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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-10-05 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/Deve lopmental Toxicity Screening Test)

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 : 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: 312-352 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

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1 basis
- Length of cohabitation: Following a minimum of 17 days of treatment for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating, until detection of mating was confirmed.
- Proof of pregnancy: 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 postcoitum. Once mating was confirmed, the males and females were separated. A maximum of 10 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Since 3 females had not shown evidence of mating (nos. 55, 58 and 75) they were re-mated with a male of the same group that has been proven to be fertile for respectively 4, 3 and 6 days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm)
- Any other deviations from standard protocol: no

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 2015), 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). Analytical results were approved by the Study director, the reserve samples were destroyed. 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 was considered acceptable if the mean measured concentrations were 90-110% of the target concentrations 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 range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Project 509772).
The concentrations analyzed in the formulations of group 2, group 3 and group 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 formulatiions 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, 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

Parental animals: Observations and examinations:

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 ditribution 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
- Time schedule: between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation, if applicable)
- parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 17 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.During pretest, this was done for 48 females. At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random1 and continued in the study. The remaining females were removed from the study, and estrous cycle results of these nonselected females were not reported but kept in the raw data. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrous. As female no. 74 (Group 4) was found dead on the morning of Day 2 of the treatment period, she was replaced by a spare female.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis; testis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on post-natal day 1 (= day the litter was found completed)
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND4; blood samples were collected from two of the surplus pups; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1:
- mortality/viability: The numbers of live and dead pups were determined on PND1 and daily thereafter. If possible, defects or cause of death were evaluated.
- clinical signs: at least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective table
- body weights: live pups were weighed on PND 1, 4, 7 and 13
- Sex: sex was determined for all pups on PND1 and 14
- anogenital distance: anogenital distance (AGD) was measured for all live pups on PND1. The AGD was normalized to the cube root of body weight.
- areola/nipple retention: on PND13, all males in each litter were examined for the number of areola/nipples

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead if possible. Pups found dead during the weekend were necropsied on the same day

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

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 25-27 (females which failed to deliver, with evidence of mating) or within 24 hours of litter loss (females with total litter loss)

GROSS NECROPSY
- 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)

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 bulbocavernosus muscle complex (LABC), Testes, Thyroid

HISTOPATHOLOGY
- 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 of all males that failed to sire and all females that failed to deliver healthy pups.
- histopathological examination of the mammary gland was also conducted for the female nos. 63 and 77 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.

Postmortem examinations (offspring):

SACRIFICE
- Pups, younger than 7 days were killed by decapitation
- The F1 offspring were sacrificed at PND 4 (by decapitation between 7.00 and 10.30 a.m.), and at PND 7-15 (using Euthasol 20% by intraperitoneal injection).

GROSS NECROPSY
- All pups were sexed both externally and internally. description of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood samplinfg, was preserved in 10% buffered formalin
- 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.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not examined

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.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/ Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) 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
Group mean values were calculated from individual litter values.
Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) 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 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.

Dermal irritation (if dermal study):

not examined

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

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 anof 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 lymphocytosis (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 were 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 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 character 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and normality of the estrous cycle were not affected by treatment.
At 10 and 30 mg eq/kg/day, most females had regular cycles of 4-5 days. Extended di-estrus during pairing occurred in two females at 10 mg eq/kg/day and one female at 30 mg eq/kg/day which all had normal litters. The incidence of this finding did not indicate a relation with treatment.
At 100 mg eq/kg/day, 5/10 females had regular cycles of 4-5 days, two had extended di-estrus during pairing, two had an irregular cycle, and one was acyclic during pairing. One of the two females with irregular cycle died before start of mating (no.72), and the other (no. 74) was mated, but not pregnant. None of the females in this high dose group had a normal litter. These findings at 100 mg eq/kg/day occurred at incidences which can also be seen in untreated females and were, therefore, considered not to be related to treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTION DATA
Mating and fertility indices and precoital time were unaffected by treatment up to 100 mg eq/kg/day. The numbers of pregnant females were 8, 10, 9 and 8 at 0, 10, 30 and 100 mg eq/kg/day, respectively.
The numbers of implantation sites were slightly (not statistically significantly) lower in females treated at 30 and 100 mg eq/kg/day (9.4 and 9.5, respectively, versus 11.9 in controls and 11.6 at 10 mg eq/kg/day).

