Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Feb 2018 - 06 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Feb 2018 - 06 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: See under "Principles of method if other than guideline"
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The testing facility 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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males); 13 weeks (females)
- Weight at study initiation: 267 - 300 g (males); 209 - 248 g (females)
- Fasting period before study: No, except during motor activity measurements (maximum of 2 hours). F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling.
- Housing: Pretest (females only) and pre-mating period: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages; During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages; Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage, females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. No known contaminants were present in the feed or in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 33-56
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 29-33 (males) or to day 52-70 (females)
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is the intended route of human exposure this is a possible route of human exposure during manufacture, handling or use of the test item.The test item and vehicle were administered to the appropriate animals once daily by oral gavage using a plastic feeding tube.
Vehicle:
other: Aqueous hydroxypropyl methylcellulose; 0.25% (w/v)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized using an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were formulated at least weekly as a suspension, prepared in daily portions which were stored in the refrigerator until use. On the day of dosing, the required formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before use. The formulations were used for dosing within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on information of the sponsor and vehicle testing performed at the test facility.
- Preparation of the vehicle: For preparation of 1000 mL of 0.25% (w/v) aqueous hydroxypropyl methylcellulose, approximately 1/3 of the required volume of Elix water was heated to at least 90°C. 2.5 gram Methocel® K4M powder was added to the heated water with agitation. The mixture was agitated until the particles were thoroughly wetted and evenly dispersed. For complete solubilization, the remainder of the Elix water as cold water to lower the temperature of the dispersion was added with agitation. Once the dispersion reached the temperature at which the Methocel® K4M powder became soluble, the powder began to hydrate and viscosity increased. Agitation was continued for at least 30 minutes. Described volumes could be adjusted proportionally when larger quantities of vehicle had to be prepared. The vehicle is stable for 14 days in a refrigerator set to maintain 4°C. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before dosing. The vehicle was also stirred continuously during dosing.
- Concentration in vehicle: 20 mg/mL, 60 mg/mL and 200 mg/mL
- Dose volume: 5 mL/kg bw
- Batch No: 3B12012N02 (Methocel K4M Premium)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. Dose formulation analysis were performed during week 1 of treatment, concentration was determined for all dose groups (including control group), homogeneity was determined for low and high dose groups. Duplicate sets of samples (approximately 500 mg) were analysed. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation of concentrations was ± 10%.
Stability analyses were performed in conjunction with the method development and validation study. The results demonstrated that the test item is stable
in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males were treated for 29-33 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 52-70 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 39 days.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A dose range finding study was conducted to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. Three females per dose group were dosed at 500 or 1000 mg/kg bw/day once daily oral gavage for 10 consecutive days. The dose levels were selected based on the information provided by the Sponsor (an acute oral toxicity study with the test item, S1 in rats; LD50 > 2000 mg/kg). No signs of toxicity were noted at any dose level.
- Fasting period before blood sampling: 24 hours (females)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily. During the dosing period, these observations were performed directly after dosing, the time of onset, grade and duration of any observed sign was recorded. In addition, clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of treatment (prior to dosing), and weekly thereafter. Terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY
Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters examined: as included in the guidance, including coagulation parameters (Prothrombin Time and Activated Partial Thromboplastin Time).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters as included in the guidance were examined. Serum of all F0 males and PND 14-16 pups was analyzed for total Thyroxine (T4). Assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment (males), during the last week of lactation (i.e. PND 6-13, females).
- Dose groups that were examined: All groups
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. Organ weights were determined.

