Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Mar 2015 - 16 Sep 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Yellow solid
- Storage condition of test material: Ambient laboratory temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: males: 9-10 weeks; females: 8-9 weeks
- Weight at study initiation: males: 254-298g; females: 152-199g
- Fasting period before study: no
- Housing: Animals were initially housed 2 or 3 per cage by sex (unless reduced by mortality) in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a solid bottom. On the first day of pairing, the individual identity of each female rat was confirmed before being placed into the corresponding male’s cage for mating. Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Day 20 of gestation. Females with litters were retained in this type of cage until termination. After mating, the males were re-housed with their original cage mates.
- Diet: SDS VRF-1 breeder diet was provided ad libitum
- Water: ad libitum from water bottles
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19-22°C
- Humidity: 38-57%
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% carboxymethylcellulose in Milli Q water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed into a pre-labelled container according to instructions from the formulation data capture system (Dispense 8). The total amount of vehicle was then added to the container, and the formulation was mixed using a magnetic stirrer. Formulations were then mixed using a high shear mixer until a homogenous suspension was achieved. The dosing formulations were prepared at least weekly, stored at ambient temperature and dispensed daily. The dosing formulations were removed from storage and were magnetically stirred for at least 30 minutes prior to dosing and continuously during dosing. Any residual volumes were discarded.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Details on mating procedure:
Pairing was on a 1 male to 1 female basis, with the exception of a 1 male to 2 female pairing in Group 2 (203M paired with 253F and 254F) due to the premature death of Animal 204M. During the evening (after 7pm) of Day 15 of dosing, females were housed with their allocated co-group male partner. The animals were paired in ascending numerical order.
Vaginal lavages were taken early each morning from the day of pairing until mating has occurred and the stage of oestrus observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation. The pairing period for each pair of animals was a maximum of 14 nights. Evidence of mating was not observed by the end of the pairing period for one animal of the intemediateh dose group, so this animal was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for this animal continued as if it had mated on the last night of pairing up to the time of littering where lactation procedures were performed as required. For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (oestruses passed without a sign of mating) was evaluated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All Week 1 and 4 formulations were found to be within the concentration acceptance criteria of ±10% of theoretical concentration (actual ranges of -3.5% to +10%) and a low relative standard deviation (<2.1%) indicated that these formulations were also homogenous. The absence of test material from all analysed control formulations was also confirmed.
Duration of treatment / exposure:
The males were dosed once daily for at least 4 weeks overall, starting from 2 weeks prior to mating.
The females were dosed once daily from 2 weeks prior to mating, then continuing until the day prior to termination.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14 day dose range findings study in which rats received the test compound at dose levels of 300 or 1000 mg/kg/day and no adverse findings were noted.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once at the start and once towards the end of the working day throughout the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning Week -1

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during pretreatment and daily during dosing period.

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, starting Week -1, for both sexes until pairing for mating. Consumption was also measured for mated females over the periods Days 0-7, 7-14 and 14-20 of gestation, and Days 0-4 of lactation, with the exception of Animal 251 (Group 2, 100 mg/kg/day) which did not have food consumption measured over Days 14-20 of gestation of Days 0-4 of lactation, in error.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles on a regular basis throughout the study. As no inter-group differences were noted, consumption was not measured by weight.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The anterior, lenticular and fundic areas were examined in all animals pretreatment (including spare animals), during Week 4 for all males and prior to sacrifice for all females – with the exception of: Animals 154F (Group 1, Control), 253F, 254F and 256F (Group 2, 100 mg/kg/day); 352F, 354F and 355F (Group 3, 300 mg/kg/day) and 453F and 457F (Group 4, 1000 mg/kg/day), where pre-terminal examination was not performed in error.

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- Animals fasted: males were fasted overnight
- How many animals: 5 animals per sex and dose group
- Parameters examined: Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin, Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils, Lymphocytes,
Monocytes, Eosinophils, Basophils, Large unstained cells, Activated partial thromboplastin time, Fibrinogen, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Animals fasted: males were fasted overnight
- How many animals: 5 animals per sex and dose group
- Parameters examined: Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Inorganic phosphate, Triglycerides, Total Bile Acids

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Detailed Functional Observations were performed pretreatment for all animals (including spare animals) and weekly during the dosing period for all animals. Functional Tests were performed: - Pretreatment (5 selected rats per sex per group); - week 4 (5 selected males per group (prior to blood sampling)); - During Lactation (the first 5 females per group which rear their litter to at least Day 3 of lactation (prior to blood sampling)).
- Battery of functions tested: Cageside Observations, observations in a Standardised Area, sensory activity, grip strength, motor activity
Sperm parameters (parental animals):
Testes of all Group 1 and Group 4 males, examined for staging of spermatogenesis (qualitative evaluation).
Litter observations:
The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined daily for the presence of milk in the stomach and for any externally visible abnormalities. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation.
Pups that were found dead or killed during lactation were sexed and appropriately examined as above. Any externally abnormal decedent pup was preserved; externally normal pups were discarded. Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Scheduled Euthanasia Day: Males: Day 29; Females: Between Days 5-7 of lactation
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis were recorded for each female. The protocol stated that the uteri of all non-pregnant females would be stained using 10% aq (v/v) ammonium sulphide solution and examined for implantation sites, however this was not performed in error prior to histopathology of these tissues.

