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Toxicity to reproduction

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Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 24 October 2008 to 13 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
2 generations toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 24 October 2008 to 13 July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
To fulfill the requirement for studying the potential effect of the test substance after repeated administration longer than 28 days, all the available information on the substance were considered all together. It appeared that the 2-generation reproduction study, in which animals were treated in both generations for 18/19 weeks (thus, longer than in a sub-chronic toxicity study) with the substance cerium and iron oxide isostearate, provided relevant information regarding the systemic toxicity of the substance. Since not all the study end-points, as measured in a conventional sub-chronic toxicity study (as in an OECD 408), were available in the 2-generation reproduction study (limited histological analysis, no haematological and biochemistry analyses, no urinalysis) but as the study was performed according to OECD GD 416 and in compliance with GLP, the study was classified reliable with restrictions. Therefore, the reliability score according to Klimisch criteria of this study was decreased, in this IUCLID section, from score 1 to score 2. However, this study is considered as sufficient for the assessment in view of the absence of effect observed in OECD the TG 407 study up to 1000 mg/kg bw (22314 TSR, CIT, 2011) as well as in all the other toxicological studies.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
See details in "Further details in study design". The deviations observed were considered not to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: (P) approximately 6 weeks old for males and approximately 5 weeks old for females; (F1) 3 weeks old (day 22 p.p.).for males and females.
- Weight at study initiation: (P) Males: 204 g to 245 g (mean value: 228 g); Females (P): 142 g to 176 g (mean value: 159 g).
- Fasting period before study: no
- Housing: the F0 males and females and the F1 generation after weaning were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). Towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet.
- Water: free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: about 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12hrs dark / 12hrs light
IN-LIFE DATES: from 23 July 2009 (arrival of the animals) to 2 April 2010 (last day of necropsy of F1 animals)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle and then passed in an ultraturax for at least 5 minutes and until the consistency appeared to be acceptable in order to achieve the nominal concentrations of 37.5, 112.5 and 250 mg/mL. The resulting suspension was left under magnetic stirring until delivery to the animal
room and, in the animal room, until treatment of the animals.
The test item dosage forms were prepared daily and were stored in brown flasks at room temperature, protected from light, prior to use.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 37.5, 112.5 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg/day
- Lot/batch no.: 017K0127, 058K0070, 049K0043, 128K0040 and MKBC6753
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the pre-study period, the homogeneity and concentration of two dosage forms prepared at concentrations which covered the lowest and highest concentrations of the study (25 and 250 mg/mL) were checked. The analytical method used was ICP-OES.

> Homogeneity and concentration:
Quadruplicate samples were taken from three levels of the container (top, middle and bottom) on the day of preparation. Two samples from each quadruplicate were analyzed and the remaining two samples were stored in case analysis was required. All samples were stored at +4°C and protected from light during shipment. On each occasion, the mean (n = 6) concentration was determined and compared to the nominal value and the coefficient of variation (CV %) was calculated. Acceptance criteria at each time-point: mean concentration = nominal value ± 20%, CV% < 10%.
The results demonstrated the satisfactory homogeneity of the two dosage forms since the differences from nominal concentration were within ± 20% and the % CV was <10%.

> Study chemical analysis - concentration:
The concentration of the test item in the dosage forms was determined in samples of each control and test item dosage form prepared for use in weeks 1, 6, 12, 14 (F0 generation), 17 (F0 and F1 g eneration), 22, 29 and 33 (F1 generation) of the study.
On each sampling day, duplicate samples were taken from each container. One sample from each duplicate was analyzed and the remaining sample was stored in case analysis was required. On each sampling day, each sample taken was stored at +4°C and protected from light until dispatch for analysis. Acceptance criterion: actual concentration: = nominal value ± 20%.
All dosage forms analyzed were within the ± 20% acceptance criterion.
Duration of treatment / exposure:
> In the F0 males:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- until sacrifice (after weaning of the pups).

> In the F0 females:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- during pregnancy,
- during lactation until day 21 post-partum (p.p.) inclusive,
- females with no delivery were treated until the day prior to sacrifice.

> In F1 males:
- from weaning (day 22 p.p.) for 10 weeks before mating,
- during the mating period (up to 3 weeks),
- until sacrifice (after weaning of the pups).

>in F1 females:
- from weaning (day 22 p.p.) for 10 weeks before mating,
- during the mating period (up to 3 weeks),
- during pregnancy,
- during lactation until day 21 p.p. inclusive,
- females with no evidence of mating or no delivery were treated until the day prior to sacrifice.
Frequency of treatment:
Once a day, 7 days a week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were selected on the basis of the results of a preliminary OECD 422 study using dose-levels of 450 and 1000 mg/kg/day (CIT/Study No. 34759 RSR, April 2010) and a 28-day repeated dose toxicity study (OCDE, 407, CIT Study N° 22314 TSR, January 2002). Neither dose elicited effects on the adult animals although the pups of the group treated at 1000 mg/kg/day had a minimally lower mean body weight gain from post natal days 1 to 5.
- Rationale for animal assignment: computerized stratification procedure, so that the average body weight of each group was similar.

Some deviations from the study plan were observed:
the temperature and relative humidity recorded in the animal room were sometimes outside the target ranges, some F1 animals had no free access to water during a short period of time because of defective water bottles, a total of 9 pups were retained in error from one F1 female (whereas each litter should have been culled to 8 pups on day 4 p.p.), two F0 females (group 1 and group 4) each gave birth to pups after delivery was thought to have been completed, one F0 male (group 2) had a left testis reduced in size which was not sampled at macroscopic examination in error, two F2 pups had scabs on the tail which were not sampled at macroscopic examination in error, statistical analyses performed on primordial follicle and corporea lutea count data were done on mean values per section per animal, instead of total number per animal.
These deviations were considered not to have compromised the validity or integrity of the study.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
In both F0 and F1generations
- Time schedule:
> Mortality or signs of morbidity: at least twice a day during the treatment period, and once a day during the acclimation period (F0 only).
> Clinical signs: From the start of treatment period, each animal was observed once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
In both F0 and F1generations
- Time schedule: once a week on all animals until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern).

BODY WEIGHT: Yes
In both F0 and F1generations
- Time schedule for examinations:
> males: on the first day of treatment (day 1), then once a week until sacrifice.
> females: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1, 4, 7, 14 and 21 p.p.. Females prematurely sacrificed were weighed prior to sacrifice.

FOOD CONSUMPTION: Yes
In both F0 and F1generations
The quantity of food consumed by each male was recorded once a week, over a 7 day period, from the first day of treatment until sacrifice.
The quantity of food consumed by each female was recorded once a week, over a 7 day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 p.c. intervals) and lactation (days 1-7, 7-14 and 14-21 p.p. intervals) until sacrifice.
During the pairing period, food consumption was not recorded for males or females.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
(F0 and F1 parental generation)
- Oestrous cyclicity (parental animals): The estrous cycle stage was determined from a fresh vaginal lavage, each morning as follows:
> during the last 3 weeks of the pre-mating period,
> during the mating period, until the females are mated.
- Sexual development (only F1 generation):
> All male animals were observed each day from day 32 of age (i.e. day 11 of F1 generation), until cleavage of the balanopreputial groove (preputial separation) was observed.
> All female animals were observed each day from day 28 of age (i.e. day 7 of F1 generation), until vaginal opening was observed.
The observation period was extended for any animals not positive by the expected end day. Body weight was recorded individually on the positive day.
- Neurobehavioral tests (only F1 generation)
> Auditory function (when the animals were 4 weeks old),
> Pupil constriction (when the animals were 4 weeks old),
> Spontaneous locomotor activity (when the animals were approximately 8 weeks old). The spontaneous locomotor activity was evaluated using an automated infra-red sensor equipment. For each trial, the activity was recorded over a 1 hour interval. The following parameters were recorded: movements within the front of the cage, movements within the back of the cage, back and forth movements.

Litter observations
STANDARDISATION OF LITTERS: performed on day 4 postpartum: maximum 8 pups/litter (4/sex/litter as nearly as possible), excess pups were killed and discarded.
The following parameters were examined in F1 / F2 offspring: litter size, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical signs, body weight, weight gain, physical or behavioural abnormalities (physical development: pinna unfolding (on day 5 p.p.), hair growth (on day 5 p.p.), tooth eruption (on day 13 p.p.), auditory canal opening (on day 17 p.p.,), eye opening (on day 17 p.p.,); and reflex development: surface righting reflex (on day 5 p.p ), cliff avoidance (on day 11 p.p), air-righting reflex (on day 17 p.p).
Sacrifice and pathology:
SACRIFICE
In both, F0 and F1generations:
On completion of the treatment period, all surviving males and females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
- F0 and F1 surviving males: after weaning of the F1 or F2 generation,
- F0 and F1 surviving females: at the weaning of the litters (on day 22 p.p.),
- F0 and F1 females which did not deliver: on day 25 p.c. after body weight recording (to check a possible un-noticed delivery),
- F0 and F1 females with litter dying entirely.
The F1 offspring not selected as parental animals and all F2 offspring were subjected to an external and internal examinations including the cervical, thoracic, and abdominal viscera.

GROSS PATHOLOGY: Yes
In both, F0 and F1generations:
- A complete macroscopic post-mortem examination was performed on all animals including on animals prematurely sacrificed of found dead. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
- The numbers of implantation sites corpora lutea and implantation sites were also recorded.
- The F1 offspring not selected as parental animals and all F2 offspring and the following pups were carefully examined externally for gross external abnormalities: pups found dead, pups prematurely sacrificed, pups culled on day 4 p.p., pups sacrificed on day 22 p.p.. In addition, for the following pups a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. Special attention was paid to the reproductive organs. pups showing external abnormalities or clinical signs, pups found dead or prematurely sacrificed, one randomly selected F1 and F2 pup/sex/litter sacrificed on day 22 p.p..