HISTOPATHOLOGY: Yes
All tissues as defined in the guidance were examined histopathologically. For the testes of all selected males of the control and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Other examinations:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but
excluded semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, Group 4 vs. Group 1 (where group 1 = controls and groups 2, 3 and 4 = low, mid and high dose groups, respectively). Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. The clinical signs noted during the treatment period, i.e. alopecia, scabs and scales and chromodacryorrhoea, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. Swelling in the throat region was observed in one males, which was prematurely sacrificed.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. One male of the 300 mg/kg bw group was euthanized in extremis on Day 17. This male showed a swelling in the throat region from Day 10 onwards. Since the animal showed no evidence of pain or other discomfort related to this finding, the male was allowed to mate (starting on Day 15) before considering sacrifice. After confirmation of mating, the male was sacrificed. Macroscopic examination showed nodules in the salivary glands (grown together with the mandibular lymph nodes) and an enlarged spleen. Histopathology revealed an adenocarcinoma of the salivary gland, which was considered a spontaneous finding to be unrelated to treatment. One female of the control group was euthanized on Lactation Day 2, because of a total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of male and female rats remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight were considered to be similar to the control level over the treatment period.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment related changes in total and differential white blood cell counts were observed in male and female rats. At the lowest dose level of 100 mg/kg bw/day, the total white blood cells (WBC) counts were decreased approximately 25-30%. In males, the WBC showed a dose related decrease and the WBC value had halved at 1000 mg/kg bw/day when compared to controls. In females the WBC value had halved at 300 mg/kg bw/day and showed no further decrease at 1000 mg/kg bw/day. Statistical significance for WBC when compared to controls was achieved in males at 1000 mg/kg bw/day and in females in all treatment groups. From the results of the differential white blood cell counts it was clear that the decreases in the absolute number of lymphocytes accounted for the decreases in the total WBC in both sexes, whereas the absolute number of neutrophils, monocytes, eosinophils and basophils remained within the same range as controls at all dose levels. In females at 100 mg/kg bw/day, the absolute number of lymphocytes had halved to that in controls and had further decreased to approximately one third of the control value in females at 300 and 1000 mg/kg bw/day, and had achieved levels of statistical significance at all dose levels in comparison with controls. In males, the changes in absolute number of lymphocytes were comparable to that in the WBC, a dose related decrease from approximately 30% at 100 mg/kg bw/day to 50% at 1000 mg/kg bw/day. The depletion of lymphocytes in the peripheral blood resulted in decreases of the percentage of lymphocytes of the WBC and consequently in increases of the percentage of neutrophils in both sexes. These changes in relative numbers of lymphocytes and neutrophils reached levels of statistical significance in females for lymphocytes at all dose levels and neutrophils at 300 and 1000 mg/kg bw/day. The relative contribution of the monocytes, eosinophils and basophils remained low in all dose groups and since their absolute numbers were unaffected in peripheral blood, the statistically significant differences for the increased percentages of monocytes at 100 and 1000 mg/kg bw/day in both sexes were of no toxicological relevance. No treatment related changes were noted in red blood cell parameters in both sexes. Relatively high values for red blood cells, haemoglobin and haematocrit were observed in the males at 300 mg/kg bw/day. Since no changes were observed in the values of the red blood cell derived parameters, MCV, MCH and MCHC in these males, the high values were considered an incidental finding and of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical biochemistry parameters of treated male and female rats were considered not to have been affected by treatment. The changes in the clinical biochemistry parameters albumin and sodium in males at 300 mg/kg bw/day, that achieved statistical significance when compared to controls, were considered fortuitous findings and to be unrelated to treatment as these occurred in the absence of a dose-related trend.