DEVELOPMENTAL DATA
Note: No postnatal developmental data were available for the 100 mg eq/kg/day group because there were no live litters in this group. Evaluation of postnatal development at 30 mg eq/kg/day was limited by the low number of surviving pups at this dose level.
The numbers of females with living pups on PND1 were 8, 9, 6 and 0 at 0, 10, 30 and 100 mg eq/kg/day, respectively, resulting in a dose-dependent decrease in the live birth index at 30 and 100 mg eq/kg/day.
The viability index (PND 1-4) was decreased at 30 mg eq/kg/day (no data available at 100 mg eq/kg/day).
At 10 mg eq/kg/day , there were no toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (postnatal endpoints are given below)

Gestation:
The gestation index was decreased to zero at 100 mg eq/kg/day (no females with live pups at this dose level) and to 67 % at 30 mg eq/kg/day (out of the nine pregnant females, one had implantations only and two delivered only dead pups). In addition, gestation was prolonged at these dose levels. At 30 mg eq/kg/day, mean gestation length was increased by about one day (statistically significant) to 22.8 days (versus 21.6 days in controls). The single female of the 100 mg eq/kg/day group that delivered (only dead pups) had a gestation length of 23 days. It is very likely that an excessively long gestation period was also the underlying cause for morbidity of the three females at 100 mg eq/kg/day that were either founddead (nos. 73 and 75) or killed in extremis (no. 80).
Parturition/maternal care:
Female nos 53 (group 2) and 61 (group 3) had signs of littering on two consecutive days. On the afternoon of the second day , female no. 61 was sacrificed due to total litter loss. Female no. 53 had no pups, but blood in her cage on the morning of the third day.
Female no. 80 (group 4) was killed in extremis due to a delayed delivery and resulting complications. She had 3 dead fetuses and 1 early resorption in her uterus. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
No deficiencies in maternal care were observed. It should be remarked that at 30 mg eq/kg/day, only two females had live pups up to scheduled termination. The few live pups of four other females at 30 mg eq/kg/day died or went missing early during lactation). At 100 mg eq/kg/day, there were no females with live pups.
Post-implantation survival index:
Post-implantation survival index was dose-dependently decreased at 30 and 100 mg eq/kg/day to 38% and 8%, respectively, versus 93% in controls. Post-implantation survival index at 10 mg eq/kg/day (83%) was also lower than in controls. This was caused mainly by female no. 53 that had 9 implantation sites only. In the absence of any other findings indicative of developmental effects at 10 mg eq/kg/day, the lower post-implantation survival index was considered not to be toxicologically relevant.
Early postnatal pups development:
Postnatal pup development could not be evaluated at 100 mg eq/kg/day because no live pups were born at this dose level (live birth index 0%).
At 30 mg eq/kg/day, the live birth index was 44% and the viability index (PND1-4) was 64% versus 93% (both indices) in controls. Survival after PND4 was not affected at 30 mg eq/kg/day (lactation index 100%, based on two small litters). Furthermore, pup body weights were lower at 30 mg eq/kg/day on PND1. Sex ratio and anogenital distance were not afected by treatment and surviving pups at 30 mg eq/kg/day showed no treatment-related clinical signs, macroscopic abnormalities or changes in serum concentration of the thyroid hormone T4 (PND13-15). Areola/nipple retention in the 3 male pups that survived until PND13 (litter 68) was not examined by error. At 10 mg eq/kg/day, no treatment-related changes were noted in the live birth, viability and lactation indices, sex ratio, anogenital distance, areola/nipple retention, clinical signs, body weight, external macroscopy and serum concentration of the thyroid hormone T4 (PND13-15)

Details on results (P0)