HISTOPATHOLOGY: Yes
In both, F0 and F1generations:
- Organ weights: The body weight of each animal was recorded before sacrifice at the end of th treatment period. The organs specified in Tissue Procedure Table 1 were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
- The tissues indicated in Table 1 were prepared for microscopic examination and weighed for the F0 and F1 animals of the control and high-dose groups (groups 1 and 4) and all macroscopic lesions. Furthermore, microscopic examinations were also performed on all animals sacrificed prematurely or found dead, and on all females sacrificed because of no delivery to investigate possible causes.
- Testicular staging: a detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with PAS-hematoxylin (groups 1 and 4) in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
- A detailed and careful microscopic examination was made of five sections of the right ovary of each F0 and F1 female, with enumeration of the total number of primordial follicles and corpora lutea.
- For the F1 offspring not selected as parental animals and all F2 offspring, the organs specified in Tissue Procedure Table 2 were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated. A microscopic examination was performed on: all the tissues listed in the Tissue Procedure Table 2 (for one randomly selected F1 pup/sex/litter not selected at weaning and for one randomly selected F2 pup/sex/litter), all macroscopic lesions and all pups with external abnormalities.

- Sperm parameters (parental animals)
Parameters examined in F0/F1 male parental generations: testis weight, epididymis weight, sperm count in testes, epididymal sperm motility, epididymal sperm morphology.
Other examinations:
The following reproductive indices were evaluated:
Pre-implantation loss, Post-implantation loss, Mating index, Fertility index, Gestation index, Number of corpora lutea, Number of implantations, Number of pups delivered.
The following offspring viability indices were evaluated:
Live birth index, Viability index on day 4 post-partum, Lactation index on day 21 post-partum, Mean litter size, Pup sex ratios.
Statistics:
Body weights, food consumption and reproductive data: mean values were compared by one-way variance analysis and Dunnett test. Percentage values were compared by Fisher exact probability test.
Organ weights: a sequence of statistical tests was used according to PathData software.
Auditory startle reflex: performed using the software SAS Enterprise Guide version 2.05.89.
Numbers of corpora lutea and primary follicles: normality and homogeneity of variances were tested using Kolmogorov Smirnov and Bartlett tests. If normality and homogeneity of variances were demonstrated (p-value>0.5 for both tests), a Student test was implemented. If normality and homogeneity of variances were not demonstrated (p-value<0.05 for one or both of the tests), a Mann-Withney-Wilcoxon test was conducted.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
P0 GENERATION:
No premature death occurred during the study in F0 male and female rats.
Some females were sacrified during lactation because of clinical signs or dead litter:
- One female at 1000 mg/kg was prematurely sacrificed on lactation day 9 because of clinical signs of piloerection, pallor, hypoactivity, abdominal breathing and half-closed eyes. At microscopic examination, the presence of bacterial colonies in a thrombus of the left cardiac atrium indicated that an infection caused the moribundity of this female.
- Two females at 450 mg/kg were prematurely sacrificed on lactation day 1 because of dead litter. Both females had dead fetuses in the uterine horns at necropsy and both females had necrosis and acute inflammation of the uterus and centrilobular necrosis in the liver at microscopic examination. Since similar lesions were observed in a control female (see below) a relationship to treatment with the test item is considered unlikely.
- One female in the control group was prematurely sacrificed on lactation day 1 because of dead litter. The female had marked centrilobular degeneration/necrosis in the liver, focal and marked necrosis in the uterus.
There were no test item treatment-related clinical signs.
Chromodacryorrhea, reflux at dosing, salivation, nodosities, hairloss, scabs and lesions were all observed at an equal or greater incidence in the controls animals, were considered to be related to the viscosity of the vehicle, corn oil, or are regularly observed in laboratory rats.
One female treated at 450 mg/kg had a mass on the 2nd right mammary gland at the end of the lactation period. This can be observed in lactating rats and given the isolated nature was considered not to be related to treatment with the test item.

F1 generation - Clinical signs during lactation:
There were few clinical signs in surviving pups, scabs and small wounds are regularly observed, and only one surviving pup from the dam S25228, given 450 mg/kg/day, had dehydration and coldness on days 6 to 8 p.p..

P1 GENERATION: no effects observed
- 1000 mg/kg/day: One female was found dead on day 48. Clinical signs of pallor, hypoactivity, piloerection, chromorhinorrhea and half-closed eyes had been observed from day 46 or day 47. Death was related to marked suppurative pyelonephritis and cystitis which were considered to be incidental and unrelated to the test item administration.
Two females were sacrificed on gestation day 23 because of difficulties to deliver evidenced by clinical signs of pallor and piloerection: one female had a fetus in the vagina but had not delivered any pups, the other female had delivered eight pups (seven live and one dead) but parturition was not complete. At microscopic examination, there were necrotic uterine lesions along with dead fetuses. A relationship to the test item administration was considered to be unlikely in view of their low incidence and presence of similar uterine lesions in a control female.
- 450 mg/kg/day: One female was found dead on day 10 of dosing. No clinical signs had been observed before death. Death was related to gavage pneumonia and was therefore unrelated to the test item.
- 150 mg/kg/day: One male was prematurely sacrificed on day 126 of treatment because of clinical signs of piloerection, round back, bent head, hypoactivity, loud breathing, half-closed eyes, swollen neck region and chromorhinorrhea. At necropsy, this male had an esophageal pouch which correlated with marked acute inflammation microscopically. This finding explained the clinical signs observed and was secondary to a dosing error.
- Control: One control female was prematurely sacrificed on gestation day 24 because of difficulties to deliver. The female had delivered 13 pups (10 live and 3 dead) but was pale and it was considered that delivery was not complete. At microscopic examination, there was a marked suppurative inflammation of the uterus. Another female was prematurely sacrificed on lactation day 7 because of dead litter: there were no macroscopic or microscopic findings.

There were no test item treatment-related clinical signs. A few signs of lesions (hairloss and nodosities) were also observed in control animals and are often seen in laboratory rats. One male treated at 1000 mg/kg/day had a mass on the left forelimb which increased in size from 2 x 1 cm on day 113 to 4 x 4 cm on the day of sacrifice (day 130). At microscopic examination, this mass correlated with a subcutaneous sarcoma which was considered to be incidental. Such malignant tumors have been reported in the literature in rats of this age (Son and Gopinath, 2004).

F2 GENERATION:
There were no test item treatment-related clinical signs. Scabs and hematomas are regularly observed in young rats and incidences of coldness were observed in control pups as well as in those from the groups treated at 450 or 1000 mg/kg/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
P0 GENERATION:
No premature death occurred during the study in F0 male and female rats.

P1 GENERATION:
see clinical signs.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
F0 GENERATION (See table 3a):
Mean male body weight gains over the study were similar to those of the controls (between 0% and +5% differences).
The female group treated at 450 mg/kg/day gained statistically significantly more body weight over the 10 week pre-mating period than the controls (+12%). This was due mostly to statistically significantly higher mean body weight gains in weeks 5 and 7. In the absence of any effects at the higher or lower dose-level, this body weight gain difference was considered not to be related to treatment with the test item.
There were no effects of treatment with the test item on mean female body weight gains during the gestation period or during the lactation period at 450 or 1000 mg/kg/day. The mean body weight gain during the lactation period of females treated at 150 mg/kg/day was lower than that of the other groups. This was due to slightly lower body weight gains during the first part of lactation and then slightly greater body weight loss during the second part. Since the groups treated with higher dose levels were not similarly affected, this lower body weight gain was considered not to be related to treatment with the test item.

Body weight of F1 pups during lactation : There were no effects of treatment with the test item on mean pup body weight or body weight gains at any dose-level.

F1 GENERATION (See table 3b):
There were no effects of treatment with the test item on mean male body weight or body weight gain during the 10-week premating treatment period. By the end of the study, the test item treated males had gained more weight than the controls and mean body weights were 3% to 4% higher.
Females treated at 150 mg/kg/day had greater mean body weight gains than the controls throughout the pre-mating and gestation periods, and had statistically significantly higher mean body weights throughout gestation and most of lactation (+6% to +9%, p<0.05, p<0.01 or p<0.001) although the overall mean body weight gain did not achieve statistical significance. The group treated at 1000 mg/kg/day had statistically significantly higher mean body weight gains during the first two weeks of gestation which contributed to a non-statistically significantly higher overall body weight gain.
Mean body weight gains over lactation were similar to that of the controls.
While treatment-related, all these changes were considered of not toxicological significance.

F2 GENERATION:
The pups from the group treated at 150 mg/kg/day had a statistically significantly greater mean body weight gain between days 7 and 14 p.p. which resulted in higher mean body weights of both male and female pups at the end of lactation. There were no effects at 450 or 1000 mg/kg/day.
In the absence of any dose-related effect, a relationship to the treatment was considered unlikely.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F0 GENERATION:
Mean male food consumption was statistically significantly higher at 1000 mg/kg/day in weeks 6 and 7 and then from weeks 9 to 16, when compared with the controls. This correlated with greater body weight gains.
The female groups treated at 450 or 1000 mg/kg/day had slightly greater food consumption than the controls at the beginning of the pre-mating period (+9%, p<0.05) and the female group treated at 1000 mg/kg/day again had slightly, but statistically significantly, greater food consumption than the controls at the end of the pre-mating period and during the middle of the gestation period.
There were no effects of treatment with the test item on food consumption during the lactation period.

F1 GENERATION:
Mean food consumption of the males treated at 1000 mg/kg/day was statistically significantly higher than that of the controls from week 7 until the end of the study. The mean food consumption of the group treated at 450 mg/kg/day was also statistically significantly higher from week 12.
The female group treated at 1000 mg/kg/day showed a similar effect, having statistically significantly higher food consumption from week 9 until mid-gestation, when compared with the controls.
In all treated groups mean food consumption of the females from treated groups was slightly, but non statistically significantly, higher than that of the controls during lactation.
While treatment-related, all these changes were considered of not toxicological significance.
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 examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F0 GENERATION (see table 7):
The test item administration was associated with increases in the mean kidney and liver weights in F0 male parents. However, these changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
There were increases in the mean ovary weights (both sides) in high-dose females compared with controls. The biological significance of these variations remained unclear.
There were minimal, statistically significant increases of the mean absolute weights of the right adrenal in mid-dose and high-dose females. In the absence of similar variations in the left adrenal, these changes were considered not to be of toxicological significance.
There were statistically significant increases of the mean prostate weights (up to 15%) in high dose males. Since there were no microscopic correlates and no changes in the prostate weights of F1 males, these variations were considered to be incidental and not to be related to the test item administration.
The test item administration in F0 parents did not induce any changes in organ weights in their pups.