The mean serum levels of T4 in treated F0 males were approximately 20-25% lower at all dose levels when compared to that in controls. No clear dose response relationship was observed and a statistically significant difference for the T4 level in males at 300 mg/kg bw/day was apparent. Moreover, all T4 values were within the historical control data range for rats of this strain an age and used in this type of studies. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and no treatment related changes in thyroid weight were recorded.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and high dose animals. The animals within all groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Possible test substance-related higher relative liver weight (statistcally significant) were noted in the 1000 mg/kg bw/day males. Mean percent liver weight differences for exposed males compared to control males were -2%, -3% and 8% (absolute) and 0%, 4% and 13% (relative). For exposed females compared to control females this was -4%, -2% and 3% (absolute) and -5%, -3% and 5% (relative). In the absence of any histopathological correlates, this finding was considered to be non-adverse. There were no other test item-related alterations in organ weights. The statistically significant differences for the relative brain weight in males at 300 mg/kg bw/day and the absolute and relative spleen weight and relative pelvic weight in females at 300 mg/kg bw/day was considered incidental findings, based on the absence of a clear dose response relationship.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross
observations encountered in rats of this age and strain. Watery fluid in the uterus, found in two mid dose and one high dose female, was related to stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-related microscopic findings after treatment the test item were noted in females. It is of note that 5 animals per dose were examined. Hypercellularity of medulla and loss of distinction cortex/medulla was observed in the thymus of all exposed females that were examined (not present in controls; 100 mg/kg bw/day: 1 slight, 4 moderate; 300 mg/kg bw/day: 5 moderate; 1000 mg/kg bw/day: 4 moderate, 1 marked). This increase of lymphoid was primarily in the medulla and consisted of an increase in amount of small lymphocytes (hypercellularity), which appeared to expand the medullary area and/or obscure the boundary with the cortex. This resulted in an (optic) smaller cortex filled with a normal (to minimal increased) density of lymphocytes (loss of junction cortex/medulla).
In the spleen, depletion of lymphoid tissue (PALS, T-cell area) was present in females from 100 mg/kg bw/day (not present in controls; 100 mg/kg bw/day: 4 slight; 300 mg/kg bw/day: 3 minimal, 1 slight; 1000 mg/kg bw/day: 4 minimal, 1 slight). This decrease in lymphocytes was located in the periarteriolar lymphoid sheaths of the white pulp, known to be populated largely by T cells (PALS, T-cell area). Vacuolation of the zona fasciculata of the adrenal gland was present in females from 300 mg/kg bw/day (not present in controls and 100 mg/kg bw/day; 300 mg/kg bw/day: 3 minimal; 1000 mg/kg bw/day: 2 minimal, 3 slight). The vacuolation was in the outer layer of the zona fasciculata of the cortex. There were no test item-related microscopic observations in males. The remainder of the recorde
d microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment.
Key result
Dose descriptor:
LOAEL
Effect level:
<= 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results dose range finder study:


No mortality occurred. At 1000 mg/kg bw/day, 1/3 female showed scabs on the upper lips on days 1 and 2 of treatment. No further clinical signs were observed. Body weights and food consumption were considered normal. No abnormalities were noted at macroscopic examination. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day. Since no treatment-related clinical signs were observed in the dose range finder, the observation of clinical signs at no specific time point after dosing was selected in the main study.


 


Results formulation analysis:


The concentrations analyzed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 98% and 100%). No test item was detected in the control group formulation. The formulations of the low dose and the high dose groups were homogeneous (i.e. coefficient of variation ≤ 3.3%).


 

Conclusions:
In a 28-Day Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening, performed according to OECD/EC guidelines and GLP principles, the LOAEL of the test material was found to be 100 mg/kg bw/day, based on effects adverse effects on the lymphocyte population in peripheral blood (in males and females) and histopathological alterations in the thymus and spleen at all dose levels (females only).
Executive summary:

A combined 28 -day repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. The test material was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were treated for 29-33 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 52-70 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 39 days. Formulation analyses confirmed that formulations of test item in aqueous hydroxypropyl methylcellulose were prepared accurately and homogenously. No test item-related mortality occurred, no clinical signs were observed related to test item exposure. In peripheral blood, marked decreases in lymphocytes were noted already at the lowest dose level of 100 mg/kg bw/day in both sexes, i.e. decreases of approximately 30% in males and 45% in females compared to control values. The total number of lymphocytes had further decreased at higher dose levels, reaching approximately 50% of the control values in males at 1000 mg/kg bw/day and 30% in females at 300 and 1000 mg/kg bw/day. The absolute values of the other differential white blood cells, neutrophils, monocytes, eosinophils and basophils remained unchanged at all dose levels. The decreases in white blood cell counts seen at each dose level in both sexes could be fully attributed to decreases in the number of lymphocytes. In the females, histopathology of the lymphoid organs thymus and spleen revealed hypercellularity of the thymic medulla, consisting of small sized lymphocytes, and a decrease in T-cells in the spleen. These findings are likely to be related to the lower lymphocyte count in the peripheral blood. Therefore, the combination of changes in thymus and spleen resulting in decreased circulating white blood cells and lymphocytes was considered to be adverse. Histopathological alterations in the thymus and spleen were not observed in males at all dose levels. Further histopathological changes observed as vacuolation of the zona fasciculata of the cortex of the adrenal gland in females treated at 300 and 1000 mg/kg bw/day were considered as non adverse, based on the minor severity, the fact that it can be seen as a spontaneous finding in untreated rats and the absence of any associated degenerative changes. Slightly higher liver weights (13% higher than in concurrent controls) were observed in males at 1000 mg/kg bw/day. In the absence of any histopathological correlates, this finding was considered to be non-adverse. The serum levels of T4 in treated F0 males were lower at all dose levels when compared to that in controls, but showed no dose response relationship. No corroborating morphological changes were observed in the thyroid gland in males. Based on the magnitude of the changes and the fact that all T4 values were within the historical control range, the changes in T4 were considered to be non-adverse. No treatment-related or toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical biochemistry investigations (including male T4 thyroid hormone levels) and macroscopic examination). Based on the adverse effects on the lymphocyte population in peripheral blood (males and females) and histopathological alterations in the thymus and spleen (females only) at 100 mg/ kg bw/day, with a dose-related increase in severity, the Lowest Observed Adverse Effect Level (LOAEL) for the test material was established to be 100 mg/kg bw/day. The outcome of this study did not allow conclusion on the No Observed Adverse Effect Level (NOAEL).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: See under "Principles of method if other than guideline"
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.
GLP compliance:
yes
Limit test:
no
Justification for study design:
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Test material

Constituent 1
Chemical structure
Reference substance name:
603-101-3
EC Number:
603-101-3
Cas Number:
125962-78-9
Molecular formula:
C21 H28 O2
IUPAC Name:
603-101-3

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The testing facility 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.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males); 13 weeks (females)
- Weight at study initiation: 267 - 300 g (males); 209 - 248 g (females)
- Fasting period before study: No, except during motor activity measurements (maximum of 2 hours). F0-males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling.
- Housing: Pretest (females only) and pre-mating period: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages; During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages; Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage, females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. No known contaminants were present in the feed or in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 33-56
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 29-33 (males) or to day 52-70 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Aqueous hydroxypropyl methylcellulose; 0.25% (w/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized using an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were formulated at least weekly as a suspension, prepared in daily portions which were stored in the refrigerator until use. On the day of dosing, the required formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before use. The formulations were used for dosing within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected based on information of the sponsor and vehicle testing performed at the test facility.
- Preparation of the vehicle: For preparation of 1000 mL of 0.25% (w/v) aqueous hydroxypropyl methylcellulose, approximately 1/3 of the required volume of Elix water was heated to at least 90°C. 2.5 gram Methocel® K4M powder was added to the heated water with agitation. The mixture was agitated until the particles were thoroughly wetted and evenly dispersed. For complete solubilization, the remainder of the Elix water as cold water to lower the temperature of the dispersion was added with agitation. Once the dispersion reached the temperature at which the Methocel® K4M powder became soluble, the powder began to hydrate and viscosity increased. Agitation was continued for at least 30 minutes. Described volumes could be adjusted proportionally when larger quantities of vehicle had to be prepared. The vehicle is stable for 14 days in a refrigerator set to maintain 4°C. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before dosing. The vehicle was also stirred continuously during dosing.
- Concentration in vehicle: 20 mg/mL, 60 mg/mL and 200 mg/mL
- Dose volume: 5 mL/kg bw
- Batch No: 3B12012N02 (Methocel K4M Premium)
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females which have not shown evidence of mating (2 control females) were separated from their males. Since less than nine (out of ten) females of the control group had shown evidence of mating after 14 days cohabitation, each non-mated female was re-mated with a male of proven fertility of the same group. From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. Dose formulation analysis were performed during week 1 of treatment, concentration was determined for all dose groups (including control group), homogeneity was determined for low and high dose groups. Duplicate sets of samples (approximately 500 mg) were analysed. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation of concentrations was ±10%. Stability analyses were performed in conjunction with the method development and validation study. The results demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males were treated for 29-33 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 52-70 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after
delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 39 days. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A dose range finding study was conducted to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. Three females per dose group were dosed at 500 or 1000 mg/kg bw/day once daily oral gavage for 10 consecutive days. The dose levels were selected based on the information provided by the Sponsor (an acute oral toxicity study with the test item, S1 in rats; LD50 > 2000 mg/kg). No signs of toxicity were noted at any dose level.
- Fasting period before blood sampling: 24 hours (females)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily. During the dosing period, these observations were performed directly after dosing, the time of onset, grade and duration of any observed sign was recorded. In addition, clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of treatment (prior to dosing), and weekly thereafter. Terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
FOOD EFFICIENCY
Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters examined: As included in the guidance, including coagulation parameters (Prothrombin Time and Activated Partial Thromboplastin Time).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group
- Parameters examined: As included in the guidance. Serum of all F0 males and PND 14-16 pups was analyzed for total Thyroxine (T4). Assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment (males), during the last week of lactation (i.e. PND 6-13, females).
- Dose groups that were examined: All groups
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex , fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Sperm parameters (parental animals):
For the testes of all selected males of the control and the high dose group, and all males that failed to sire a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Clinical observations were performed at least once daily for all pups. Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS
- Unscheduled Deaths: Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated. On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. Organ weights were recorded.