Parental results:
No treatment-related changes in parental parameters were observed at 10 mg eq/kg/day. Treatment at 100 mg eq/kg/day and, to a lesser extent at 30 mg eq/kg/day, was associated with changes in several parameters, particularly in females. Most of the changes in females were related to the affected pregnancies at these dose levels as explained below. A few changes occurring in both sexes (salivation, microscopic changes in the liver and thyroid) were not toxicologically relevant.
Three out of 10 females at 100 mg eq/kg/day were found dead or humanely killed a few days after their expected delivery date. This mortality/morbidity was considered to be related to their (test item-related) affected pregnancy. The main cause of death of the two females found dead was moderate centrilobular coagulative necrosis in the liver, which was most likely related to the presence of dead fetuses in their uterus. The morbidity of the third female was probably related to complications due to delayed parturition (fetuses were present in the uterus, no specific cause of morbidity was evident from histopathology).
during the post coitum period, particularly in the last week, females at 30 and 100 mg eq/kg/day gained less weight than controls. This reduced body weight gain was related to the test item-related post implantation loss at 30 and 100 mg eq/kg/day, reslting in less developing fetuses and hence lower gross body weight gain. This reduced body weight gain was, therefore, considered not to be a direct adverse effect of the test item on maternal body weight.
Many clinical pathology values of females at 100 mg eq/kg/day differed from those of control females. Haematological changes in females at 100 mg eq/kg/day included a higher percentage of lymphocytes, lower percentages of neutrophils and monocytes, lower number of red blood cells, lower haemoglobin, lower haematocrit, and higher percentage of reticulocytes. Clinical biochemistry changes comprised lower values of enzyme activities (ALAT, ASAT, ALP), total bilirubin, urea, bile acids, and inorganic phosphate, and higher values of total protein, albumin, sodium and chloride. A few of the above changes were also seen at 30 mg eq/kg/day (changes in differential white blood cells, lower bile acids). When interpreting these findings, it should be taken into account that control values were all from lactating females whereas none of the 100 mg eq/kg/day females were lactating. Stiudies comparing blood parameters in lactating and non-pregnant female rats have shown that changes occur in hematology and clinical biochemistry parameters during lactation. Most of the blood changes seen in the present study were in line with reported differences between lactating and non-pregnant rats. Exceptions are the changes in the red blood cell parameters (possibly resulting from blood loss), and the lower plasma levels of bile acids and inorganic phosphate which remained within the physiological range of non-pregnant females. THe above clinical pathology findings were considered to be related to the affected pregnancies at 30 and 100 mg eq/kg/day and not to reflect specific target organ toxicity of the test item.
Females treated at 100 mg eq/kg/day also showed changes in the weights of several organs (higher weights of the thymus, adrenals, spleen, ovaries, and uterus). These changes were related to the affected pregnancies of these females and not directly related to treatment with the test item. Lactation is known to be associated with a decrease in thymus weight, explaining the difference in thymus weight between the non-lactating 100 mg eq/kg/day females and lactating controls. Stress due to affected pregnancy probably resulted in enlargement of the adrenals. The higher spleen weight (increased extramedullary haematopoiesis) was considered to be related to blood loss due to the affected pregnancy. The increased ovary weight (compared to control females which were all necropsied 14-16 days after delivery) could be explained by the different stages of estrous cycle and the large size of the corpora lutea due to pregnancy or necropsy shortly after the expected delivery date. The difference in uterus weight could also be explained by the difference in time of necropsy. The selected females of the control group were all euthanized 14-16 days after delivery and their uterus showed therefore more involution with an accompanying lower organ weight than 100 mg eq/kg/day females which were necropsied a few days after their expected delivery date.
Treatment-related microscopic changes were present in the liver (hepatocellular centrilobular hypertrphy) of both sexes at 100 mg eq/kg/day , and in the thyroid (hypertrophy of follicular cells) of males at 30 and 100 mg/eq/kg/day and females at 100 mg eq/kg/day. These findings were considered to be non-adverse based on the minor increase in incidence and/or severity and the absence of any degenerative findings. Females that did not deliver healthy pups showed microscopic findings (with correlating macroscopic findings and/or changes in organ weights) which were considered to be related to the poor health conditions/stress of the females that had total litter loss (mostly at 30 mg eq/kg/day) or didn't deliver their pups after a normal pregnancy duration (mostly at 100 mg eq/kg/day). These findings were considered not to be directly related to the treatment with the test item. They occurred in the thymus (increased lymphocytolysis and lymphoid depletion), liver (coagulative centrilobular necrosis), spleen (increased extramedullary hematopoiesis) and adrenal gland (increased extramedullary hematopoiesis and cortical hypertrophy).
No treatment-related or toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, food consumption, serum concentration of the thyroid hormone T4 (males only), and macroscopic examination).

Reproductive results:
No reproduction toxicity was observed at 10 mg eq/kg/day.
Slightly lower numbers of implantation sites were noted at 30 and 100 mg eq/kg/day.
No test item-related changes were noted in any of the remaining reproductive parameters investogatd in this study (i.e. mating and fertility indices, precoital time, spermatogenic profiling, and histopathological examination of reproductive organs).