F1 GENERATION (see table 8):
The test item administration was associated with statistically significant increases in the mean kidney and liver weights in F1 male parents. However, these changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose related in magnitude, and/or were not consistent for the sexes.
There were increases of the mean absolute (statistically significant) and relative weights of seminal vesicles in mid-dose and low-dose males compared with controls. In the absence of similar variations in high-dose males and since there were no macroscopic and microscopic correlates, this finding was considered to be incidental and unrelated to the test item administration.
The test item did not induce any significant change in all treated groups of F1 females as compared to controls.
The test item administration in F1 parents did not induce any changes in organ weights in their pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F0 GENERATION:
Premature deaths
- 1000 mg/kg/day: in the female sacrificed moribund on lactation day 9, there was a diffuse white discoloration of the right cardiac ventricle. In addition, the liver had a marked lobular pattern, and the adrenals and the spleen were enlarged.
- 450 mg/kg/day: two females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns. In one of them, the thymus was gelatinous and the spleen was enlarged.
- Control: In the female sacrificed on lactation day 1, the thymus was gelatinous, the liver was diffusely white, and the spleen was enlarged.
The other macroscopic findings in these animals had no microscopic correlates or correlated with common histologic findings in control rats, and were considered to be incidental.

Terminal sacrifice:
The few macroscopic findings noted at the end of the treatment period in F0 parents as well as in their pups were of those commonly recorded in the Sprague-Dawley rat, and none were considered to be related to the test item administration. Among them was a mass in the subcutis of one female (450 mg/kg/day) which correlated with marked, multilobular hyperplasia associated with subacute inflammation of the mammary gland.

F1 GENERATION:
Premature deaths:
- 1000 mg/kg/day: one found dead female (day 48) had many white discolored foci in the kidneys, unilaterally dilated renal pelvis, dilated urinary bladder with red liquid content, and enlarged adrenals. In two females sacrificed because of difficulties to deliver (day 23), there were dead fetuses and black content within both uterine horns and red content in the vagina ; in one female, all 17 fetuses remaining in the uterine horns were found dead, whereas in the other female, four fetuses were found dead and one fetus was still alive.
- 450 mg/kg/day: In one female, found dead on day 10, the lungs were enlarged.
- 150 mg/kg/day: a male sacrificed moribund on day 126 had a large pouch (2.5 cm long) around the esophagus with white thick content.
- Control: In one control female sacrificed because of difficulties to deliver (day 24), there was still one fetus within the uterus and two placentas in the vagina. The spleen was enlarged. In another female sacrificed on lactation day 7 because of dead litter, there were no macroscopic findings.
Terminal sacrifice:
The few macroscopic findings noted at the end of the treatment period in F1 parents, as well as in their pups were of those commonly recorded in the Sprague-Dawley rat, and none were considered to be related to the test item administration. Among them was a large white mass of the forelimb of high-dose parent male S24963, which correlated microscopically with a subcutaneous sarcoma. Such malignant tumors have been reported in the literature in rats of this age (Son and Gopinath, 2004).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
F0 GENERATION:
Premature deaths:
- 1000 mg/kg/day: In one female sacrificed moribund on lactation day 9, the presence of severe cardiac lesions of septic origin explain the poor clinical status of this animal. This animal had a severe thrombosis of the left cardiac atrium, along with secondary hypertrophy of the right ventricle, and multifocal degeneration/necrosis of the myocardium, the latter correlated with white discoloration at necropsy. Numerous bacterial colonies were found within the thrombus and were most likely secondary to genital infection during delivery. Therefore the cardiac lesions were considered to be incidental and unrelated to the test item administration. In addition, there was a moderate centrilobular necrosis in the liver, correlated with marked lobular pattern at necropsy, and which may also have contributed to the poor clinical condition. Although the pathogenesis is unclear, the presence of a similar lesion in a control female makes the relationship to the test item administration very unlikely. There was a marked, diffuse cortical hypertrophy in the adrenals, correlated with macroscopic enlargement. In the spleen, there was a marked extramedullary hemopoiesis correlated with macroscopic enlargement, and which was considered to be secondary to the ongoing infection and inflammatory lesions. A slight lymphoid atrophy of the spleen and increased porphyrin pigment in the Harderian glands, correlated with black discoloration at necropsy, were considered to be secondary to the stress of the severe cardiac lesions. There were no abnormalities in the genital organs. Placentas were seen in the uterus. The vagina showed marked mucification, as it is awaited during pregnancy and days after delivery.
- 450 mg/kg/day: two females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns.
In one female, moderate acute neutrophilic inflammation and slight multifocal necrosis were found in the uterus (mucosa, lumen and placenta) and correlated with the presence of a dead fetus at necropsy. Slight acute centrilobular necrosis was seen in the liver and likely also contributed to the poor clinical status of this rat. There was a marked lymphoid atrophy of the thymus, which correlated with the gelatinous aspect, and was considered to be secondary to the inflammatory and necrotic lesions in the uterus and liver. In the spleen, there was a marked extramedullary hemopoiesis correlated with macroscopic enlargement, and which was considered to be secondary to the ongoing inflammatory lesions. The second female had similar lesions of necrosis and acute inflammation in the uterus, and of centrilobular necrosis in the liver. In both females, it is unclear whether the uterine findings were primary or secondary to the presence of dead fetuses. Both the uterine and hepatic lesions contributed to their poor clinical status. For both animals, although the pathogenesis of the hepatic necrosis is unclear, the presence of similar necrotic uterine and hepatic lesions in a control female makes the relationship to the test item administration very unlikely. The vagina of both females showed marked mucification, as it is awaited during pregnancy and within days after delivery.
- Control: In one female sacrificed on lactation day 1 with a dead litter, marked centrilobular hepatic degeneration/necrosis correlated with white color of the liver. There was a focal, marked necrosis in the uterus in one horn and within the underlying mucosa and placenta, along with multifocal vascular necrosis. Both the hepatic and genital lesions were considered to have contributed to the poor clinical condition of this animal. In the spleen, there was a marked extramedullary hemopoiesis, correlated with macroscopic enlargement, and considered to be secondary to the ongoing inflammatory lesions. There was a diffuse marked lymphoid atrophy of the thymus, correlated with gelatinous aspect at necropsy, and which was secondary to the stress of the lesions. The vagina showed marked mucification, as it is awaited during pregnancy and within days after delivery. There were neutrophils within the vaginal epithelium and lumen, which is not considered to be abnormal after delivery.

Terminal sacrifice:
F0 generation:
> Qualitative evaluation of the genital organs in F0 parents:
There were no significant differences between control and high-dose groups in the incidences and severity of microscopic findings in the genital organs from F0 parents. In particular, at histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
In females which were not pregnant (one at 150 mg/kg, one at 450 mg/kg and three at 1000 mg/kg), there were no microscopic findings in the genital organs attributed to the test item.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
> Quantitative evaluation of the ovaries in F0 females:
There was a slight, statistically significant increase in the mean numbers of corpora lutea in high dose females (12.82 ± 3.047 compared to 10.74 ± 4.631 in control, p<0.01), which correlated with the increase in the mean ovary weights in this group. This variation was not toxicologically significant based on the direction of the change.
There was a slight, not statistically significant, decrease in the mean numbers of primordial follicles in high-dose F0 females. Since such variation was not found in high-dose F1 females, this variation was not considered to be toxicologically significant.
> Microscopic evaluation of the liver and kidneys in F0 parents:
The test item administration at 1000 mg/kg/day induced minimal centrilobular hypertrophy in the liver from 16/25 males, correlated with the increased liver weights. This finding was not adverse. There were no significant changes in the liver from high-dose females.
At this dose-level, the test item administration also induced a minimal increase of the incidence and severity of hyaline droplets in the proximal renal tubules from males, correlated with the increased kidney weights. There were no significant microscopic changes in the kidneys from high-dose females. This change, spontaneously seen in normal mature rats, was reported to be exacerbated by some chemicals but is known to be male rat-specific and therefore has no human relevance.

F1 GENERATION:
Premature deaths:
- 1000 mg/kg/day: In found dead female (day 48), there was a marked suppurative inflammation of the kidneys (pyelonephritis) correlated with the macroscopic white foci and pelvic dilation, urinary bladder (cystitis correlated with dilation and red content) and tissue adjacent to the vagina. Numerous bacterial colonies were seen in the kidneys. These suppurative lesions were the cause of death of this female. There was a moderate diffuse hypertrophy of the cortex correlated with enlargement, and which was considered to be secondary of the stress of the multiple suppurative lesions in this animal. Pyelonephritis and cystitis are common findings in female rats, and in this case were considered to be incidental and unrelated to the test item administration. In two females sacrificed because of difficulties to deliver (day 23), most fetuses were dead. In the uterus, there was slight acute focal necrosis on the endometrial surface at the level of one placenta in both females, along with slight multifocal hemorrhage in one female and minimal multifocal neutrophilic inflammation in the other female. It is unclear whether the uterine lesions were primary or secondary to the presence of dead fetuses. All these findings likely contributed to the clinical signs of the females. A relationship to the test item administration of the fetal deaths was considered to be unlikely in view of their low incidence and presence of similar uterine lesions in a control F0 female.
- 450 mg/kg/day: In the female found dead on day 10, there was a diffuse gavage pneumonia characterized by the presence of yellow material interpreted as compound within the alveoli. This pneumonia, correlated with the macroscopic enlargement, was considered to be the cause of death.
- 150 mg/kg/day: the esophageal pouch of the male sacrificed moribund on day 126 correlated with marked acute inflammation microscopically. This finding explained the clinical signs observed and was secondary to a dosing error.
- Control: in the control female sacrificed because of difficulties to deliver (day 24), there was a marked acute neutrophilic inflammation of the uterus related to the presence of bacterial colonies, associated with multiple necrotic areas in the mucosa/placentas and focal thrombosis. It is difficult to determine whether the septic inflammation was the cause or the consequence of the difficulties to deliver. There was marked hemopoiesis in the spleen which correlated with splenic enlargement, and was considered to be compensatory to the ongoing inflammatory lesions. In the control female sacrificed on lactation day 7 because of dead litter, there were no significant microscopic findings in the genital organs.

