Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2015 to 04 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
various deviations with no impact on integrity of study results (see below)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Description: clear colourless liquid
Expiry date: 29 June 2013
Storage conditions: room temperature, in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- A total of 124 Sprague-Dawley RjHan:SD rats (62 males and 62 females) were received at CiToxLAB France on 03 March 2015.
- On the first day of treatment, males were approximately 10 weeks old and females were approximately 9 weeks old.
- At the start of treatment, mean body weight was 422 g (range: 375 g to 458 g) for males and 255 g (range: 224 g to 277 g).
- Males and females were sexually mature and were not siblings.
- Females were virgin.
- Animals were clinically examined on arrival to ensure they were in good health.
- The animals were acclimated to the study conditions for a period of 8 days before the beginning of the treatment period. A larger number of animals than necessary were acclimated to permit selection and/or replacement of individuals.
- During the acclimation period, the required number of animals (60 males and 60 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerised randomisation procedure based on body weight so that the average body weight of each group was similar.
- Each animal was identified by an individual ear tattoo or on the tail. At the beginning of the study, each animal received a unique CiToxLAB France identity number.

ENVIRONMENTAL CONDITIONS
- Animals were housed in a barriered rodent unit from the point of receipt.
- Temperature: 22 ± 2 °C
- Relative humidity: 50 ± 20 %
- Lighting: 12 hours light and 12 hours dark
- Ventilation: 8 to 15 changes/hour of filtered, non-recylced, air
- The corresponding instrumentation and equipemtn were checked and calibrated at regular intervals.
- Temperature and relative humidity were recorded continuously (recording devices equipped with alarm systems).
- The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.

HOUSING
- Except during pairing and lactation, animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order to not jeopardise gestations and to avoid aggressive behaviour around mating between males
- Towards the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were placed in each cage of females.
- Each cage contained an object (rat hut) to provide environmental enrichment.
- The cages were placed in numerical order on the racks.

FOOD AND WATER
- All animals had free access to SSNIFF R/M-H pelleted maintenance diet (batch 4482794; SSNIFF Spezialdiaten GmbH, soest, Germany), which was distributed weekly.
- Animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).

CONTAMINANT ANALYSES
- Batches of diet, sawdust and wood shaving were analysed by the suppliers for composition and contaminant levels.
- Bacterial and chemical analyses of water were performed regularly by external laboratories. These analyses included the detection of possible contaminants (pesticides and heavy metals).
- No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with or prejudice the the outcome of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Batch number MKBQ9948V
Details on exposure:
DURATION
- Dose formulations were administered to males daily for 2 weeks before mating, during the mating period and until sacrifice or treatment-free period (at least 5 weeks of treatment in total).
- Dose formulations were administered to females for 2 weeks before mating, during the mating period, during gestation, during lactation until day 5 post-partum inclusive or until sacrifice for females with no delivery.
- At the end of the treatment period, the animals were sacrificed expect for certain animals in groups 1 and 5 (the last five surviving males and the first five surviving lactating females). Animals that were not sacrificed were kept for a 2 week treatment-free period.

ADMINISTRATION
- Dose formulations were administered once daily, at approximately the same time of day, by gavage using a plastic syringe fitted with a metal gavage tube.
- The quantity of dose formulationadministered to each animal was adjusted according to the most recently recorded body weight.
- A constant dose volume of 4 mL/kg bw/day was used.
- Control animals (group 1) received the vehicle alone.
- The control dose formulation was stirred just before administration and the test item dose formulations for 15 minutes before administration.
- Formulations were maintained under continuous magnetic stirring throughout the dosing procedure.
Details on mating procedure:
MATING
- Females were paired with males from the same dose-level group.
- One female was placed with one male in the latter's cage during the night.
- Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
- The day of confirmed mating was designated day 0 post-coitum.
- Each female was placed with the same male until mating occurred.
- The pre-coital time was calculated for each female.