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Parental

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

Remarks:

Parental

Effect level:

30 other: mg eq/kg/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: secondary to affected pregnancies

3

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

10 other: mg eq/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg eq/kg/day, there were no live litters (clinical signs could not be evaluated).
All four missing pups at 30 mg eq/kg/day (of litters no. 63, 66, 67) had no milk in their stomach on the day(s) before they went missing (first litter check and, in one pup, PND 2). Additionally, one pup was cold (litter no. 66) and another pup was dehydrated (litter no. 67) at first litter check. The pup of litter 62 that was found dead on PND 4 showed no clinical signs on PND 2 and 3 (for this pup no adequate results were recorded at first litter check). Surviving pups at 30 mg eq/kg/day (litter nos. 68 and 69) showed no abnormalities.
Clinical signs of pups of 10 mg eq/kg/day group were limited to scabs (on the tail, head, neck or snout) and blue spot (on the neck or snout). These incidental findings represent normal background findings and not related to treatment with the test item.
The pups of the control female (no. 49) that had total litter loss were cold and had no milk in the stomach and one of these pups had abnormally hard hind legs. No clinical signs were seen in the other missing or surviving pups of the control group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of dead pups at first litter check was increased at 30 mg eq/kg/day (18 dead pups from 4/8 litters with total litter loss in two litters, versus 6 dead pups from 2/8 litters in the control group). At 100 mg eq/kg/day, all six pups from the single litter in this group were found dead at first litter check. This mortality was considered to be related to treatment. There were no dead pups at first litter check at 10 mg eq/kg/day.
Mortality at 30 mg eq/kg/day continued during early lactation. Four out of the six lactating females had total litter loss. These litters consisted of only one (litters no. 62, 63, 66) or two (litter no. 67) live pups that were found dead on PND 4 (single pup of litter no. 62) or went missing on PND 2 or 3 (four pups in total). Pups missing were most likely cannibalised. In the remaining two litters at 30 mg eq/kg/day all pups survived until scheduled necropsy. At 100 mg eq/kg/day there were no lactating females. At 10 mg eq/kg/day, no pups died or went missing during lactation. In the control group one female (no. 49) had total litter loss (two pups were killed in extremis on PND 2 and three pups went missing on this day) and a single pup of two other females went missing (on PND 2 and 5, respectively).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg eq/kg/day, there were no live pups and therefore no data on body weights could be collected. At 30 mg eq/kg/day, evaluation of pup body weights was compromised by the low number of surviving pups (i.e. two small litters that survived until scheduled necropsy and four litters consisting of only one or two pups which died soon after birth). On PND 1, mean pup weights at 30 mg eq/kg/day were lower (males 14%, females 11%) compared to controls. The difference for the two sexes combined was statistically significant. From PND4 onwards, body weight (gain) of pups at 30 mg eq/kg/day was not adversely affected by treatment (based on only two small litters consisting of 4 or 5 pups). Pup body weights at 10 mg eq/kg/day were similar to those in the control group.

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:

not specified

Description (incidence and severity):

At 100 mg eq/kg/day, T4 levels could not be determined because there were no live pups at this dose level.
Treatment at 10 mg eq/kg/day had no effect on the serum level of the thyroid hormone T4 in male and female pups. At 30 mg eq/kg/day, only two litters had live pups at PND 13-15. Increased concentration of the thyroid hormone T4 was noted in the single male pup and one of the three female pups at 30 mg eq/kg/day (PND 13-15). However, due to the very limited data available no definitive conclusion can be drawn on possible treatment-related effects on T4 values at this mid dose level.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg eq/kg/day, macroscopic findings in the pups that were found dead at first litter check (all of the single litter at this dose level) consisted of cannibalism, absence of milk in the stomach and advanced autolysis. Macroscopic findings in pups of the 30 mg eq/kg/day group that were found dead at first litter check included cannibalism, absence of milk in the stomach and beginning autolysis. No macroscopic findings were noted in the single 30 mg eq/kg/day pup found dead during lactation and the surviving pups of the 10 and 30 mg eq/kg/day groups.
The pups of the control group that were found dead at first litter check or killed in extremis during lactation had no milk in the stomach. No macroscopic findings were noted the in surviving pups of the control group.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

ANOGENITAL DISTANCE
At 100 mg eq/kg/day, there were no live pups (anogenital distance could not be evaluated). Anogenital distance in male and female pups was not affected by treatment up to 30 mg eq/kg/day. It is, however, important to note that at this mid dose level only 6 live male and 8 live female pups were available for examination on PND 1. This in contrast to 45 male and 37 female pups in the control group and 50 male and 46 female pups in Group 2. Therefore, these results should be evaluated with care.

