cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-05 to 2016-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: Guidelines, EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA Guideline OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

Principles of method if other than guideline:

No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

EC Number:

282-252-3

EC Name:

cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

Cas Number:

84145-27-7

Molecular formula:

C15H16Cl2N2O5S.HCl

IUPAC Name:

[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methyl methanesulfonate hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of the test material (as cited in study reports): JNJ-17336007-AAC (T001202)
- Physical state : solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I14KB4717
- Expiration date of the lot/batch: 2016-11-25 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Project 509772).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution (groups 2, 3 and 4)

OTHER SPECIFICS: CORRECTION FACTOR 1.09

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch 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: Crl:WI(Han) (outbred, SPF-Quality) from Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 11 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 301-352 g (males) and 193-235 g (females)
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of a maximum of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 2015-10-05 To: 2016-01-26

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose

Vehicle:

propylene glycol

Remarks:

specific gravility 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity of the test item. A correction factor of 1.09 was used. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1), 2 mg/mL (group 2), 6 mg/mL (group 3), 20 mg/mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight (Actual dose volumes were calculated according to the latest body weight)
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses were conducted on a single occasion during the treatment phase (24 November 2016), according to a validated method (Project 509772). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored as reserve samples. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of +/-10% compared to those obtained during the method validation. The accuracy of preparation were considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was <= 10%.
Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Project 509772).
The concentrations analysed in the formulations of group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90 and 110%). No test item was detected in the group 1 formulation. The formulations of group 2 and group 4 were homogeneous (i.e. coefficient of variation <= 10%).

Duration of treatment / exposure:

31 days (males) except for male no. 38: 38 days (due to second mating); 53-67 days (females that delivered); 41-48 days (females with total litter loss or failed to deliver). Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

0 mg eq/kg/day, Group 1

Doses / concentrations 2

Dose / conc.:

10.9 mg/kg bw/day (nominal)

Remarks:

10 mg eq/kg/day, Group 2

Doses / concentrations 3

Dose / conc.:

32.7 mg/kg bw/day (nominal)

Remarks:

30 mg eq/kg/day, Group 3

Doses / concentrations 4

Dose / conc.:

109 mg/kg bw/day (nominal)

Remarks:

100 mg eq/kg/day, Group 4

No. of animals per sex per dose:

10 animals/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 508951) in which animals were dosed for 10 days at 10, 30 and 100 mg eq/kg/day
- Rationale for animal assignment (if not random): randomized

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early in the morning and close to the end of the working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Body weight and calculated body weight gain were reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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 on PND 1, 4, 7 and 13.
Both absolute food consumption and food consumption relative to body weight were reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: Yes
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K3-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- thyroid hormone analysis

FUNCTIONAL OBSERVATIONS
Functional observation were conducted on only three females of Group 3 (30 mg eq/kg/day) and one female of Group 4 (100 mg eq/kg/day) because there were insufficient females with live pups in these groups.
- parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity

Sacrifice and pathology:

SACRIFICE
- Male animals: all surviving animals, following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: all surviving animals, on PND 14-16 (females which delivered), on days 26-28 ( females which failed to deliver, with evidence of mating) or within 24 hours of litter loss (females with total litter loss)

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possible after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes- mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve ( M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic,lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus(M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F) Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin:Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

HISTOPATHOLOGY: Yes
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin
- The following slides were examined by a pathologist: The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4; The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis. All gross lesions of all animals (all dose groups); Thyroid glands and liver of all selected 5 males and females of Groups 2 and 3, and the spleen, ovaries and sternal bone marrow of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs (including the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina) of all males that failed to sire and all females that failed to deliver healthy pups.
- Histopathological examination of the mamary gland was also conducted for the females that had a total litter loss
- All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings
- A peer review on the histopathology data was performed by a second pathologist

Other examinations:

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavern osus muscle complex (LABC), Testes, Thyroid