PARTRUITION
- Females were allowed to litter normally and rear their progeny until day 5 post-partum.
- Any sign of a difficult or prolonged partruition was recorded.
- The morning when partruition was completed was designated day 1 post-partum.
- The length of gestation was calculated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Gas Chromatography with FID detection (GC-FID)
- Analytical method provided by the sponsor and validated at CiToxLAB France prior to dose formulation analysis.
- Test item concentration in dose formulations was determined once on formulations used in weeks 1, 3 and 6. A sample was taken from control and test item dose formulations and analysed using the validated method.
- Measured test item concentration was found to equal nominal concentration ± 10 %.
Duration of treatment / exposure:
- At least 5 weeks (males)
- Until day 6 post-partum (females)
Frequency of treatment:
Once daily
Details on study schedule:
- See chronology of study (attached)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- Group 1: 0 mg/kg bw/day (15 males including 5 treatment free males and 15 females including 5 treatment-free females )
- Group 2: 100 mg/kg bw/day (10 males and 10 females)
- Group 3: 250 mg/kg bw/day (10 males and 10 females)
- Group 4: 500 mg/kg bw/day (10 males and 10 females)
- Group 5: 1000 mg/kg bw/day (15 males including 5 treatment free males and 15 females including 5 treatment-free females )
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CLINICAL EXAMINATIONS (F0 ANIMALS)

Morbidity and mortality: Each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment and treatment-free periods (including weekends and public holidays).

Clinical signs: From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal
was observed once a day, at approximately the same time each day, for the recording of clinical signs.

MEDICAL CARE
- Cothivet, an antiseptic and healing product, was applied on lesions of animals D27526 (group 1), D27474 (group 3) and D27477 (group 4) for 2 or 5 days.

BODY WEIGHT

- The body weight of each male was recorded on the first day of treatment (day 1) and then once per week until sacrifice.
- The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 post-coitum, and on days 1 and 5 post-partum. Body weights of treatment-free females were recorded on days 12 and 19.

FOOD CONSUMPTION
- The quantity of food consumed by each male was measured once a week over a 7 day period from the first day of treatment until the start of the mating period, and then once a week during the treatment-free period for treatment-free males.
- The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the mating period, during gestation for the intervals days 0-7, 7-14 and 14-20 post-coitum, and during lactation days 1-5 post-partum. Food consumption of treatment-free females was also measured during days 5-12 and 12-19 post-partum.
- Food consumption was not measured for males or females during the mating period.
- Food intake per animal and per day was calculated by noting the difference between the food given and that in the food hopper the next time.

Oestrous cyclicity (parental animals):
- The estrous cyclce stage was determined from a fresh vaginal lavage (stained with methylene blue).
- The procedure was repeated each morning during the mating period until the females were mated.
Sperm parameters (parental animals):
- During microscopic examination following sacrifice, special emphasis was placed on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Litter observations:
LITTER SIZE
- each live pup was identified individually on day 1 post-partum by subcutaneous injection of Indian ink.
- The toatl litter size and sex of each pup were recorded as soon as possible after birth.
- Any gross external malformations in pups were noted.
- The litters were observed daily in order to note the number of live, dead and cannibalised pups.

CLINICAL SIGNS
- Pups were observed daily for clinical signs, abnormal behaviour and external abnormalitied.

BODY WEIGHT
- The body weight of each pup was recorded on days 1 and 5 post-partum.
- Body weight of pups from treatment-free females was also recorded on days 12 and 19.
Postmortem examinations (parental animals):
SACRIFICE
- On completion of the treatment or treatment-free period, after at least 14 hours fasting, all surviving F0 animals were deeply anaesthetised by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
(a) Males: after the end of the mating period (at least 5 weeks of treatment in total) except treatment-free males (sacrificed at the end of the treatment-free period).
(b) Females on day 6 post-partum except for treatment-free females (sacrificed at the end of the treatment-free period).
(c) Females that did not deliver were sacrificed on days 25-26 using the same method without overnight fasting. A body weight recording was made before sacrifice to check for a possibly unnoticed delivery.
- Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital.
- Sacrifice took place on day 5 post-partum for surviving pups except pups from treatment-free females (scarificed on day 19 post-partum).