AREOLA/NIPPLE RETENTION
At 100 mg eq/kg/day, areola/nipple retention could not be evaluated because there were no live litters at this dose level. At 30 mg eq/kg/day, no areola/nipple retention was determined for the three male pups (of litter no. 68) available in this group. Treatment at 10 mg eq/kg/day had no effect on areola/nipple retention. For none of the examined male pups of the control and 10 mg eq/kg/day groups nipples were observed at PND13.

MACROSCOPY
At 100 mg eq/kg/day, macroscopic findings in the pups that were found dead at first litter check (all of the single litter at this dose level) consisted of cannibalism, absence of milk in the stomach and advanced autolysis. Macroscopic findings in pups of the 30 mg eq/kg/day group that were found dead at first litter check included cannibalism, absence of milk in the stomach and beginning autolysis. No macroscopic findings were noted in the single 30 mg eq/kg/day pup found dead during lactation and the surviving pups of the 10 and 30 mg eq/kg/day groups.
The pups of the control group that were found dead at first litter check or killed in extremis during lactation had no milk in the stomach. No macroscopic findings were noted in the surviving pups of the control group.
SEX RATIO
Sex ratio was not affected by treatment. Data from the 30 mg eq/kg/day group should be evaluated with care due to the low number of pups available for evaluation. In the absence of live pups at 100 mg eq/kg/day, no sex ratio could be determined at this high dose level.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Developmental results
Test item-related developmental toxicity occurred at 30 and 100 mg eq/kg/day. This toxicity was characterized by increased post-implantation loss, decreased gestation index, increased gestation length, decreased live birth index, decreased viability index, and decreased pup weight at birth. At 30 mg eq/kg/day, eight of the nine pregnant females had litters which were all small (1-7 pups) and mostly short-lived (two litters had no live pups at first litter check; four litters, consisting of only one or two live pups, were lost entirely during the first few days of lactation). Only two 30 mg eq/kg/day litters (containing 4 or 5 pups) survived until scheduled sacrifice. At the higher dose of 100 mg eq/kg/day, only one female delivered pups (all dead). Of the eight pregnant 100 mg eq/kg/day females that failed to deliver three had evidence of former implantations and another three had (dead) fetuses in their uterus, along with (mostly early) resorptions (the latter three females died or were sacrificed a few days after their expected delivery date).
Clinical signs and macroscopic findings associated with pup mortality included cannibalized pups, absence of milk in the stomach and beginning/advanced autolysis.
Increased concentration of the thyroid hormone T4 was noted in the single male pup and one of the three female pups at 30 mg eq/kg/day (PND 13-15). However, due to the very limited data available no definitive conclusion can be drawn on possible treatment-related effects on T4 values at this mid dose level.
At 30 mg eq/kg/day, there were no treatment-related changes in parturition, maternal care, sex ratio, anogenital distance (PND1), lactation index and clinical signs and macroscopic findings in surviving pups. However, it should be remarked that these conclusions were based on small numbers of pups and litters.
Postnatal development at 100 mg eq/kg/day could not be evaluated because there were no live litters at this dose level.
At 10 mg eq/kg/day, no treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care, sex ratio, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND1), areola/nipple retention (males PND13), concentration of thyroid hormone T4 (PND13-15) and macroscopy).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

10 other: mg eq/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

other: increased post-implantation loss, decreased gestation index, increased gestation length, decreased live birth index, decreased viability index

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment with the test item by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg eq/kg/day revealed parental toxicity in females at 100 mg eq/kg/day (peripartum mortality related to affected pregnancy). No parental toxicity was noted in males up to 100 mg eq/kg/day. Reproduction toxicity was indicated by slightly lower numbers of implantation sites at 30 and 100 mg eq/kg/day. Developmental toxicity was observed at 30 and 100 mg eq/kg/day (increased post-implantation loss, decreased gestation index, increased gestation length, decreased live birth index, decreased viability index, decreased pup weight at birth, clinical signs, and macroscopic findings associated with pup mortality).
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 100 mg eq/kg/day for males; 30 mg eq/kg/day for females (secondary to affected pregnancies).
Reproduction NOAEL: 10 mg eq/kg/day.
Developmental NOAEL: 10 mg eq/kg/day (note: only two small litters at 30 mg eq/kg/day survived until scheduled necropsy; no litters with live pups were born at 100 mg eq/kg/day).