Terminal sacrifice:
> Qualitative evaluation of the genital organs in F1 parents:
There were no significant differences between control and high-dose groups in the incidences and severity of microscopic findings in the genital organs from F1 parents. . In particular, at histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
In a female at 150 mg/kg/day which was not pregnant and was sacrificed on day 99, there was evidence of a persistent estrus, characterized by multiple follicular cysts and absence of corpora lutea in the ovaries, tall columnar endometrial epithelium and squamous metaplasia in the uterus, and epithelial hyperplasia and cornification in the vagina (Westwood, 2008). This isolated condition, not observed in high-dose females, was considered to be incidental and unrelated to the test item administration.
In females which were not pregnant (one in control, three at 150 mg/kg, two at 450 mg/kg and two at 1000 mg/kg), there were no microscopic findings in the genital organs attributed to the test item.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
> Quantitative evaluation of the ovaries in F1 females:
There were no significant differences in the mean number of primordial follicles between control and high-dose F1 females. In the right ovary of one high-dose female, there were no primordial follicles and there was a small number of corpora lutea (n=9), which correlated with a reduced size macroscopically. Since the left ovary of this female was not affected, this unilateral change was considered to be incidental and unrelated to the test item administration.
There was a slight, not statistically significant increase in the mean number of corpora lutea in high-dose F1 females compared with controls. This variation was not considered to be toxicologically significant based on the direction of the change.
> Microscopic evaluation of the liver and kidneys in F1 parents:
The test item administration at 1000 mg/kg/day induced minimal centrilobular hypertrophy in the liver from 10/25 males, correlated with the increased liver weights. This finding was not adverse. There were no significant changes in the liver from high-dose females.
There were no significant differences in the incidences of hyaline droplets in the kidneys from high-dose males compared with controls. There were no significant findings in the kidneys from high-dose females.

All microscopic findings noted in treated F0/F1 parent animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.-
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
F0 and F1 parents:
Reproductive function:
- oestrous cycle: There were no effects of treatment with the test item on estrous cycles of F0 and F1 females.
- sperm measures: There were no effects of treatment with the test item on sperm parameters in the F0 and F1 males.
Reproductive performance: There were no treatment-related effect on mean reproductive parameters and indices in F0 and F1 parents.
(see detailed results in section 7.8.1: "Source DPX10 2-Gen. Oral Tox V1 2011 CIT")

F1 pups - Sexual development:
There was no treatment-related effect on sexual development: There were no treatment-related effects on balanopreputial separation at any dose-level. There were no test item treatment-related effects on vaginal opening at any dose-level. The later mean age of vaginal opening at 150 and 450 mg/kg/day was due to one female per group which was considered not to have achieved complete opening before mating.

F1 pups - neurobehavioral tests:
- Auditory function: There was no treatment-related effect on auditory function. All animals had a positive response to the auditory startle test. The latency and amplitude of the response was similar in all groups therefore it was concluded that no group showed impairment of hearing.
- Pupil constriction and locomotor activity: There was no treatment-related effect on pupil constriction and locomotor activity. All groups had similar numbers of horizontal and rearing movements during the 1-hour period as the controls. All animals were positive for pupil constriction reflex at 4 weeks of age.

F1 pups - Physical and reflex development during lactation:
All pups were positive for pinna unfolding and hair growth on post-natal day 5, tooth eruption on day 13 p.p. and auditory canal opening on day 17 p.p.. Not all pups were positive for eye opening on day 17 p.p., however the incidence was identical to the control group (one pup per group in groups 1 and 4).
The number of pups passing the surface righting and air righting tests was similar to or greater than in the control group at all dose-levels. The number of pups failing the cliff avoidance test on day 11 p.p. was slightly higher at 450 and 1000 mg/kg/day than in the control group. In the absence of any effects on any of the other physical or reflex development tests, this difference was considered to be incidental.


F2 generation
There were no test item treatment-related clinical signs and no effects on physical or reflex development. All pups were positive for pinna unfolding and hair growth on day 5 p.p. and tooth eruption on day 13 p.p.. Not all pups were positive for eye opening or auditory canal opening on day 17 p.p., however the
incidence was nearly identical to the control group. The number of pups passing the surface righting, cliff avoidance and air righting tests was similar
to or greater than in the control group at all dose-levels.
There were no test item-related changes in organ weights or macroscopic findings in the pups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant systemic toxicity observed in all animals of the study
Key result
Critical effects observed:
no

Table 3a: Body weightand body weight change(F0 generation)

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Premating

 

 

 

 

 

 

 

 

Days 1 - 71

+317

+331

+312

+331

+139

+144

+156**

+147

Days 1 - 127

+396

+416

+395

+416

/

/

/

/

Gestationa

 

 

 

 

 

 

 

 

GD 0 - 20

/

/

/

/

+144

+151

+143

+150

Lactation

 

 

 

 

 

 

 

 

LD 1 - 21

/

/

/

/

+16

+6

+18

+18

GD: gestation day, LD: lactation day, /: not applicable, a: only includes pregnant females with live fetuses.

Statistically significant **: p<0.01.

Table 3b: Body weight and body weight change (F1 generation)

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Premating

 

 

 

 

 

 

 

 

. Days 1 - 71

+462

+473

+477

+475

+222

+232

+226

+222

. Days 1 - 127

+566

+585

+594

+581

/

/

/

/

Gestation

 

 

 

 

 

 

 

 

. GD 0 - 20

/

/

/

/

+148

+161

+156

+166

Lactation

 

 

 

 

 

 

 

 

. LD 1 - 21

/

/

/

/

+19

+13

+17

+23

GD: gestation day, LD: lactation day, /: not applicable.

Table 4a : Estrous cycles (F0 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of cycles per female

4.7

4.8

4.8

4.7

Mean cycle length

(days)

4.0

4.0

4.1

4.1

Number of abnormally cycling females

1

2

2

2

An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.

Table 4b : Estrous cycles (F1 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of cycles per female

4.6

4.5

4.8

4.6

Mean cycle length

(days)

4.2

4.4

4.1

4.2

Number of abnormally cycling females

1

2

1

2

An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.

Table 5a: Mating, fertility and parturition (F0 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Number of males + females paired

25 +25

25 + 25

25 + 25

25 + 25

Number of pairs mated

25

25

25

25

Mating index

100%

100%

100%

100%

Mean number of days taken to mate

2.0

2.9

3.4

3.0

Number of pregnant females

25

24

24

23

Fertility index

100%

96%

96%

92%

Number of females delivering live pups

25

24

24

22

Gestation index

100%

100%

100%

96%

Mean duration of gestation (days)

22.0

21.8

21.8

21.9

Table 5b: Mating, fertility and parturition (F1 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Number of males paired

25

25

24

24

Number of males mated

25

23

24

24

Male fertility index

100%

92%

100%

100%

Number of females paired

25

25

24

24

Number of females mated

25

25

24

24

Mating index

100%

100%

100%

100%

Mean number of days taken to mate

3.0

4.3

2.7

2.8

Number of pregnant females

24

21

22

22

Fertility index

96%

84%

92%

92%

Number of females delivering live pups

23a

21

22

20a

Gestation index

96%

100%

100%

91%

Mean duration of gestation (days)

21.9

21.9

21.9

21.9

a: females sacrificed mid-parturition because of poor clinical condition.

Table 6a: Mating, fertility and parturition (F0 generation) Cont’

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of implantation sites

13.8

14.8

14.4

14.1

Mean number of pups born

11.8

12.7

12.7

12.3

Post-implantation loss (calculated manually)

14.2

14.2

11.7

12.4

Number of entire litters dead (no. of pups)

1 (14)

0

2 (24)

1 (14)

Number of dead pups: days 1 - 4

22

6**

38*

34*a

Number of dead pups: days 5 - 21

0

1

0

1

Number of litters with dead pups

6

4

6

11

% of male pups at birth

51.7%

46.7%

48.8%

50.0%

Statistically significant *: p<0.05, **: p<0.01.  a: does not include one pup which was cannibalized and could not be sexed.

Table 6b: Mating, fertility and parturition (F1 generation) Cont’

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of implantation sites

12.1

14.4

13.1

14.4

Mean number of pups born

11.1

12.3

12.0

12.1

Post-implantation loss (calculated manually)

10.1

14.0

8.2

16.1

Number of entire litters dead

1

0

0

0

Number of dead pups: days 1 - 4

19

12

12

14

Number of dead pups: days 5 - 21

1

4

1

0

Number of litters with dead pups

11

9

5

8

% of male pups at birth

46.6

50.8

39.5

51.4

Table 7:Relevant changes in mean absolute and relative organ weights in treated F0 parents (% changes from controls)

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

150

450

1000

150

450

1000

Exam. animals / Num. of animals

25/25

25/25

25/25

25/25

23/25

24/25

Body weight

+3

-1

+3

0

+2

+2

- Kidney, left

 

 

 

 

 

 

  . absolute

+6*

+7*

+12**

+2

+6*

+3

  . relative

+3

+7*

+9**

+2

+3

+1

- Kidney, right

 

 

 

 

 

 

  . absolute

+3

+6*

+10**

+5

+5

+4

  . relative

0

+7**

+7**

+4*

+3

+2

- Liver

 

 

 

 

 

 

  . absolute

+10**

+9**

+17**

+2

+4

+1

  . relative

+7**

+10**

+14**

+2

+1

-1

- Ovary, left

 

 

 

 

 

 

  . absolute

 

 

 

+16*

+14

+17*

  . relative

 

 

 

+16

+11

+15*

- Ovary, right

 

 

 

 

 

 

  . absolute

 

 

 

+9

+12

+18**

  . relative

 

 

 

+9

+10

+16*

Statistically significant from controls: *: p<0.05, **: p<0.01.

Statistical significance determined for organ weights values and not percent changes.

Table 8:Relevant changes in mean absolute and relative organ weights in treated F1 parents (% changes from controls)

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

150

450

1000

150

450

1000

Exam. animals / Num. of animals

24/25

25/25

25/25

25/25

24/25

22/25

Body weight

+4

+5

+3

+4

+1

+2

- Kidney, left

 

 

 

 

 

 

  . absolute

+8*

+10**

+9**

+3

-2

+4

  . relative

+4

+5

+7*

-1

-3

+2

- Kidney, right

 

 

 

 

 

 

  . absolute

+7

+10**

+6*

+3

0

+5

  . relative

+3

+5

+4

-1

-2

+2

- Liver

 

 

 

 

 

 

  . absolute

+8*

+9*

+13**

+1

+2

0

  . relative

+4

+4

+10**

-3

+1

-2

Statistically significant from controls: *: p<0.05, **: p<0.01.

Statistical significance determined for organ weights values and not percent changes.