HISTOPATHOLOGY: Yes
All tissues as defined in the guidance were examined histopathologically. For the testes of all selected males of the control and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, Group 4 vs. Group 1 (where group 1 = controls and groups 2, 3 and 4 = low, mid and high dose groups, respectively). Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wall is. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.
Mating index (%): (Number of females mated/Number of females paired)x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females/Number of females mated)x 100
Gestation index (%):(Number of females with living pups on Day 1/Number of pregnant females)x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites)x 100
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/ Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. The clinical signs noted during the treatment period, i.e. alopecia, scabs and scales and chromodacryorrhoea, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. Swelling in the throat region was observed in one males, which was prematurely sacrificed.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. One male of the 300 mg/kg bw group was euthanized in extremis on Day 17. This male showed a swelling in the throat region from Day 10 onwards. Since the animal showed no evidence of pain or other discomfort related to this finding, the male was allowed to mate (starting on Day 15) before considering sacrifice. After confirmation of mating, the male was sacrificed. Macroscopic examination showed nodules in the salivary glands (grown together with the mandibular lymph nodes) and an enlarged spleen. Histopathology revealed an adenocarcinoma of the salivary gland, which was considered a spontaneous finding to be unrelated to treatment. One female of the control group was euthanized on Lactation Day 2, because of a total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of male and female rats remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight were considered to be similar to the control level over the treatment period.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment related changes in total and differential white blood cell counts were observed in male and female rats. At the lowest dose level of 100 mg/kg bw/day, the total white blood cells (WBC) counts were decreased approximately 25-30%. In males, the WBC showed a dose related decrease and the WBC value had halved at 1000 mg/kg bw/day when compared to controls. In females the WBC value had halved at 300 mg/kg bw/day and showed no further decrease at 1000 mg/kg bw/day. Statistical significance for WBC when compared to controls was achieved in males at 1000 mg/kg bw/dayand in females in all treatment groups. From the results of the differential white blood cell counts it was clear that the decreases in the absolute number of lymphocytes accounted for the decreases in the total WBC in both sexes, whereas the absolute number of neutrophils, monocytes, eosinophils and basophils remained within the same range as controls at all dose levels. In females at 100 mg/kg bw/day, the absolute number of lymphocytes had halved to that in controls and had further decreased to approximately one third of the control value in females at 300 and 1000 mg/kg bw/day, and had achieved levels of statistical significance at all dose levels in comparison with controls. In males, the changes in absolute number of lymphocytes were comparable to that in the WBC, a dose related decrease from approximately 30% at 100 mg/kg bw/day to 50% at 1000 mg/kg bw/day. The depletion of lymphocytes in the peripheral blood resulted in decreases of the percentage of lymphocytes of the WBC and consequently in increases of the percentage of neutrophils in both sexes. These changes in relative numbers of lymphocytes and neutrophils reached levels of statistical significance in females for lymphocytes at all dose levels and neutrophils at 300 and 1000 mg/kg bw/day. The relative contribution of the monocytes, eosinophils and basophils remained low in all dose groups and since their absolute numbers were unaffected in peripheral blood, the statistically significant differences for the increased percentages of monocytes at 100 and 1000 mg/kg bw/day in both sexes were of no toxicological relevance. No treatment related changes were noted in red blood cell parameters in both sexes. Relatively high values for red blood cells, haemoglobin and haematocrit were observed in the males at 300 mg/kg bw/day. Since no changes were observed in the values of the red blood cell derived parameters, MCV, MCH and MCHC in these males, the high values were considered an incidental finding and of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical biochemistry parameters of treated male and female rats were considered not to have been affected by treatment. The changes in the clinical biochemistry parameters albumin and sodium in males at 300 mg/kg bw/day, that achieved statistical significance when compared to controls, were considered fortuitous findings and to be unrelated to treatment as these occurred in the absence of a