ANIMALS PREMATURELY SACRIFICED OR FOUND DEAD
- There were no males prematurely sacrified or found dead during the study.
- There were no females prematurely sacrificed or found dead during the pre-mating period.
- There were no females prematurely sacrificed or found dead during the mating period.
- During the gestation period, prematurely sacrificed female D27529 (group 1) was euthanised by inhalation of carbon dioxide gas followed by cervical dislocation and was submitted for a complete macroscopic post-mortem examination. The pregnancy status was determined and the numbers of corpora lutea and implantation sites were recorded and classified as liveor dead concepti, early or late resorptions, or scars.
- There were no females prematurely sacrificed or found dead during the lactation period.

ORGAN WEIGHTS (F0 ANIMALS)
- The body weight was recorded before sacrifice for each animal sacrificed as scheduled at the end of the treatment period (after the end of the mating period for males or on day 6 post-partum for females) or after the end of the treatment-free period.
- Organs of those animals specified in the tissue procedure table (attached) were weighed wet as soon as possible after dissection.
- The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

MACROSCOPIC POST-MORTEM EXAMINATION (F0 ANIMALS)
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all F0 animals including the female prematurely sacrificed.
- Special attention was paid to the reproductive organs.
- The numbers of corpora lutea and implantation sites were recorded for females sacrificed as scheduled on day 6 post-partum and sacrificed on days 25-26 due to no delivery.
- For non-pregnant females the absence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964).
- The number of implantation sites were recorded for females sacrificed as scheduled on day 20 post-partum.

PRESERVATION OF TISSUES (F0 ANIMALS)
- The tissues specified in the tissue procedure table (attached) were preserved in 10 % buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's fixative).

PREPARATION OF HISTOLOGICAL SLIDES
- All tissues required for microscopic examination were trimmed based on the RITA guidelines (Ruehl-Fehlert et al, 2003; Kittel et 2004; Morawietz et al 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stianed with hematoxylin/PAS).

MICROSCOPIC EXAMINATION (F0 ANIMALS)
- A microscopic examination was performed on all tissues listed in the tissue procedure table (attached) from all animals of the control and high dose groups (groups 1 and 5) sacrificed at the end of the treatment period (at the end of the mating period for males and on day 6 post-partum for females).
- Liver and kidneys from all males from groups 1 to 5 sacrificed at the end of the treatment period were examined microscopically.
- Liver and kidneys from all males from groups 1 to 5 sacrificed at the end of the treatment-free period were examined microscopically.
- Microscopic examinations were conducted on all macroscopic lesions for all groups.
Postmortem examinations (offspring):
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups found dead. Special attention was paid to the reproductive organs and to whether the pup had fed (for example presence of milk in the stomach).
- A macroscopic post-mortem examination was also performed on the principal thoracic and abdominal organs for all pups sacrificed on days 5 and 19 post-partum.
- No tissues were preserved.
Statistics:
- See details of data assessment and calculation of parameters (attached)
Reproductive indices:
- See details of data assessment and calculation of parameters (attached)
Offspring viability indices:
- See details of data assessment and calculation of parameters (attached)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not related to test item or of no toxicological significance (see below)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
incidental or of no toxicological significance (see below)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
incidental or of no toxicological significance (see below)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
test item-related effects not relevant to humans (see below)
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
High number of corpora lutea in one animal considered to be incidental (see below)

Details on results (P0)

CHEMICAL ANALYSIS OF DOSE FORMULATIONS
- Test item concentrations in the administered dose formulations analysed in weeks 1, 3 and 6 were within an acceptable range of variation (-4.9 % to +3.9%) when compared to nominal values (± 10 % of nominal concentration).
- No test item was observed in the control dose formulation in week 1 although a small peak (below the limit of quantification) was observed in weeks 3 and 6.