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted up to 100 mg eq/kg/day.
Slight salivation occurred after dosing in most animals at 30 and 100 mg eq/kg/day, generally starting after two days of treatment. At 10 mg eq/kg/day salivation was seen only in a single male and female on a few days in the first treatment week. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Incidental findings that were noted included alopecia, scabs and wound in the neck, rales, sneezing (female no. 73; taken from study daybook), hunched posture, piloerection and a thickened area on the flews. These findings 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. At the incidence observed, these were considered signs of no toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were six unscheduled deaths in this study.
Three females of Group 4 (no. 73, 75 and 80) did not survive until scheduled necropsy. Clinical observations, body weight and food consumption of these animals did not indicate signs of toxicity. Their untimely death was considered to be related to (test item-related) affected pregnancies rather than directly caused by the test item.
Females no. 73 and 75 were found dead on Day 42 (mating overlooked) and Day 57 on test (Day 24 post-coitum), respectively. A moderate centrilobular coagulative necrosis in the liver was considered the cause of death. This probably resulted from the presence of dead fetuses in the uterus of both animals. Main additional findings, which were probably related to the poor health condition due to the affected pregnancies, were minimal/slight cortical hypertrophy of the adrenal glands, moderate/marked increased extramedullary hematopoiesis in the spleen and in female no. 73 markedly increased lymphocytolysis in the thymus. These deaths were considered to be related to the presence of dead fetuses in the uterus, and not directly related to the treatment with the test item.
Female 80 was sacrificed moribund on Day 24 post-coitum (46 days on test). Necropsy findings of note were enlarged spleen, markedly increased extramedullary hematopoiesis in the spleen, slight cortical hypertrophy of the adrenal glands, the presence of 3 dead fetuses in the uterus and reddish contents in the vagina. No cause of morbidity could be determined from the sections examined. The morbidity was most likely related to the delayed delivery of pups. The untimely death of three additional animals was considered to be gavage-related and not related to the test item (male no. 30 of Group 3, control female no. 42 and female no. 72 of Group 4). These animals were found dead on study Day 16 without prior clinical signs of toxicity or reduced body weight gain or food consumption. At necropsy, watery-clear fluid was noted in the thoracic cavity of these animals and both females had a perforation of the esophagus, suggesting a gavage related accident. There were no other findings of note in these animals.
In addition there was one replacement in this study: Female no. 74 (Group 4) was found dead on Day 2 of the study. At necropsy, macroscopic abnormalities of the lungs (dark-red discolouration, enlargement, presence of hemorrhagic watery-clear fluid) were noted, which were all indicative for an oral gavage accident. In addition, this female showed beginning autolysis, and her gastro-intestinal tract was distended with gas.13 This animal was replaced by a spare female.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males no toxicologically relevant changes in body weight and body weight gain were noted. At Day 12 of the mating period, mean body weight and cumulative weight gain were lower in males treated at 100 mg eq/kg/day than in controls. This finding was considered not to be toxicologically relevant because the differences were slight and not statistically significant.
Females showed no toxicologically relevant changes in body weight or body weight gain during the pre-mating period. At the end of this period (Day 1 mating period), mean body weight was statistically significantly higher in females treated at 100 mg eq/kg/day than in controls. Body weight gain was statistically significantly higher at 30 mg eq/kg/day (Day 1 mating period) and 100 mg eq/kg/day (Day 8 pre-mating period and Day 1 mating period). No toxicological significance was attached to these higher body weight (gain) values.
During gestation, body weight gain was decreased in females treated at 30 and 100 mg eq/kg/day. The differences were statistically significant from post-coitum Day 11 (except for post-coitum Day 14). Mean body weights of these females were also decreased but not statistically significantly. Gestational body weight (gain) of females treated at 10 mg eq/kg/day was not affected by treatment.
During lactation, body weight and body weight gain of females treated at 10 mg eq/kg/day were not affected by treatment. At 30 mg eq/kg/day, there were only two lactating females which both had normal body weight and weight gain. At 100 mg eq/kg/day, body weight (gain) during lactation could not be evaluated because there were no lactating females at this dose level.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