Table 9a : Sperm analysis (F0 generation)

Dose-level (mg/kg/day)

0

1000

Mean number of epididymal sperm (106/cauda)

171

173

% of motile sperm

96

93

% of morphologically normal sperm

96

96

Mean number of sperm heads (106/g testis)

125

125

 

Table 9b : Sperm analysis (F1 generation)

Dose-level (mg/kg/day)

0

1000

Mean number of epididymal sperm (106/cauda)

177

191

% of motile sperm

90

99

% of morphologically normal sperm

83

92

Mean number of sperm heads (106/g testis)

122

121

Table 10: Body weight and body weight change in F1 generation during lactation (pups) 

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Body weight (g)

 

 

 

 

 

 

 

. Day 1

7.8

7.7

7.5

7.6

7.3

7.3

7.1

7.1

. Day 21

57.1

60.6

58.6

57.8

56.1

58.5

56.7

56.3

Body weight gain (g)

 

 

 

 

 

 

 

. Days 1 - 21

+49.3

+52.9

+51.1

+50.2

+48.8

+51.2

+49.6

+49.2

Conclusions:
Due to absence of effect after repeated administration of the test substance for up to 19 weeks, the No Observed Adverse Effect Level for the F0 and F1 generations for systemic toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

To fulfill the requirement for studying the potential effect of the test substance after repeated administration longer than 28 days, all the available information on this substance were considered. It appeared that the 2-generation reproduction study, performed according to OECD GD 416 and in compliance with GLP, in which animals were treated in the 2 generations for 18/19 weeks (thus, longer than in a sub-chronic toxicity study such as an OECD GD 408) with the substance cerium and iron oxide isostearate, provided relevant information regarding the systemic toxicity of the substance. Since not all the study end-points measured in a conventional sub-chronic toxicity study was available in the 2-generation reproduction study, the reliability score according to Klimisch criteria of this study was decreased in this IUCLID section from score 1 to score 2.

In this study, four groups of 25 male and 25 female Sprague-Dawley rats received the test item Cerium and iron oxide isostearate, daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups (corresponding to up to 18/19 weeks of treatment). The test item was administered as a suspension in corn oil, by oral gavage, at dose-levels of 0 (control), 150, 450 or 1000 mg/kg/day. A constant dosage volume of 4 mL/kg/day was used.

The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once a week. Body weight and food consumption were recorded weekly. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening and, surface righting, cliff avoidance and air righting reflexes).

After weaning of the pups, the males and females of the F0 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs of the control and high-dose group animals and on all macroscopic lesions.

After weaning (day 22 post-partum) of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of four groups of 25 male and 25 female rats. Three groups received the test item, Cerium and iron oxide isostearate, and the fourth group (control) received the vehicle only (corn oil)daily for 10 weeks prior to mating and, during mating, gestation and lactation until weaning of the pups. The test item was administered as a suspension in corn oil, by oral gavage, at dose-levels of 150, 450 or 1000 mg/kg/day under a constant dosage‑volume of 4 mL/kg/day.

The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once a week. Body weight and food consumption were recorded weekly. Each animal was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight was recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, andweeks of age, spontaneous locomotor activity was measured using an automated infra-red sensor equipment. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 16 days.

The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development was assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening and, surface righting, cliff avoidance and air righting reflexes).

At weaning of the pups, the males and females of the F1 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs of the control and high-dose groups and on all macroscopic lesions.

In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.

There were no found dead animals in F0 generation. A total of four females (one at 1000 mg/kg/day, two at 450 mg/kg/day and one in controls) were prematurely sacrificed during lactation, and microscopic examination findings excluded a relationship to the test item.

The F1 generation showed no effects of treatment while pups; there were no test item treatment‑related clinical signs, no effects on body weight and no differences in physical or reflex development when compared with the controls.There were no treatment-related findings at external examination of the F1 pups. There were no test item treatment-related mortalities In F1 parents.

No test item treatment-related clinical signs were observed in F0 parents and there were no treatment-related effects on body weight or body weight gain. There was a statistically significant increase in mean food consumption in males and females treated at 1000 mg/kg/day during the pre-mating period and mid-gestation and in females treated at 450 mg/kg/day at the beginning of the pre-mating period. As for the F0 generation, the F1 males treated at 450 or 1000 mg/kg/day and the F1 females treated at 1000 mg/kg/day had statistically significantly increased food consumption which correlated with a tendency towards a non statistically significant increase in body weight gains. The F1 females treated at 150 or 450 mg/kg/day also had slightly increased food consumption during lactation.

There were no effects on estrous cyclicity, mating or fertility parameters at any dose-level in F0 and F1 parents. F1 pup mortality was statistically significantly higher at 450 and 1000 mg/kg/day, but concentrated in a few litters, probably related to poor maternal nesting/nursing behavior since one dead litter was also found in the control group. Thus, it was considered unlikely that these deaths were related directly to test item treatment. Further, no such effect was seen in F2 pups.

No effects on spermatogenesis were detected after analysis of epididymidal sperm motility and morphology and count and, after analyis of testicular sperm count in the males of the 2 generations. At histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.

At all dose-levels, there were a few organ weight changes in F0 parents which were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes. In F1 males, the test item administration at all dose-levels induced statistically significant dose-related increases in mean kidney and liver weights. On microscopic examiniation, the increase in mean liver weight was correlated with minimal centrilobular hypertrophy in males at 1000 mg/kg/day. There were no microscopic correlates in the kidneys. There were no effects on organ weights and no relevant microscopic findings in the liver and kidneys in F1 females. There were no test item-related macroscopic findings in F1 parents.

There were no test item‑related microscopic findings in the genital organs from F0 and F1 parents.

There was a statistically significant increase in the mean ovary weight in F0 females treated at 1000 mg/kg/day, correlating with an increase in the mean number of corpora lutea. This finding was not considered to be toxicologically significant based on the direction of the change. In this group, mean number of primordial follicles was slightly decreased. Since such variation was not found F1 females, this variation was not considered to be of toxicological significance. There were increases in the mean number of corpora lutea in high-dose F1 females. This finding was not considered to be toxicologically significant based on the direction of the change. There were no significant differences in the mean number of primordial follicles between control and high‑dose F1 females.

There were no test item treatment-related clinical signs in F2 pups and no effects on physical or reflex development. The pups from the groups treated at 150 mg/kg/day had statistically significantly higher mean body weight gains mid-lactation but there were no effects at 450 or 1000 mg/kg/day. Therefore a relationship to the treatment with the test-item was considered unlikely. There were no test item-related changes in organ weights or macroscopic findings in the pups.

The No Observed Adverse Effect Level for systemic toxicity of F0 and F1 generations was therefore considered to be 1000 mg/kg/day.

No classification for systemic toxicity is warranted based on the absence of relevant effects in this study, according to the criteria of UN/EU GHS.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
See details in "Further details in study design". The deviations observed were considered not to have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
753480-32-9
Cas Number:
753480-32-9
IUPAC Name:
753480-32-9

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: (P) approximately 6 weeks old for males and approximately 5 weeks old for females; (F1) 3 weeks old (day 22 p.p.).for males and females.
- Weight at study initiation: (P) Males: 204 g to 245 g (mean value: 228 g); Females (P): 142 g to 176 g (mean value: 159 g).
- Fasting period before study: no
- Housing: the F0 males and females and the F1 generation after weaning were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). Towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet.
- Water: free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: about 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: from 23 July 2009 (arrival of the animals) to 2 April 2010 (last day of necropsy of F1 animals)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle and then passed in an ultraturax for at least 5 minutes and until the consistency appeared to be acceptable in order to achieve the nominal concentrations of 37.5, 112.5 and 250 mg/mL. The resulting suspension was left under magnetic stirring until delivery to the animal room and, in the animal room, until treatment of the animals.
The test item dosage forms were prepared daily and were stored in brown flasks at room temperature, protected from light, prior to use.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 37.5, 112.5 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg/day
- Lot/batch no.: 017K0127, 058K0070, 049K0043, 128K0040 and MKBC6753
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days had elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: towards the end of the gestation period and with their litter during lactation, the females were housed in polycarbonate cages (43.0 x 21.5 x 20.0 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the pre-study period, the homogeneity and concentration of two dosage forms prepared at concentrations which covered the lowest and highest concentrations of the study (25 and 250 mg/mL) were checked. The analytical method used was ICP-OES.

> Homogeneity and concentration:
Quadruplicate samples were taken from three levels of the container (top, middle and bottom) on the day of preparation. Two samples from each quadruplicate were analyzed and the remaining two samples were stored in case analysis was required. All samples were stored at +4°C and protected from light during shipment. On each occasion, the mean (n = 6) concentration was determined and compared to the nominal value and the coefficient of variation (CV %) was calculated. Acceptance criteria at each time-point: mean concentration = nominal value ± 20%, CV% < 10%.
The results demonstrated the satisfactory homogeneity of the two dosage forms since the differences from nominal concentration were within ± 20% and the % CV was <10%.

> Study chemical analysis - concentration:
The concentration of the test item in the dosage forms was determined in samples of each control and test item dosage form prepared for use in weeks 1, 6, 12, 14 (F0 generation), 17 (F0 and F1 generation), 22, 29 and 33 (F1 generation) of the study.
On each sampling day, duplicate samples were taken from each container. One sample from each duplicate was analyzed and the remaining sample was stored in case analysis was required. On each sampling day, each sample taken was stored at +4°C and protected from light until dispatch for analysis. Acceptance criterion: actual concentration: = nominal value ± 20%.
All dosage forms analyzed were within the ± 20% acceptance criterion.
Duration of treatment / exposure:
> In the males:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- until sacrifice (after weaning of the pups).

> In the females:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- during pregnancy,
- during lactation until day 21 post-partum (p.p.) inclusive,
- females with no delivery were treated until the day prior to sacrifice.
Frequency of treatment:
Once a day, 7 days a week
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation on day 22 post partum.
- Age at mating of the mated animals in the study: between 13 and 14 weeks old.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were selected on the basis of the results of a preliminary OECD 422 study using dose-levels of 450 and 1000 mg/kg/day (CIT/Study No. 34759 RSR, April 2010) and a 28-day repeated dose toxicity study (OCDE, 407, CIT Study N° 22314 TSR, January 2002). Neither dose elicited effects on the adult animals although the pups of the group treated at 1000 mg/kg/day had a minimally lower mean body weight gain from post natal days 1 to 5.
- Rationale for animal assignment: computerized stratification procedure, so that the average body weight of each group was similar.