CLINICAL EXAMINATIONS
- There were no premature deaths in test item-treated groups.
- Control female D27529 was prematurely sacrificed on day 22 post-coitum due to the presence of a mass in the urogenital region that made a normal delivery impossible. Swollen urogenital area was noted from day 12 post-coitum and piloerection, round back, hypoactivity and absence of feces were recorded on day 22 post-coitum. Another mas, in the thoracic region, was noted in this female from day 9 post-coitum.
- The only test item-related clinical sign was ptyalism noted in 4/15 males at 1000 mg/kg bw/day. In view of the low incidence (4/30 animals in the high dose group) and the absence of a similar finding in females, it was considered to be of no toxicological significance.
- Other clinical signs recorded in test item-treated groups (ptyalism, chromodacryorrhea, hair loss, cutaneous lesion, reddish vaginal discharge, piloerection, pallor) were not attributed to the test item treatment as there was no dose relationship and observations were recorded in only a few animals or in a similar incidence to that in controls.

BODY WEIGHT
- Mean body weights and mean body weight changes (g) are summarised in the tables attached.
- There were no effects on mean body weights and mean body weight changes in males at any dose levels during treatment and treatment-free periods. The statistically significant higher mean body weights gain at 250 mg/kg bw/day over the first 15 days of treatment in males was considered to be incidental and not dose-related.
- There were no toxicologically significant effects on mean body weight in females at any dose levels during treatment and treatment-free periods.
- Statistically significant lower mean body weight gains were recorded at 500 and 1000 mg/kg bw/day in the second week of treatment, and statistically significant higher mean body weight gains were recorded at 1000 mg/kg bw/day in the first week of the treatment-free period. As the effects were transient and did not have significant impact on mean body weights, they were considered to be of no toxicologicical significance.

FOOD CONSUMPTION
- Details of mean food consumption are attached.
- There were no toxicologically significant effects on mean food consumption during treatment and treatment-free periods.
- At 1000 mg/kg bw/day, the statistically higher mean food consumption in females during the first week of treatment and the last week of gestation was considered to be of no toxicological significance. The increases were small, transient and more variable during the first week of treatment than in controls. Moreover, there was no relevant impact on mean body weights.
- At 500 mg/kg bw/day in males, the statistically significant lower mean food consumption during the second week of treatment was not ascribed to treatment with the test item as it was not dose related.

REPRODUCTIVE DATA
- Pairing, mating, fertility and delivery data are summarised in the attached tables.
- There were no effects on mean pairing, mating and fertility data at any dose level.
-Female D27537 (100 mg/kg bw/day) and females D27557 and D27564 (500 mg/kg bw/day) were blocked in diestrous during most of the mating period and thus took more days to mate compared with the other animals. This was not considered to be related to test item treatment as it was not dose related and occurred in a limited number of animals.
- Control females D27525, D27526 and D27533 and female D27544 from the low dose group were sacrificed on days 25/26 post-coitum because they did not deliver (not ascribed to test item treatment for D27544 and the other animals were controls). The animals were found to be non-pregnant except control female D27525 which had one late resorption in the uterus at necropsy.
There were no test item-related effects on mean delivery data at any dose levels.
- The higher mean number of corpora lutea at 500 mg/kg bw/day was due to female D27557 with an excessive number of corpora lutea. As this was isolated, the finding was considered to be incidental. Nevertheless, the very high number of corpora lutea (40) likely did not correspond to gestational corpora lutea only (total number abnormally high).

PARENTAL PATHOLOGY

Mortality

There were no premature deaths except the premature sacrifice of one control female with two large masses which correlated with mammary gland adenocarcinoma. This finding is not uncommon in female rats of this strain and age.

Organ weights

At the end of the treatment period there were test item-related liver and kidney mean weight differences in males. The final mean body weights were considered to be unchanged by test item administration.