ORGAN WEIGHTS
The following organs were weighed at necropsy for all scheduled euthanasia animals:
Brain, Epididymisa, Gland, adrenala, Gland, pituitary, Gland, prostate, Gland, thyroidab, Heart, Kidney , Liver, Ovarya, Spleen, Testisa, Thymus, Uterus

HISTOPATHOLOGY: Yes
The following tissues were examined:
- Full tissues of unscheduled deaths
- Full tissues of the 5 male and 5 female Group 1 and Group 4 animals used for laboratory investigations
- The reproductive organs of all Group 1 and Group 4 animals and Animal 254F (Group 2) which failed to produce a litter.
- Testes of all Group 1 and Group 4 males, examined for staging of spermatogenesis (qualitative evaluation).
- Gross lesions of all animals (all groups).
Tissues examined:
Animal identification, Artery, aorta, Bone marrow smear x 2, femur bone marrow, sternum bone marrow, femur (with articulating surface), rib, sternum, Brain, Cervix, Epididymis x 2, Eye x 2, adrenal gland x 2, clitorial gland, hardarian gland x 2, lacrimal gland x 2, mammary gland, parathyroid x 2, pituitary gland, prostate gland, preputial gland, salivary (mandibular) gland x 2, seminal vesicle including coagulating gland, thyroid x 2, Gross lesions/masses, Gut-associated lymphoid tissue (Peyer’s Patches), Heart, Kidney x 2, caecum, colon, rectum, Larynx, Liver, Lung, axillary lymph node x 2, mesenteric lymph node, skeletal muscle, Nasal cavity, optic nerve x 2, sciatic nerve x 2, Oesophagus, Ovary x 2, Oviduct x 2, Pancreas, Pharynx, Skin, duodenum, ileum, jejunum, Spinal cord, Spleen, Stomach, Testis x 2, Thymus, Tongue, Trachea, Ureter x 2, Urinary bladder, Uterus, Vagina
Postmortem examinations (offspring):
Where practicable, animals found dead or killed were sexed, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal pups were discarded.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately. Pairwise comparisons were only performed against the control group. The following pairwise comparisons were performed:
Control Group vs Group 2
Control Group vs Group 3
Control Group vs Group 4
Body weight (males throughout the study and females prior to mating), food consumption (prior to mating), selected functional observational battery and motor activity data, haematology, coagulation and clinical chemistry were analysed for homogeneity of variance using the ‘F Max' test. If the group variances appear homogeneous, a parametric ANOVA was used and pairwise comparisons made using Fisher’s F protected LSD method via Student's t test i.e. pairwise comparisons were made only if the overall F test is significant. If the variances are heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remain heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
In circumstances where it was not possible to perform the F Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results are reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis. In circumstances where the variances in the ANCOVA remain heterogeneous following log or square root transformations, the data was subjected to a rank transformation prior to analysis. Where it is not possible to perform the F-Max test due to the small sample size, the untransformed parametric ANCOVA results are reported.
Reproductive indices:
Fertility Index (male), Fertility Index (female), Gestation Index
Offspring viability indices:
Birth Index, Live Birth Index, Viability Index

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Animal 204M (Group 2, 100 mg/kg/day) was killed prematurely on Day 9 after biting and swallowing part of the gavage tube during dosing. There were no clinical observations, body weight or food consumption effects, or any necropsy or histopathological findings in this animal which were considered to be treatment related. The death of this animal was therefore considered to be incidental and unrelated to treatment. There were no clinical observations which were considered to be related to treatment with the test article. All clinical observations were considered to be incidental background findings commonly observed in this species.

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights followed similar patterns to Controls in both sexes throughout study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Group mean food consumption was generally comparable to Controls in both sexes prior to mating and in females during gestation and lactation.
On Day 15, mean values in females at 100 or 1000 mg/kg/day were statistically different to Controls; however, as these differences were minor and did not follow a dose related pattern, they were considered to be incidental and unrelated to treatment.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopy findings which were considered to be related to treatment with the test item.