Some deviations from the study plan were observed:
the temperature and relative humidity recorded in the animal room were sometimes outside the target ranges, some F1 animals had no free access to water during a short period of time because of defective water bottles, a total of 9 pups were retained in error from one F1 female (whereas each litter should have been culled to 8 pups on day 4 p.p.), two F0 females (group 1 and group 4) each gave birth to pups after delivery was thought to have been completed, one F0 male (group 2) had a left testis reduced in size which was not sampled at macroscopic examination in error, two F2 pups had scabs on the tail which were not sampled at macroscopic examination in error, statistical analyses performed on primordial follicle and corporea lutea count data were done on mean values per section per animal, instead of total number per animal.
These deviations were considered not to have compromised the validity or integrity of the study.
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
(F0 and F1 parental generation)
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
> Mortality or signs of morbidity: at least twice a day during the treatment period, and once a day during the acclimation period (F0 only).
> Clinical signs: once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week on all animals until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern).

BODY WEIGHT: Yes
- Time schedule for examinations:
> males: on the first day of treatment (day 1), then once a week until sacrifice.
> females: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1, 4, 7, 14 and 21 p.p.. Females prematurely sacrificed were weighed prior to sacrifice.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each male was recorded once a week, over a 7 day period, from the first day of treatment until sacrifice.
The quantity of food consumed by each female was recorded once a week, over a 7 day period, from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 p.c. intervals) and lactation (days 1-7, 7-14 and 14-21 p.p. intervals) until sacrifice.
During the pairing period, food consumption was not recorded for males or females.

WATER CONSUMPTION: No

OTHER:
- Sexual development (only F1 generation):
> All male animals were observed each day from day 32 of age (i.e. day 11 of F1 generation), until cleavage of the balanopreputial groove (preputial separation) was observed.
> All female animals were observed each day from day 28 of age (i.e. day 7 of F1 generation), until vaginal opening was observed.
The observation period was extended for any animals not positive by the expected end day. Body weight was recorded individually on the positive day.
- Neurobehavioral tests (only F1 generation)
> Auditory function (when the animals were 4 weeks old),
> Pupil constriction (when the animals were 4 weeks old),
> Spontaneous locomotor activity (when the animals were approximately 8 weeks old).
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage, each morning as follows:
- during the last 3 weeks of the pre-mating period,
- during the mating period, until the females are mated.
(F0 and F1 parental generation)
Sperm parameters (parental animals):
Parameters examined in F0/F1 male parental generations: testis weight, epididymis weight, sperm count in testes, epididymal sperm motility, epididymal sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: litter size, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical signs, weight gain, physical or behavioural abnormalities (physical development: pinna unfolding (on day 5 p.p.), hair growth (on day 5 p.p.), tooth eruption (on day 13 p.p.), auditory canal opening (on day 17 p.p.,), eye opening (on day 17 p.p.,); and reflex development: surface righting reflex (on day 5 p.p ), cliff avoidance (on day 11 p.p), air-righting reflex (on day 17 p.p).

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
On completion of the treatment period, all surviving males and females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
- F and F1 surviving males: after weaning of the F1 or F2 generation,
- P and F1 surviving females: at the weaning of the litters (on day 22 p.p.),
- P and F1 females which did not deliver: on day 25 p.c. after body weight recording (to check a possible un-noticed delivery),
- P and F1 females with litter dying entirely.

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all animals including on animals prematurely sacrificed of found dead. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of implantation sites were also recorded for females sacrificed on day 22 p.p..
The numbers of corpora lutea and implantation sites were recorded if possible for females sacrificed on day 25 p.c. due to no delivery. For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively. Furthermore, microscopic examinations were also performed on all animals sacrificed prematurely or found dead, and on all females sacrificed because of no delivery to investigate possible causes.
Testicular staging: a detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with PAS-hematoxylin (groups 1 and 4) in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
A detailed and careful microscopic examination was made of five sections of the right ovary of each F0 and F1 female, with enumeration of the total number of primordial follicles and corpora lutea.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed on day 4 p.p. or on day 22 p.p..
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
The following pups were carefully examined externally for gross external abnormalities:
- pups found dead,
- pups prematurely sacrificed,
- pups culled on day 4 p.p.,
- pups sacrificed on day 22 p.p..
In addition, for the following pups a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. Special attention was paid to the reproductive organs.
- pups showing external abnormalities or clinical signs,
- pups found dead or prematurely sacrificed,
- one randomly selected F1 and F2 pup/sex/litter sacrificed on day 22 p.p..

HISTOPATHOLOGY / ORGAN WEIGTHS
The organs specified in Tissue Procedure Table 2 were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
A microscopic examination was performed on:
- all the tissues listed in the Tissue Procedure Table 2 for one randomly selected F1 pup/sex/litter not selected at weaning and for one randomly selected F2 pup/sex/litter,
- all macroscopic lesions,
- all pups with external abnormalities.
Statistics:
Body weights, food consumption and reproductive data: mean values were compared by one-way variance analysis and Dunnett test. Percentage values were compared by Fisher exact probability test.
Organ weights: a sequence of statistical tests was used according to PathData software.
Auditory startle reflex: performed using the software SAS Enterprise Guide version 2.05.89.
Numbers of corpora lutea and primary follicles: normality and homogeneity of variances were tested using Kolmogorov Smirnov and Bartlett tests. If normality and homogeneity of variances were demonstrated (p-value>0.5 for both tests), a Student test was implemented. If normality and homogeneity of variances were not demonstrated (p-value<0.05 for one or both of the tests), a Mann-Withney-Wilcoxon test was conducted.
Reproductive indices:
The following parameters were evaluated:
- Pre-implantation loss,
- Post-implantation loss,
- Mating index,
- Fertility index,
- Gestation index,
- Number of corpora lutea,
- Number of implantations,
- Number of pups delivered
Offspring viability indices:
The following parameters were evaluated:
- Live birth index,
- Viability index on day 4 post-partum,
- Lactation index on day 21 post-partum,
- Mean litter size,
- Pup sex ratios

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No premature death occurred during the study in F0 male and female rats.
Some females were sacrified during lactation because of clinical signs or dead litter:
- One female at 1000 mg/kg was prematurely sacrificed on lactation day 9 because of clinical signs of piloerection, pallor, hypoactivity, abdominal breathing and half-closed eyes. At microscopic examination, the presence of bacterial colonies in a thrombus of the left cardiac atrium indicated that an infection caused the moribundity of this female.
- Two females at 450 mg/kg were prematurely sacrificed on lactation day 1 because of dead litter. Both females had dead fetuses in the uterine horns at necropsy and both females had necrosis and acute inflammation of the uterus and centrilobular necrosis in the liver at microscopic examination. Since similar lesions were observed in a control female (see below) a relationship to treatment with the test item is considered unlikely.
- One female in the control group was prematurely sacrificed on lactation day 1 because of dead litter. The female had marked centrilobular degeneration/necrosis in the liver, focal and marked necrosis in the uterus.
There were no test item treatment-related clinical signs.
Chromodacryorrhea, reflux at dosing, salivation, nodosities, hairloss, scabs and lesions were all observed at an equal or greater incidence in the controls animals, were considered to be related to the viscosity of the vehicle, corn oil, or are regularly observed in laboratory rats.
One female treated at 450 mg/kg had a mass on the 2nd right mammary gland at the end of the lactation period. This can be observed in lactating rats and given the isolated nature was considered not to be related to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
No premature death occurred during the study in F0 male and female rats.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
F0 generation (See table 3a):
Mean male body weight gains over the study were similar to those of the controls (between 0% and +5% differences).
The female group treated at 450 mg/kg/day gained statistically significantly more body weight over the 10 week pre-mating period than the controls (+12%). This was due mostly to statistically significantly higher mean body weight gains in weeks 5 and 7. In the absence of any effects at the higher or lower dose-level, this body weight gain difference was considered not to be related to treatment with the test item.
There were no effects of treatment with the test item on mean female body weight gains during the gestation period or during the lactation period at 450 or 1000 mg/kg/day. The mean body weight gain during the lactation period of females treated at 150 mg/kg/day was lower than that of the other groups. This was due to slightly lower body weight gains during the first part of lactation and then slightly greater body weight loss during the second part. Since the groups treated with higher dose-levels were not similarly affected, this lower body weight gain was considered not to be related to treatment with the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean male food consumption was statistically significantly higher at 1000 mg/kg/day in weeks 6 and 7 and then from weeks 9 to 16, when compared with the controls. This correlated with greater body weight gains.
The female groups treated at 450 or 1000 mg/kg/day had slightly greater food consumption than the controls at the beginning of the pre-mating period (+9%, p<0.05) and the female group treated at 1000 mg/kg/day again had slightly, but statistically significantly, greater food consumption than the controls at the end of the pre-mating period and during the middle of the gestation period.
There were no effects of treatment with the test item on food consumption during the lactation period.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Premature deaths:
- 1000 mg/kg/day: In one female sacrificed moribund on lactation day 9, the presence of severe cardiac lesions of septic origin explain the poor clinical status of this animal. This animal had a severe thrombosis of the left cardiac atrium, along with secondary hypertrophy of the right ventricle, and multifocal degeneration/necrosis of the myocardium, the latter correlated with white discoloration at necropsy. Numerous bacterial colonies were found within the thrombus and were most likely secondary to genital infection during delivery. Therefore the cardiac lesions were considered to be incidental and unrelated to the test item administration. In addition, there was a moderate centrilobular necrosis in the liver, correlated with marked lobular pattern at necropsy, and which may also have contributed to the poor clinical condition. Although the pathogenesis is unclear, the presence of a similar lesion in a control female makes the relationship to the test item administration very unlikely. There was a marked, diffuse cortical hypertrophy in the adrenals, correlated with macroscopic enlargement. In the spleen, there was a marked extramedullary hemopoiesis correlated with macroscopic enlargement, and which was considered to be secondary to the ongoing infection and inflammatory lesions. A slight lymphoid atrophy of the spleen and increased porphyrin pigment in the Harderian glands, correlated with black discoloration at necropsy, were considered to be secondary to the stress of the severe cardiac lesions. There were no abnormalities in the genital organs. Placentas were seen in the uterus. The vagina showed marked mucification, as it is awaited during pregnancy and days after delivery.
- 450 mg/kg/day: two females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns.
In one female, moderate acute neutrophilic inflammation and slight multifocal necrosis were found in the uterus (mucosa, lumen and placenta) and correlated with the presence of a dead fetus at necropsy. Slight acute centrilobular necrosis was seen in the liver and likely also contributed to the poor clinical status of this rat. There was a marked lymphoid atrophy of the thymus, which correlated with the gelatinous aspect, and was considered to be secondary to the inflammatory and necrotic lesions in the uterus and liver. In the spleen, there was a marked extramedullary hemopoiesis correlated with macroscopic enlargement, and which was considered to be secondary to the ongoing inflammatory lesions. The second female had similar lesions of necrosis and acute inflammation in the uterus, and of centrilobular necrosis in the liver. In both females, it is unclear whether the uterine findings were primary or secondary to the presence of dead fetuses. Both the uterine and hepatic lesions contributed to their poor clinical status. For both animals, although the pathogenesis of the hepatic necrosis is unclear, the presence of similar necrotic uterine and hepatic lesions in a control female makes the relationship to the test item administration very unlikely. The vagina of both females showed marked mucification, as it is awaited during pregnancy and within days after delivery.
Control: In one female sacrificed on lactation day 1 with a dead litter, marked centrilobular hepatic degeneration/necrosis correlated with white color of the liver. There was a focal, marked necrosis in the uterus in one horn and within the underlying mucosa and placenta, along with multifocal vascular necrosis. Both the hepatic and genital lesions were considered to have contributed to the poor clinical condition of this animal. In the spleen, there was a marked extramedullary hemopoiesis, correlated with macroscopic enlargement, and considered to be secondary to the ongoing inflammatory lesions. There was a diffuse marked lymphoid atrophy of the thymus, correlated with gelatinous aspect at necropsy, and which was secondary to the stress of the lesions. The vagina showed marked mucification, as it is awaited during pregnancy and within days after delivery. There were neutrophils within the vaginal epithelium and lumen, which is not considered to be abnormal after delivery.