Moderately increased, slightly dose-related, absolute and relative-to-body mean kidney weights were noted in males at all dose levels (up to +34 % in absolute weights with P < 0.01 in males at 1000 mg/kg bw/day (see table attached). These changes were considered to be related to test item administration since they correlated with the hepatocellular hypertrophy and the increased periportal vacuolation seen microscopically.

It was considered that the minimally higher liver relative-to-body weights in females at 250 and 500 mg/kg bw/day were probably unrelated to test item administration since those changes were poorly dose related and of very low magnitude. The slides of liver from females were not examined.

The minimal epididymides and spleen absolute and relative-to-body mean weight changes in males at all dose-levels were not considered to be test item-related because they were of small magnitude, not dose-related and uncorrelated with microscopic findings.

At the end of the treatment-free period there were no mean liver weight differences while minimally increased kidney weights were seen in males previously treated at 1000 mg/kg bw/day (+17 % in relative-to-body weights with p < 0.05), with microscopic correlates as mentioned at the end of the treatment period. This suggested total reversibility for the liver changes but not for the kidney changes for which reversibility was incomplete.

Macroscopic observation

There was one unscheduled death when one control female was sacrificed with two large subcutaneous white masses (3 x 4 and 2 x 3 cm) which correlated with mammary gland adenocarcinoma.

Terminal sacrifice

At the end of the treatment period there were test itme-related irregulat colour and/or tan discolouration in the kidneys from 6/10 males treated wt 100 mg/kg bw/day and from all males treated at 250, 500 or 1000 mg/kg bw/day. These changes were considered to be related to test item administration since they correlated with the increased weights and hyaline droplets in tubular epithelium, tubular dilation and dilated tubules with eosinophilic content seen microscopically in the kidneys of these males.

There were increased incidence of accentuated lobular pattern in the liver from males treated at 100, 250, 500 or 1000 mg/kg bw/day, together with tan discolouration. These findings correlated with the increased weights and the hepatocellular hypertrophy plus the increased periportal vacuolation seen microscopically in these animals.

The few other gross observations (see attached table) were considered to be consistent with spontaneous findings encountered in the rats of this strain and age because they were not dose-related, isolated and/or correlated with common background lesions.

At the end of the treatment-free period there were no test item-related macroscopic findings, suggesting a complete reversibility of these changes.

Microscopic examination

The control female sacrificed on day 38 had a mammary gland adenocarcinoma. This finding is not uncommon in female rats of this strain and age.

At the end of the treatment period a series of dose-related test item-related microscopic findings were seen in the kidneys and liver of males treated from 100 mg/kg bw/day, which were considered adverse at 1000 mg/kg bw/day.

Kidney
- Minimal degeneration/necrosis in the kidneys from two males treated at 1000 mg/kg bw/day, suggesting an adverse effect at this dose level.
- Increased incidence and severity of minimal to moderate tubular basophilia at all dose levels, considered not to be adverse in view of the low magnitude of the increase.
- Minimal to marked hyaline droplets in tubular epithelium in all test item-treated males, considered to be a species-specific change not relevant in humans.
- Minimal to slight tubular dilation (i.e. with empty lumen), together with minimal to moderate dilated tubules containing a granular eosinophilic content, considered not to be adverse in view of the absence of associated degeneration/necrosis.
- Increased incidence of minimal infiltrate of mononuclear cells in males treated at 1000 mg/kg bw/day although poorly dose-related, considered not to be adverse in view of the minimal severity of this change.

Liver
- Minimal hepatocellular hypertrophy, considered not to be adverse in view of the minimal severity of this change.
- Increased incidence and severity of minimal to slight periportal vacuolation, considered not to be adverse in view of the low magnitude of this increase.

These findings correlated with gross irregular colour and tan discolouration in kidneys and accentuated lobular pattern with tan discolouration in liver, together with increased weights.

There were no test item-related findings in the testes, epididymides or ovaries at microscopic examination.

There were a few other microscopic observations, which were seen at low incidence, at minimal severity, were not dose-related and correlated with background lesions seen in rats of this strain and age.