In males, food consumption before allowance for body weight was consistently lower at 30 and 100 mg eq/kg/day. As the differences from controls were small and did not increase in the course of the study, these findings (and corresponding differences in relative food consumption) were considered not to be toxicologically relevant.
The only finding of note in females was the lower food consumption (before and after allowance for body weight) at 30 mg eq/kg/day during the lactation period. This could be explained by the small litter sizes of the two lactating females at 30 mg eq/kg/day and was not a direct adverse effect of the test item on food consumption.
At 100 mg eq/kg/day, food consumption during lactation could not be evaluated because there were no lactating females at this dose level.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males treated with the test item showed no changes in haematological parameters.
Females treated at 100 mg eq/kg/day differed from controls with respect to differential white blood cells (higher percentage of lymphocytes, lower percentages of neutrophils and monocytes) and red blood cell parameters (lower number of red blood cells, haemoglobin and haematocrit, higher percentage of reticulocytes).These differences were statistically significant, except for those in monocytes and reticulocytes. It should be remarked that all control females were lactating whereas none of the 100 mg eq/kg/day females were lactating.
Further it was noted that two females treated at 100 mg eq/kg/day (no. 76 and 77) had higher numbers of platelets. In the absence of corroborative changes, these isolated findings were considered not to represent an adverse effect of the test item on the number of platelets.
Females treated at 30 mg eq/kg/day had normal values for red blood cell and coagulation parameters and total white blood cell counts. They had higher percentages of lymphocytes and lower percentages of neutrophils and monocytes compared with concurrent controls (these differences were not statistically significant). It should be noted that haematology was conducted on two females treated with 30 mg eq/kg/day (i.e. the only lactating females at this dose level).
No findings of note were observed in females treated at 10 mg eq/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males treated with the test item showed no changes in clinical biochemistry parameters. A statistically significantly higher sodium level noted in males treated at 30 mg eq/kg/day was not attributed to treatment because there was no dose-related response.
Females treated at 100 mg eq/kg/day (none of which was lactating) showed the following differences compared to controls (all lactating). The differences were statistically significant unless indicated otherwise. Relative changes in mean values as compared to the concurrent control group are indicated between parentheses.
- Lower (not statistically significant) enzyme activities for alanine aminotransferase (ALAT; 27%), aspartate aminotransferase (ASAT; 28%) and alkaline phosphatase (ALP; 55%).
- Higher total protein (15%) and albumin (20%).
- Lower total bilirubin (32%).
- Lower urea (39%).
- Lower (not statistically significant) bile acids (73%).
- Higher sodium (3%) and chloride (5%).
- Lower inorganic phosphate (47%).
Lower plasma levels of bile acids were also observed in females treated at 30 mg eq/kg/day (two of the three females examined at this dose level were lactating). Bile acid levels at 30 mg eq/kg/day were similar to those at 100 mg eq/kg/day.
The other statistically significant differences noted in females (higher fasting glucose at 30 mg eq/kg/day, higher calcium at 10 mg eq/kg/day) were not attributed to treatment because the values remained within normal limits and showed no dose related response.
Thyroid hormone analyses: Serum levels of T4, measured in F0 males, were not affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
Note: Functional observation were conducted on only three females of Group 3 (30 mg eq/kg/day) and one female of Group 4 (100 mg eq/kg/day) because there were insufficient females with live pups in these groups. The functional observational results obtained for these females were within normal limits. Moreover, the daily clinical observations revealed no clinical signs indicative of neurobehavioural effects.
Therefore, it was considered very likely that functional observational parameters were not affected by treatment in females treated at 30 or 100 mg eq/kg/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following test item differences in organ weights were observed between treated and control animals:
The high spleen weight (increased extramedullary hematopoiesis) was considered to be related to blood loss due to the affected pregnancies. The high adrenal gland weight was considered to be related to stress due to the affected pregnancies. The increased ovary weight, which showed a high variability, was related to different stages of estrous cycle and the large size of the corpora lutea due to pregnancy. In contrast, the females of the control group and females treated at 10 mg eq/kg/day were all necropsied 14-16 days after delivery. The high uterus weight noted for some selected females at 30 and 100 mg eq/kg/day was related to the affected pregnancies and consequent necropsy shortly after the expected delivery date. The selected females of the control group and females treated at 10 mg eq/kg/day were all euthanized 14-16 days after delivery and the uterus showed therefore more involution with an accompanying lower organ weight.
The statistically significant higher weight of the thymus in females at 100 mg eq/kg/day is considered to be the consequence of a lactation-related lower thymus weight in the females of the control group. The other statistically significant relative organ weight changes were in line with the slight decrease in terminal body weight.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross observations.
There were macroscopic findings in female rats which were considered to be related to affected pregnancies/total litter loss: An enlarged spleen (microscopic correlate increased extramedullary hematopoiesis) was recorded in 1/10 females at 10 mg eq/kg/day, 3/10 females at 30 mg eq/kg/day and in 4/10 females at 100 mg eq/kg/day, reddish/red-brown foci or accentuated lobular pattern was recorded in the liver of 1/10 females at 30 mg eq/kg/day and in 2/10 females at 100 mg eq/kg/day (microscopic correlate centrilobular coagulative necrosis), and reduced size of the thymus (microscopic correlate lymphoid depletion) was recorded in 1/10 females at 10 mg eq/kg/day, 1/10 females at 30 mg eq/kg/day and 1/10 females of the control group. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings which were considered to be related to the treatment with the test item were noted in the thyroid gland of males at 30 and 100 mg eq/kg/day and in females at 100 mg eq/kg/day, consisting of a slightly increased incidence and severity of follicular cell hypertrophy, and in the liver of a few males and females at 100 mg eq/kg/day, consisting of low grades of centrilobular hepatocellular hypertrophy.
In the females that didn't deliver healthy pups the following microscopic findings were noted which were considered to be related to the poor health condition of the females that had total litter loss or didn't deliver their pups after a normal pregnancy duration. These findings were considered not to be directly related to the treatment with the test item.
In the thymus microscopic findings were considered to be related to stress/poor health condition and consisted of increased lymphocytolysis (two females at 100 mg eq/kg/day) and lymphoid depletion (one female of the control group, one female at 10 mg eq/kg/day, one female at 30 mg eq/kg/day and two females at 100 mg eq/kg/day).
In the liver coagulative necrosis of the centrilobular area was recorded in one female at 30 mg eq/kg/day and in three females at 100 mg eq/kg/day and was considered to be related to the presence of dead pups or inflammatory/hemorrhagic contents in the uterus.
Increased extramedullary hematopoiesis was recorded in the spleen at an increased incidence and severity in females showing affected pregnancies and total litter loss.
In addition, extramedullary hematopoiesis was recorded in the adrenal gland of one female at 10 mg eq/kg/day and two females at 100 mg eq/kg/day. In the adrenal gland cortical hypertrophy was recorded in one female of the control group which had a total litter loss and in eight females with total litter loss or affected pregnancies at 100 mg eq/kg/day. This was considered to be a stress-related microscopic finding.
The remainder of the recorded 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 chracter of those incidental tissue alterations.
Couples without offspring : There was a test item-related increase in affected pregnancies in females at 30 mg eq/kg/day and at 100 mg eq/kg/day. From 8 couples at 30 mg eq/kg/day and 10 couples at 100 mg eq/kg/day the reproductive organs were examined because they didn't deliver healthy pups, compared to 3 couples of the control group and 1 couple at 10 mg eq/kg/day. In most couples, at 30 and 100 mg eq/kg/day there was evidence of (former) pregnancies (fetuses in uterus or presence of implantation sites in the uterus).
No abnormalities were seen in the reproductive organs, which could account for their lack of healthy offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 100 other: mg eq/kg/day

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

30 other: mg eq/kg/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: secondary to affected pregnancies

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

other: Hepatobiliary system, endocrine system, peripartum mortality

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results, the following NOAEL was derived for parental animals: 100 mg/kg/day for males; 30 mg eq/kg/day for females (based on hepatocellular centrilobular vacuolation at 10, 30 and 100 mg/kg). The test item was classified as STOT RE 2 according to CLP Regulation.