Terminal sacrifice:
F0 generation:
> Qualitative evaluation of the genital organs in F0 parents:
There were no significant differences between control and high-dose groups in the incidences and severity of microscopic findings in the genital organs from F0 parents. In particular, at histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
In females which were not pregnant (one at 150 mg/kg, one at 450 mg/kg and three at 1000 mg/kg), there were no microscopic findings in the genital organs attributed to the test item.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
> Quantitative evaluation of the ovaries in F0 females:
There was a slight, statistically significant increase in the mean numbers of corpora lutea in high dose females (12.82 ± 3.047 compared to 10.74 ± 4.631 in control, p<0.01), which correlated with the increase in the mean ovary weights in this group. This variation was not toxicologically significant based on the direction of the change.
There was a slight, not statistically significant, decrease in the mean numbers of primordial follicles in high-dose F0 females. Since such variation was not found in high-dose F1 females, this variation was not considered to be toxicologically significant.
> Microscopic evaluation of the liver and kidneys in F0 parents:
The test item administration at 1000 mg/kg/day induced minimal centrilobular hypertrophy in the liver from 16/25 males, correlated with the increased liver weights. This finding was not adverse. There were no significant changes in the liver from high-dose females.
At this dose-level, the test item administration also induced a minimal increase of the incidence and severity of hyaline droplets in the proximal renal tubules from males, correlated with the increased kidney weights. There were no significant microscopic changes in the kidneys from high-dose females. This change, spontaneously seen in normal mature rats, was reported to be exacerbated by some chemicals but is known to be male rat-specific and therefore has no human relevance.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
It was considered that there were no effects of treatment with the test item on estrous cyclicity in F0 parental generation. (see table 4a)
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on sperm parameters in F0 parental generation. (See table 9a)
Reproductive performance:
no effects observed
Description (incidence and severity):
F0 generation (see table 5a and 6a):
Overall, there were no test item treatment-related effects on mean reproductive parameters and indices.
All males and females mated although the mean number of days taken to mate was greater in all test item-treated groups than for the controls. It is considered however, that a mean of up to 4 days of pairing before mating is within normal range since the rat estrous cycle usually lasts for 4 days and rats mate while in estrus.
There were no more than two non-pregnant females per group which is also considered to be within normal range.
One female treated at 1000 mg/kg/day was pregnant but did not deliver.
The mean duration of gestation was similar in all groups.

The mean number of implantation sites was similar in all groups as were the mean number of pups born and the post-implantation loss. The percentage of male pups was similar in all groups.
The number of dead pups was statistically significantly greater in the groups treated at 450 or 1000 mg/kg/day when compared with the controls. Even when taking into account the litters where all the pups died, pup mortality was still increased at the two highest dose-levels.
At 1000 mg/kg/day, the majority of the dead pups (23/36) were from two litters. One Female had no clinical signs during lactation yet 9/15 of the pups were dead or cannibalized on lactation day 1. the second female had 13 dead pups on lactation day 1 and only one live pup. The pup was still alive on lactation day 9 when the dam was prematurely sacrificed because of poor clinical condition (piloerection, pallor, hypoactivity, abdominal breathing and half-closed eyes). The dam had shown no clinical signs prior to lactation day 9 however microscopic examination revealed the presence of a bacterial infection which is considered to have caused the moribundity of the dam.
At 450 mg/kg/day, the majority of the dead pups were from three litters. The 1st female was pale and had piloerection on lactation day 1 and on the same day all the pups were found dead (7 pups) or cannibalized (5 pups). It is likely that the poor clinical condition of the dam caused lack of nursing or nesting behavior resulting in death of the pups. The 2nd female also had an entire dead litter on lactation day 1 but had no clinical signs. The 3rd female lost 10/14 pups on lactation day 1 (9 were found dead and 1 was cannibalized) but had no clinical signs. The remaining four pups survived until weaning. The 2nd and 3rd females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns, along with necrotic uterine and hepatic lesions which were most probably the main cause of their clinical signs and/or secondary lack of nursing. In view of their low incidence and presence of similar lesions in a control female, the relationship to the test item was considered to be unlikely.
One control female also had a dead litter on lactation day 1 (13 found dead pups and 1 cannibalized pup) and had piloerection and pallor. The clinical signs could indicate poor maternal condition after delivery. At microscopic examination, there were necrotic hepatic and uterine lesions which were considered to have contributed to its poor clinical condition.
This distribution of pup mortality (the majority of the dead pups being from a small number of litters) is more indicative of poor maternal care rather than a direct effect of the test item on pup mortality, which would probably cause more widespread pup death rather than being concentrated in some litters.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
(parental F0)
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effect observed in the reproductive performance and systemic toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
1000 mg/kg/day: One female was found dead on day 48. Clinical signs of pallor, hypoactivity, piloerection, chromorhinorrhea and half-closed eyes had been observed from day 46 or day 47. Death was related to marked suppurative pyelonephritis and cystitis which were considered to be incidental and unrelated to the test item administration.
Two females were sacrificed on gestation day 23 because of difficulties to deliver evidenced by clinical signs of pallor and piloerection: one female had a fetus in the vagina but had not delivered any pups, the other female had delivered eight pups (seven live and one dead) but parturition was not complete. At microscopic examination, there were necrotic uterine lesions along with dead fetuses. A relationship to the test item administration was considered to be unlikely in view of their low incidence and presence of similar uterine lesions in a control female.
- 450 mg/kg/day: One female was found dead on day 10 of dosing. No clinical signs had been observed before death. Death was related to gavage pneumonia and was therefore unrelated to the test item.
- 150 mg/kg/day: One male was prematurely sacrificed on day 126 of treatment because of clinical signs of piloerection, round back, bent head, hypoactivity, loud breathing, half-closed eyes, swollen neck region and chromorhinorrhea. At necropsy, this male had an esophageal pouch which correlated with marked acute inflammation microscopically. This finding explained the clinical signs observed and was secondary to a dosing error.
- Control: One control female was prematurely sacrificed on gestation day 24 because of difficulties to deliver. The female had delivered 13 pups (10 live and 3 dead) but was pale and it was considered that delivery was not complete. At microscopic examination, there was a marked suppurative inflammation of the uterus. Another female was prematurely sacrificed on lactation day 7 because of dead litter: there were no macroscopic or microscopic findings.
There were no test item treatment-related clinical signs. Signs of lesions, hairloss and nodosities were also observed in control animals and are often seen in laboratory rats. One male treated at 1000 mg/kg/day had a mass on the left forelimb which increased in size from 2 x 1 cm on day 113 to 4 x 4 cm on the day of sacrifice (day 130). At microscopic examination, this mass correlated with a subcutaneous sarcoma which was considered to be incidental. Such malignant tumors have been reported in the literature in rats of this age (Son and Gopinath, 2004).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
see clinical signs
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on mean male body weight or body weight gain during the 10-week premating treatment period. By the end of the study, the test item treated males had gained more weight than the controls and mean body weights were 3% to 4% higher.
Females treated at 150 mg/kg/day had greater mean body weight gains than the controls throughout the pre-mating and gestation periods, and had statistically significantly higher mean body weights throughout gestation and most of lactation (+6% to +9%, p<0.05, p<0.01 or p<0.001) although the overall mean body weight gain did not achieve statistical significance. The group treated at 1000 mg/kg/day had statistically significantly higher mean body weight gains during the first two weeks of gestation which contributed to a non-statistically significantly higher overall body weight gain.
Mean body weight gains over lactation were similar to that of the controls.
While treatment-related, all these changes were considered of not toxicological significance.
(See table 3b)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption of the males treated at 1000 mg/kg/day was statistically significantly higher than that of the controls from week 7 until the end of the study. The mean food consumption of the group treated at 450 mg/kg/day was also statistically significantly higher from week 12.
The female group treated at 1000 mg/kg/day showed a similar effect, having statistically significantly higher food consumption from week 9 until mid-gestation, when compared with the controls.
In all treated groups mean food consumption of the females from treated groups was slightly, but non-statistically significantly, higher than that of the controls during lactation.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The test item administration was associated with statistically significant increases in the mean kidney and liver weights in F1 male parents. However, these changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.
There were increases of the mean absolute (statistically significant) and relative weights of seminal vesicles in mid-dose and low-dose males compared with controls. In the absence of similar variations in high-dose males and since there were no macroscopic and microscopic correlates, this finding was considered to be incidental and unrelated to the test item administration.
The test item did not induce any significant change in all treated groups of F1 females as compared to controls.
The test item administration in F1 parents did not induce any changes in organ weights in their pups.
(see table 8)
Gross pathological findings:
no effects observed
Description (incidence and severity):
Premature deaths:
- 1000 mg/kg/day: one found dead female (day 48) had many white discolored foci in the kidneys, unilaterally dilated renal pelvis, dilated urinary bladder with red liquid content, and enlarged adrenals. In two females sacrificed because of difficulties to deliver (day 23), there were dead fetuses and black content within both uterine horns and red content in the vagina ; in one female, all 17 fetuses remaining in the uterine horns were found dead, whereas in the other female, four fetuses were found dead and one fetus was still alive.
- 450 mg/kg/day: In one female, found dead on day 10, the lungs were enlarged.
- 150 mg/kg/day: a male sacrificed moribund on day 126 had a large pouch (2.5 cm long) around the esophagus with white thick content.
- Control: In one control female sacrificed because of difficulties to deliver (day 24), there was still one fetus within the uterus and two placentas in the vagina. The spleen was enlarged. In another female sacrificed on lactation day 7 because of dead litter, there were no macroscopic findings.
Terminal sacrifice:
The few macroscopic findings noted at the end of the treatment period in F1 parents, as well as in their pups were of those commonly recorded in the Sprague-Dawley rat, and none were considered to be related to the test item administration. Among them was a large white mass of the forelimb of high-dose parent male S24963, which correlated microscopically with a subcutaneous sarcoma. Such malignant tumors have been reported in the literature in rats of this age (Son and Gopinath, 2004).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Premature deaths:
- 1000 mg/kg/day: In found dead female (day 48), there was a marked suppurative inflammation of the kidneys (pyelonephritis) correlated with the macroscopic white foci and pelvic dilation, urinary bladder (cystitis correlated with dilation and red content) and tissue adjacent to the vagina. Numerous bacterial colonies were seen in the kidneys. These suppurative lesions were the cause of death of this female. There was a moderate diffuse hypertrophy of the cortex correlated with enlargement, and which was considered to be secondary of the stress of the multiple suppurative lesions in this animal. Pyelonephritis and cystitis are common findings in female rats, and in this case were considered to be incidental and unrelated to the test item administration. In two females sacrificed because of difficulties to deliver (day 23), most fetuses were dead. In the uterus, there was slight acute focal necrosis on the endometrial surface at the level of one placenta in both females, along with slight multifocal hemorrhage in one female and minimal multifocal neutrophilic inflammation in the other female. It is unclear whether the uterine lesions were primary or secondary to the presence of dead fetuses. All these findings likely contributed to the clinical signs of the females. A relationship to the test item administration of the fetal deaths was considered to be unlikely in view of their low incidence and presence of similar uterine lesions in a control F0 female.
- 450 mg/kg/day: In the female found dead on day 10, there was a diffuse gavage pneumonia characterized by the presence of yellow material interpreted as compound within the alveoli. This pneumonia, correlated with the macroscopic enlargement, was considered to be the cause of death.
- 150 mg/kg/day: the esophageal pouch of the male sacrificed moribund on day 126 correlated with marked acute inflammation microscopically. This finding explained the clinical signs observed and was secondary to a dosing error.
Control: in the control female sacrificed because of difficulties to deliver (day 24), there was a marked acute neutrophilic inflammation of the uterus related to the presence of bacterial colonies, associated with multiple necrotic areas in the mucosa/placentas and focal thrombosis. It is difficult to determine whether the septic inflammation was the cause or the consequence of the difficulties to deliver. There was marked hemopoiesis in the spleen which correlated with splenic enlargement, and was considered to be compensatory to the ongoing inflammatory lesions. In the control female sacrificed on lactation day 7 because of dead litter, there were no significant microscopic findings in the genital organs.