At the end of the treatment-free period there were no test item-related findings in the liver whilse lesions similar to those recorded at the end of the treatment period were seen in the kidneys and correlated with the increased mean renal weights.
- Increased incidence and severity of slight to marked tubular basophilia.
- Minimal hyaline droplets in tubular epithelium from one test item-treated male.
- Increased severity of minimal to moderate tubular dilation (i.e. with empty lumen) together with slight to moderate dilated tubules containing a granular eosinophilic content.
- Increased incidence of minimal infiltrate of mononuclear cells.

These findings suggested complete reversibility for the liver changes while there was incomplete reversal of the renal findings.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: adverse microscopic effects in kidneys of male rats given 1000 mg/kg bw/day but not relevant to humans (see below)
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: lack of adverse test item-related effects in the 100, 250, 500 and 1000 mg/kg bw/day groups
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test item-related effects on pairing, mating, fertility or delivery at any dose level.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not related to test item (see below)
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
not related to test item (see below)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
not toxicologically significant (see below)
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):
not related to test item (see below)
Histopathological findings:
not examined

Details on results (F1)

OBSERVATION OF PUPS AFTER BIRTH
- Viability indexes are described in the attached table.
- There were no test item-related effects on pup viability. The slightly lower viability index at 250 and 1000 mg/kg bw/day was mainly due to one litter in each group which lost respectively 10 pups (D27549 by day 1 post-partum) or 8 pups (D27566 between day 1 and 2 post-partum).
- No pups were found dead during the treatment-free period.
- Clinical signs observed in pups during lactation are shown in the attached table.
- Poor health condition (D27571-8 on day 5 post-partum) can sometimes be observed in such studies and a relationship with the test item treatment of the mother was considered to be unlikely.
- Hematoma, scabs and wound recorded in the study with low incidence can be observed in rat pups in this type of investigation and were considered incidental.
- There were no clinical signs after the treatment period in treatment-free pups.
- Mean pup body weights and mean body weight changes are shown in the attached table.
- There were no toxicologically significant effects on pup mean body weight and mean body weight gains at any dose levels during treatment and treatment-free periods.
- At 1000 mg/kg bw/day and when compared to controls, the slightly higher mean body weight gains and mean body weights during the treatment-free period was mainly due to the lower mean number of pups kept after the end of maternal treatment
- The percentage of male pups at birth is shown in the attached table.
- There were no dose-related effects on percentage of male pups at birth.

MACROSCOPIC POST-MORTEM EXAMINATION OF PUPS
- Necropsy findings noted after scheduled sacrifice of pups are summarised in the attached table.
- Pale liver and dilated renal pelvis in pups of test item-treated groups at the end of the treatment period were not considered to be related to treatment because they can occure spontaneously in Sprague-Dawley rats and had a very low incidence.
- There were no necropsy findings at 1000 mg/kg bw/day at the end of the treatment-free period.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of test item-related effects on pup viability, clinical condition, body weights and body weight gains or sex ratio at any dosage level and no test-item related findings at necropsy.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

PATHOLOGY DISCUSSION

Presence of hyaline droplets in kidneys of males correlated with increases in mean kidney weights at necropsy. The hyaline droplets were consistent with what is described in the literature as a2u-globulin nephropathy, which is known to increase after treatment with a wide range of drugs or chemicals (Greaves, 2012). These hyaline droplets were observed with a dose related trend and were associated with a higher incidence and/or severity of tubular basophilia. The prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover, which may explain the tubular basophilia seen in all test item-treated groups and the degeneration/necrosis seen at 1000 mg/kg bw/day. These findings were therefore considered to be adverse at 1000 mg/kg bw/day. However, humans excrete proteins of a similar nature in only trace amounts and these findings are therefore considered not to be relevant to humans (Hard et al, 1993; Swenberg 1993).

