for Cohort 1A. Surplus offspring (10/sex/group) not included in any of the F1 cohorts, were selected for thyroid hormone analysis and organ weights on PND21. F1 females from Cohort 1B were mated and allowed to litter and development of the offspring was observed up to weaning on PND21. The following reproduction/developmental parameters were determined: Mating performance and fertility, and delivery and litter data. For Cohort 1B, F1 males were sacrificed after at least 20 weeks of exposure and F1 females after at least 19 weeks of exposure.


Method: F1 generation: F2 offspring (from Cohort 1B) were observed for early postnatal development, mortality, clinical signs, body weights, sex, anogenital distance, number of aerola/nipples, organ weights and macroscopy. F2 offspring were sacrificed on PND21-23.


 


Results: Samples collected from formulations at nominal concentrations of 470, 825 and 1650 ppm on Day 1, Week 11 and Week 22 of treatment and from nominal concentrations of 205 and 1650 ppm on Week 16 and on Week 32 were accurate and homogeneous. No test item was present in the vehicle sample. Samples collected at the rodent unit at concentration of 205 and 1650 ppm were stable after 4 days refrigerated or at room temperature but a loss of 19.9% and 25.1% of the test item in the diet was observed when formulations were stored for 8 days at room temperature. This was consistent with the overestimation of the dose levels (+25%) when compared with the target doses.


Results: F0 Generation: There were no test item-related death in any group for F0 animals. There was no test item-related clinical sign in any group for F0 animals. There was no test item-related effect in any group in the mean F0 body weight gain or food consumption for either sex with the exception of a slightly lower mean body weight gain for females during the gestation period at 1650 ppm considered related to the lower mean pup weight in this group. There was no test item-related effect on TSH serum concentration either sex or on total T4 serum concentration for females.


Necropsy: At necropsy of F0 animals, bilateral diffuse follicular cell hypertrophy of the thyroid gland was observed in males and females at all doses and a minor dose-related decrease in mean total T4 serum concentration for males that remained within the HCD range. This finding was dose-related and was minimal or mild, correlated with an increased organ weight and was considered not adverse in view of the low magnitude of the change. There was no test item-related macroscopic in any group for either sex.


Fertility: There was no test item-related effect in any group on F0 male and female mating and fertility, male copulation, mean number of implantations, mean estrous cycle lengths, mean precoital intervals, gestation lengths and sperm parameters. There were no test item-related effects on hematology, coagulation, serum clinical chemistry and urinary parameters for F0 animals at any dose for either sex.


Results: F1 Generation: Pups: There was no test item-related effect on F1 postnatal survival in any group. The mean number of pups delivered was comparable with that of the control group and the historical control data mean in all treated groups. There were no test item-related effects on F1-pup clinical condition, anogenital distance, or areolae/nipple at any dose level. There was a dose-related lower mean pup body weight in all groups when compared with the control group and the HCD mean on PND1 which was persistent throughout the lactation period at 1650 ppm. This finding was considered not adverse at 1650 ppm since the mean pup values at 1650 ppm remained within or very close to the HCD range on PND21. In addition, there was no consistency with respect to the lower mean pup body weight on PND1 with the second generation. There were no test item-related effects on T4 and TSH levels in F1-pups on PND4 and PND21.


Adults: There was no test item-related effect on the mean ages at attainment of balanopreputial cleavage and vaginal opening for F1 animals in any group. The duration from vaginal opening to first estrous in the treated groups was comparable to the control group. There was no test item-related clinical sign in any group for either sex. There was a dose-related effect in all groups in the mean body weight gain prior to mating (males and females) and during the gestation period (Cohort 1B) associated with a slight reduction in mean food consumption considered not toxicologically significant in view of the low magnitude of the changes and considering that the terminal mean body weight for males at 1650 ppm on Day 120 was comparable with the HCD. There were no test item-related effects on hematology, coagulation, serum clinical chemistry and urinary parameters for the F1 males and females after approximately 13 weeks of dosing (Cohort 1A) at any dose. There was no test item-related effect on the splenic lymphocyte population for the F1 males and females after approximately 13 weeks of dosing (Cohort 1A) at any dose.


Necropsy: At necropsy, higher mean absolute and relative thyroid weights were noted for male and female groups (Cohorts 1A and 1B) corelated with a minor increase in TSH level for females at 1650 ppm and a slight dose-related decrease in T4 levels for males when compared with the concurrent control. A relationship to HHCB was likely at 1650 ppm for males but considered not adverse in view of the low magnitude of thyroid weight increase, where it correlated with follicular cell hypertrophy. For females, there was no histopathologic correlate in the thyroid gland, and a relationship to HHCB administration was therefore considered equivocal. In the liver, higher mean relative organ weights were noted in male given 1650 ppm from both Cohorts 1A and 1B considered not adverse in view of the low magnitude of the change and in the absence of histology findings. There were no test item-related macroscopic or other microscopic findings and no other difference in organ weights for F1 animals at any dose level. There was no difference in TSH serum levels at 470 and 825 ppm or in T4 serum levels in any group for females or in TSH serum levels for males in any group.


Fertility: There was no test item-related effect in any group on F1 males and females mating and fertility, male copulation, mean number of implantations, mean precoital intervals, gestation lengths (Cohort 1B). There were no test item-related effects on mean F1 estrous cycle lengths or spermatogenic parameters noted in any group (Cohort 1A).


 


F2 Generation: There was no test item-related effect on F2 postnatal survival in any group and no test item-related effect on the mean number of pups delivered. There were no test item-related effects on F2-pup clinical condition, anogenital distance, or areolae/nipple at any dose level. There was a dose-related lower mean pup body weight at 825 and 1650 ppm for both sexes when compared with the control group and the HCD mean. This finding was considered not adverse given that there was no differences in mean pup body weight on PND1 and that mean pup values on PND21 at 825 and 1650 ppm remained within or very close to the HCD range. Necropsy: At necropsy of F2 animals, findings were limited to a test item-related increase liver weight for males and females at 1650 ppm considered not adverse in view of the low magnitude of the change. There was no macroscopic finding in any group.


 


Conclusion: A parental and reproduction NOAEL of at least 1650 ppm was derived, corresponding to a minimum achieved intake of 94.1 and 91.7 mg/kg/day in males and females, respectively (taken into account the lowest HHCB intake prior to mating or during gestation and lactation). Test item-related preweaning developmental effects consisted in a dose-related lower mean pup body weight in all groups from PND1 for the F1 generation or at 825 and 1650 ppm on PND21 for both sexes for the F2 generation. This finding was considered not adverse as all mean values on PND21 for both generations remained within or very close to the HCD range. Postweaning developmental effects included a dose-related effect in all groups on mean body weight gain prior to mating (males and females) and during the gestation period (Cohort 1B) associated with a slight reduction in mean food consumption considered not toxicologically significant in view of the low magnitude of the changes and given that terminal mean body weight for males was comparable with the HCD. In addition, the minor differences in mean pup body weight or postweaning mean body weight gains were not associated with any other test item-related developmental effect. A developmental NOAEL of at least 1650 ppm was derived, corresponding to a minimum achieved intake of 123.2 and 95.2 mg/kg/day in males and females, respectively (taken into account the lowest HHCB intake during development of the pups).

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