dose-related trend. The mean serum levels of T4 in treated F0 males were approximately 20-25% lower at all dose levels when compared to that in controls. No clear dose response relationship was observed and a statistically significant difference for the T4 level in males at 300 mg/kg bw/day was apparent. Moreover, all T4 values were within the historical control data range for rats of this strain an age and used in this type of studies. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were no ted in F0-males, both sexes showed a similar degree of thyroid follicular hypertrophy and no treatment related changes in thyroid weight were recorded.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and high dose animals. The animals within all groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period a decreasing trend in activity over the duration of the test period.
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):
Dose-related microscopic findings after treatment the test item were noted in females. It is of note that 5 animals per dose were examined. Hypercellularity of medulla and loss of distinction cortex/medulla was observed in the thymus of all exposed females that were examined (not present in controls; 100 mg/kg bw/day: 1 slight, 4 moderate; 300 mg/kg bw/day: 5 moderate; 1000 mg/kg bw/day: 4 moderate, 1 marked). This increase of lymphoid was primarily in the medulla and consisted of an increase in amount of small lymphocytes (hypercellularity), which appeared to expand the medullary area and/or obscure the boundary with the cortex. This resulted in an (optic) smaller cortex filled with a normal (to minimal increased) density of lymphocytes (loss of junction cortex/medulla).
In the spleen, depletion of lymphoid tissue (PALS, T-cell area) was present in females from 100 mg/kg bw/day (not present in controls; 100 mg/kg bw/day: 4 slight; 300 mg/kg bw/day: 3 minimal, 1 slight; 1000 mg/kg bw/day: 4 minimal, 1 slight). This decrease in lymphocytes was located in the periarteriolar lymphoid sheaths of the white pulp, known to be populated largely by T cells (PALS, T-cell area). Vacuolation of the zona fasciculata of the adrenal gland was present in females from 300 mg/kg bw/day (not present in controls and 100 mg/kg bw/day; 300 mg/kg bw/day: 3 minimal; 1000 mg/kg bw/day: 2 minimal, 3 slight). The vacuolation was in the outer layer of the zona fasciculata of the cortex. There were no test item-related microscopic observations in males. The remainder of the recorde
d microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. The females of all groups had regular cycles of 4 to 5 days. From start mating until positive pairing continuous di-estrus was noted in several females. The distribution over the groups, i.e. 3/10, 1/10, 0/10 and 4/10 in the control group and the groups exposed to 100, 300 and 1000 mg/kg bw/day, respectively, did not indicate a relation to treatment and this phenomenon was therefore considered not test item related.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
All females showed evidence of mating, resulting in mating indices of 100% for all groups. Precoital time was considered not to be affected by treatment. In the control and the high dose group, relatively high numbers of respectively 3/10 and 4/10 females showed evidence of mating approximately two weeks after start mating. For two control females mating did not occur within the pre-determined 14-days mating period and were re-mated with a male of proven fertility of the same group. Evidence of mating was observed for these two females within 1-3 days after start of re-mating. However, based on the results of estrous cycle determination, it was concluded that mating was not related to the (new) male, but to the fact that these females reached the estrus stage of the cycle just a few days later than the end of the 14-day mating period after a prolonged period of di-estrus.
Number of implantation sites was considered not to be affected by treatment. There were a few females with relatively low number of implantation sites (IS). The distribution of these females involved was 2 (with 2 and 5 IS), 0, 1 (with 8 IS) and 1 (with 6 IS) for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The control female with two implantation sites (no.45) had a total litter loss on LD 2. As the occurrences were in absence of a dose response relationship, these events were considered to be unrelated to treatment.