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.
Executive summary:

GUIDELINE

The study design was based on OECD 421 of 27 July 1995. The objective was to evaluate the portential toxic effects of the test item following daily oral (gavage) administration to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum. On completion of the treatment period, additional dams and litters were held for a two week treatment-free period in the control and high dose groups in order to evaluate the reversibility of any findings. The study provided initial information on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and partrutition, and on reversibility of potential findings on development of the offspring or gonadal weight and morphology.

METHODS

Four groups of 10 male and 1o female (groups 2 to 4) or 15 male and 15 female (group 5) Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for females, through gestation until day 5 post-partum. The test item was administered as a solution in the vehicle, corn oil, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day. Another group of 15 males and 15 females received the vehicle alone, under the same experimental conditions, and acted as the control group. A constant dose volume of 4 mL/kg bw/day was used. On completion of the treatment period, the animals in each group were sacrificed, except for five animals per sex in the control and high dose groups which were kept for a 2 -week recovery period. The concentrations of the dose formulations were checked in study weeks 1, 3 and 6.

The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment and treatment-free periods. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on days 0, 7, 14 and 20 post-coitum plus lactation on days 1 and 5 post-partum. These data were also recorded on days 12 and 19 post-coitum for treatment-free females and weekly after the mating period for treatment-free males.

Animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 5 post-partum or until day 19 post-partum for treatment-free females. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 post-partum. Pups of treatment-free females were also weighed on days 12 and 19 post-partum.

Males were sacrificed after at least 5 weeks of treatment and females on day 6 post-partum except for treatment-free animals, which were sacrificed 2 weeks after the end of treatment (thus on day 20 post-partum for females). Final body weights and selected organ weights (epididymides, testes, kidneys, liver, spleen) were recorded and a macroscopic post-mortem examination of the prinicpal thoracic and abdominal organs was performed. Particular attention was paid to the reproductive organs. A microscopic examination was performed on ovaries(with oviducts) from all females sacrificed on day 6 post-partum in the control and high dose groups and epididymides and/or testes from all males sacrificed at the end of the treatment period in the control and high dose groups. A microscopic examination was also performed on the kidneys and liver from all males sacrificed at the end of the treatment or treatment-free period in all groups and on all macroscopic lesions. Pups were sacrificed on day 5 post-partum (or day 19 post-coitum for treatment-free pups) and submitted for post-mortem examination of the principal thoracic and abdominal organs.

RESULTS

Chemical analysis: No test item was detectable above the limit of quantification in the control dose formulation and the test item concentrations in the analysed dose formulations were within an acceptable range.

F0 animals: The following findings were reported:

- No premature deaths in the test-item treated groups during treatment and treatment-free periods.

- No toxicologically significant clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weights, mean body weight changes and mean food consumption during treatment and treatment-free periods.

- No effects on mean pairing, mating, fertility and delivery data.

- Test item-related kidney and liver mean weight increases were seen in males treated with 100 mg/kg bw/day or above.

- Test item-related irregular colour and or tan discolouration of kidneys and increased incidence of accentuated lobular pattern together with tan discolouration was observed in males treated with 100 mg/kg bw/day or above. At the end of the treatment-free period, there were no test item-related gross findings in either the liver or the kidneys.

- A series of test-item related microscopic findings was found in the kidneys and liver of males treated with 100 mg/kg bw/day or above. These findings were degradation/necrosis of tubules (adverse) in 2/10 males treated at 1000 mg/kg bw/day, hyaline droplets in tubular epithelium, tubular dilation with or without eosinophilic content in kidneys, and hepatocellular hypertrophy plus increased periportal vacuolation in the liver. At the end of the treatment-free period, the reversibility of the liver changes was complete whilst reversibility of kidney changes was incomplete.

Pups: The following findings were reported:

- No test item-related effects on pup viability during treatment and treatment-free periods.

- No test-item related clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weight and mean body weight gains during treatment and treatment-free periods.

- No clear indication of a test item treatment effect on percentage of male pups at birth.

- No test item-related necropsy findings during treatment and treatment-free periods.

CONCLUSION

The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.