HAEMATOLOGY
In treated males, mean white blood cell and lymphocyte counts were lower than Controls at the end of the treatment period. These findings followed a treatment related pattern, with values at 1000 mg/kg/day being approximately 45% lower than controls and being statistically significant at 300 or 1000 mg/kg/day. There were no other haematology or coagulation findings in males or any findings in females which were considered to be related to treatment. Occasional other statistically significant differences from controls were considered to be incidental and unrelated to treatment since the differences were minor and lacked a dose related response.

CLINICAL CHEMISTRY
In treated groups, mean lactate dehydrogenase and creatine phosphokinase were lower than Controls at the end of the treatment period. In males, the differences followed a treatment related pattern and were statistically significant, with values at 1000 mg/kg/day being approximately 45% lower than Controls. In females, the changes did not follow a treatment related pattern therefore these findings were not positively attributed to treatment in this sex. There were no other clinical chemistry findings in either sex which were considered to be related to treatment. Higher chloride concentrations in males were considered to be incidental due to the lack of a dose-related response.

NEUROBEHAVIOUR
Throughout the study, the type and distribution of neurotoxicity observations did not indicate an association with treatment with test material. Detailed functional observations were generally similar between Controls and treated groups. Slight intergroup difference did not follow dose related patterns and were, therefore, considered to be incidental and unrelated to treatment. There were no differences in motor activity between treated animals and Controls which were considered to be related to treatment. In males at 1000 mg/kg/day, lower mean X and Y ambulation values during Week 4, when compared to Controls, were considered to be incidental as these parameters were also lower than Controls during the pretrial period.

ORGAN WEIGHTS
There were no weight changes at any dose level, in either sex. Isolated values that differed from controls did not follow and patterns or trends or have any correlating data to suggest that they were toxicologically relevant, and as such, were considered be incidental and unrelated to treatment.

GROSS PATHOLOGY
There were no treatment related gross necropsy findings at any dose level, in either sex. All findings were considered to be incidental or of the nature commonly observed in this species, and/or were of similar incidence and severity in controls and treated groups, therefore, they were considered not to be associated with treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment related microscopic findings at any dose level, in either sex. All findings were considered to be incidental or of the nature commonly observed in this species, and/or were of similar incidence and severity in controls and treated groups, therefore, they were considered not to be associated with treatment.

MATING PERFORMANCE
Mating performance, fertility, duration of gestation and litter size were similar in Controls and treated groups.
One female at 100 mg/kg and one male and female at 1000 mg/kg failed to produce/sire a litter, however, these were considered to be incidental as there was no dose-related pattern or correlating data to suggest any relation to treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related clinical observations, body weight, food consumption, detailed functional observation or ophthalmoscopy changes, or any gross necropsy or histopathological findings.

Results: F1 generation

Details on results (F1)

Survival indices and litter and pup weights were generally similar between Controls and treated groups. Slight intergroup differences did not follow a dose related pattern and were therefore not attributed to treatment.
The type and distribution of observations noted in dams and pups during lactation did not indicate any association with treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Fertility
Generation:
F1
Effect level:
1 000 mg/kg bw (total dose)
Sex:
male/female
Basis for effect level:
other: Fertility, mating and pregnancy performance and pup/litter performance were similar between all groups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It was considered that 1000 mg/kg/day was the parental No Observed Adverse Effect Level (NOAEL) and the reproductive/developmental No Observed Effect Level (NOEL).
Executive summary:

The objective of this study was to assess the toxicity of the test item in the Han Wistar rat after oral administration and aimed to provide initial information on possible effects on reproduction and/or development and neurotoxicity. Three test groups (100, 300 and 1000 mg/kg bw) and one control group (vehicle), each containing 10 males and 10 females were used. Males were treated for 2 weeks prior to mating until necropsy after 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation. The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, detailed functional observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), fertility, mating and pregnancy performance, maternal care, pup performance (litter survival and litter/pup weight), gross necropsy findings, organ weights and histopathological examinations. Dosing with the test item at dose levels of up to 1000 mg/kg/day was not associated with any treatment related clinical observations, body weight, food consumption, detailed functional observation or ophthalmoscopy changes, or any gross necropsy or histopathological findings. In addition, fertility, mating performance, pregnancy performance and pup/litter performance were similar between all groups. Clinical pathology findings in treated males of lower white blood cell and lymphocyte counts (up to approximately 40% at 1000 mg/kg/day) and lower lactate dehydrogenase and creatine phosphokinase (up to approximately 45% at 1000 mg/kg/day) were considered not to be adverse as there were no correlating histopathology or organ weight findings and changes were of a small enough magnitude that they may indicate a chance related finding. In conclusion, it was considered that 1000 mg/kg/day was the parental No Observed Adverse Effect Level (NOAEL) and the reproductive/developmental No Observed Effect Level (NOEL)