Fertility index was considered not to be affected by treatment. The fertility indices were 100% for the control and 100 mg/kg bw/day group and 80% and 90% for the 300 and 1000 mg/kg bw/day groups, respectively. A total of two females at 300 mg/kg and one female at 1000 mg/kg were not pregnant. Since the incidence of non-pregnancy was within normal limits and no dose-response relationship was apparent, this finding was considered not to be related to treatment.

Gestation index and duration of gestation were considered not to be affected by treatment. All pregnant females had live offspring. The gestation indices were 100% for all groups. The mean duration of gestation was 21.6, 21.6, 21.8 and 21.3 days for the control, 100, 300
and 1000 mg/kg bw/day groups, respectively.

Litter size was considered not affected by treatment. The mean number of living pups per litter were 9.3, 11.9, 11.0 and 11.9 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The relatively low mean value for litter size in the controls group, was due to the presence of two females with relatively low number of live pups in this group (one dam with one pup and one dam with three pups at birth).

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 99%, 100%, 100% and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One pup in a control litter and one in a litter at 1000 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproductive toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. It was apparent that in several litters, all pups showed (whole body) alopecia. These litters were equally distributed over all dose groups, including the control group. The incidence of those litters was, 2, 3, 3 and 1 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The reasons for alopecia in these pups could not be elucidated from the results of the study, but because of the absence of a dose relationship, this phenomenon was considered not related to treatment. The nature and incidence of the other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 98%, 98%, 97% and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups,
respectively. Two pups of the control group, two pups at 100 mg/kg bw/day, three pups at 300 and two pups at 1000 mg/kg bw/day were found dead or missing between PND 2 and 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. One of the dead pups in the control group was the only one in that litter, thus indicating total litter loss for this dam.
No pups were found dead or went missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At birth, the body weights of the pups were considered similar in all groups. During the first week of lactation, body weight gain of the male and female pups at 1000 mg/kg bw/day was lower compared to that in the other groups, resulting in approximately 15% lower body weights on PND 7 in comparison with controls. Over the second week of lactation, the growth of the high dose pups ran parallel to that of controls and the difference in mean body weights between high dose pups and controls had remained approximately 15% on PND 13. Statistically significant differences were observed for male pups and sexes combined on PND 7 and PND 13 and for females on PND 13 when compared to controls. The body weights and body weight gain of the pups at 100 and 300 mg/kg bw/day were similar to that of controls during lactation.
Food consumption and compound intake (if feeding study):
not examined
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:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly lower serum T4 levels were observed in male and female PND 14-16 pups at 1000 mg/kg bw/day, i.e. approximately 16% in male pups and 10% in female pups, respectively, not reaching levels of statistical significance for both sexes in comparison with controls. The changes in T4 levels in these high dose pups in comparison with control pups remained within normal limits for pups of this strain and age. The absolute T4 value for high dose female pups was close to the lower limit of the historical control range, but the difference compared to the control value in the current study is well within the normal variation.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.
Histopathological findings:
not examined
Description (incidence and severity):
Sex ratio of the pups was considered not to be affected by treatment (percentage of males: 48, 52, 47 and 54 for the control group and the groups exposed to 100, 300 and 1000 mg/kg bw/day, respectively).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Results dose range finder study:


No mortality occurred. At 1000 mg/kg bw/day, 1/3 female showed scabs on the upper lips on days 1 and 2 of treatment. No further clinical signs were observed. Body weights and food consumption were considered normal. No abnormalities were noted at macroscopic examination. Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day. Since no treatment-related clinical signs were observed in the dose range finder, the observation of clinical signs at no specific time point after dosing was selected in the main study.


 


Results formulation analysis:


The concentrations analyzed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 98% and 100%). No test item was detected in the control group formulation. The formulations of the low dose and the high dose groups were homogeneous (i.e. coefficient of variation ≤ 3.3%).

Applicant's summary and conclusion

Conclusions:
In a 28-Day Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening, performed according to OECD/EC guidelines and GLP principles, the LOAEL of the test material was found to be 100 mg/kg bw/day, based on effects adverse effects on the lymphocyte population in peripheral blood (in males and females) and histopathological alterations in the thymus and spleen at all dose levels (females only). The NOAEL for reproduction was found to be at least 1000 mg/kg bw/day (no effects seen at highest dose tested). The NOAEL for development was established to be at least 300 mg/kg bw/day, based on the fact that reduced body weights of the pups at 1000 mg/kg bw/day were observed.
Executive summary:

A combined 28 -day repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. The test material was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were treated for 29-33 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 52-70 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 39 days. Formulation analyses confirmed that formulations of test item in aqueous hydroxypropyl methylcellulose were prepared accurately and homogenously. No test item-related mortality occurred, no clinical signs were observed related to test item exposure. In peripheral blood, marked decreases in lymphocytes were noted already at the lowest dose level of 100 mg/kg bw/day in both sexes. The severity of the effect increased in a dose-related fashion. The decreases in white blood cell counts seen at each dose level in both sexes could be fully attributed to decreases in the number of lymphocytes. In the females, histopathology of the lymphoid organs thymus and spleen revealed hypercellularity of the thymic medulla, consisting of small sized lymphocytes, and a decrease in T-cells in the spleen. These findings are likely to be related to the lower lymphocyte count in the peripheral blood. Therefore, the combination of changes in thymus and spleen resulting in decreased circulating white blood cells and lymphocytes was considered to be adverse. Histopathological alterations in the thymus and spleen were not observed in males at all dose levels. No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). During lactation, body weight gain of the male and female pups at 1000 mg/kg bw/day was lower compared to that in the other groups, resulting in approximately 15% lower body weights on PND 13 for both sexes. Furthermore, slightly lower serum T4 levels were observed in male and female pups at 1000 mg/kg bw/day, i.e. approximately 16% in male pups and 10% in female pups respectively. These decreased T4 values might be related to the lower body weights in these high dose pups, possibly indicating a slight retardation in development. However a definitive conclusion could not be drawn from the results obtained in this study. Based on the magnitude of the changes, the changes in T4 were considered to be non-adverse. Based on the adverse effects on the lymphocyte population in peripheral blood (males and females) and histopathological alterations in the thymus and spleen (females only) at 100 mg/ kg bw/day, with a dose-related increase in severity, the Lowest Observed Adverse Effect Level (LOAEL) for the test material was established to be 100 mg/kg bw/day. The NOAEL for reproduction was found to be at least 1000 mg/kg bw/day (no effects seen at highest dose tested). The NOAEL for development was established to be at least 300 mg/kg bw/day, based on the fact that reduced body weights of the pups at 1000 mg/kg bw/day were observed.