Terminal sacrifice:
> Qualitative evaluation of the genital organs in F1 parents:
There were no significant differences between control and high-dose groups in the incidences and severity of microscopic findings in the genital organs from F1 parents. . In particular, at histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.
In a female at 150 mg/kg/day which was not pregnant and was sacrificed on day 99, there was evidence of a persistent estrus, characterized by multiple follicular cysts and absence of corpora lutea in the ovaries, tall columnar endometrial epithelium and squamous metaplasia in the uterus, and epithelial hyperplasia and cornification in the vagina (Westwood, 2008). This isolated condition, not observed in high-dose females, was considered to be incidental and unrelated to the test item administration.
In females which were not pregnant (one in control, three at 150 mg/kg, two at 450 mg/kg and two at 1000 mg/kg), there were no microscopic findings in the genital organs attributed to the test item.
All microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
> Quantitative evaluation of the ovaries in F1 females:
There were no significant differences in the mean number of primordial follicles between control and high-dose F1 females. In the right ovary of one high-dose female, there were no primordial follicles and there was a small number of corpora lutea (n=9), which correlated with a reduced size macroscopically. Since the left ovary of this female was not affected, this unilateral change was considered to be incidental and unrelated to the test item administration.
There was a slight, not statistically significant increase in the mean number of corpora lutea in high-dose F1 females compared with controls. This variation was not considered to be toxicologically significant based on the direction of the change.
> Microscopic evaluation of the liver and kidneys in F1 parents:
The test item administration at 1000 mg/kg/day induced minimal centrilobular hypertrophy in the liver from 10/25 males, correlated with the increased liver weights. This finding was not adverse. There were no significant changes in the liver from high-dose females.
There were no significant differences in the incidences of hyaline droplets in the kidneys from high-dose males compared with controls. There were no significant findings in the kidneys from high-dose females.

All microscopic findings noted in treated F0/F1 parent animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
It was considered that there were no effects of treatment with the test item on estrous cyclicity in F1 parental generation. (see table 4b)
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on sperm parameters in F1 parental generation. (See table 9b)
Reproductive performance:
no effects observed
Description (incidence and severity):
See tables 5b and 6b.
There were no treatment-related effect on mean reproductive parameters and indices.
Two males treated at 150 mg/kg/day did not mate but all other animals mated and the mean number of days of pairing before mating was considered not to have been affected by treatment with the test item. There were four non-pregnant females at 150 mg/kg/day compared with one in the control group. There were no microscopic findings in the genital organs of these females or the males which were attributed to the test item. One female from the control group and two females from the high-dose group were sacrificed mid-parturition because of poor clinical condition.
The mean duration of gestation was identical in all groups.

There were no statistically significant differences between control and treated groups regarding mean numbers of implantations, delivered pups and post-implantation loss.
Pup mortality was not increased in the test item-treated groups and there was no effect of treatment on the mean percentage of male pups.
Generally, the pups which were later cannibalized or found dead did not have clinical signs.

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effect observed in the reproductive performance and systemic toxicity

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were few clinical signs in surviving pups during lactation, scabs and small wounds are regularly observed, and only one surviving pup from the dam S25228, given 450 mg/kg/day, had dehydration and coldness on days 6 to 8 p.p..
Scabs and hematomas are regularly observed in young rats and incidences of coldness were observed in control pups as well as in those from the groups treated at 450 or 1000 mg/kg/day.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of dead pups was statistically significantly greater in the groups treated at 450 or 1000 mg/kg/day when compared with the controls. Even when taking into account the litters where all the pups died, pup mortality was still increased at the two highest dose-levels.
At 1000 mg/kg/day, the majority of the dead pups (23/36) were from two litters. One Female had no clinical signs during lactation yet 9/15 of the pups were dead or cannibalized on lactation day 1. The second female had 13 dead pups on lactation day 1 and only one live pup. The pup was still alive on lactation day 9 when the dam was prematurely sacrificed because of poor clinical condition (piloerection, pallor, hypoactivity, abdominal breathing and half-closed eyes). The dam had shown no clinical signs prior to lactation day 9 however microscopic examination revealed the presence of a bacterial infection which is considered to have caused the moribundity of the dam.
At 450 mg/kg/day, the majority of the dead pups were from three litters. The 1st female was pale and had piloerection on lactation day 1 and on the same day all the pups were found dead (7 pups) or cannibalized (5 pups). It is likely that the poor clinical condition of the dam caused lack of nursing or nesting behavior resulting in death of the pups. The 2nd female also had an entire dead litter on lactation day 1 but had no clinical signs. The 3rd female lost 10/14 pups on lactation day 1 (9 were found dead and 1 was cannibalized) but had no clinical signs. The remaining four pups survived until weaning. The 2nd and 3rd females, sacrificed moribund on lactation day 1, had dead fetuses in the uterine horns, along with necrotic uterine and hepatic lesions which were most probably the main cause of their clinical signs and/or secondary lack of nursing. In view of their low incidence and presence of similar lesions in a control female, the relationship to the test item was considered to be unlikely.
One control female also had a dead litter on lactation day 1 (13 found dead pups and 1 cannibalized pup) and had piloerection and pallor. The clinical signs could indicate poor maternal condition after delivery. At microscopic examination, there were necrotic hepatic and uterine lesions which were considered to have contributed to its poor clinical condition.
This distribution of pup mortality (the majority of the dead pups being from a small number of litters) is more indicative of poor maternal care rather than a direct effect of the test item on pup mortality, which would probably cause more widespread pup death rather than being concentrated in some litters.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on mean pup body weight or body weight gains at any dose-level.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
In F1 generation, there were no treatment-related effects on balanopreputial separation at any dose-level.
There were no test item treatment-related effects on vaginal opening at any dose-level. The later mean age of vaginal opening at 150 and 450 mg/kg/day was due to one female per group which was considered not to have achieved complete opening before mating.
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The test item administration in F0 parents did not induce any changes in organ weights in F1 pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
On external examination of F1 pups during lactation period, one female pup from one litter had generalized palor, scab and nodositie on abdomen on day 4 p.p. and, another female pup from another litter had a knicked tail on day 22 p.p. in the 1000 mg/kg/day group, one female pup had scab on head on day 4 p.p. in the 450 mg/kg/day group and one female pup had scab on abdomen on day 4 p.p. in the 150 mg/kg/day group. All these findings were isolated and a relationship to the treatment was considered unlikely.
Histopathological findings:
not examined
Description (incidence and severity):
As no macroscopic lesion was observed in F1 pups at external examination, no microscopic examination was performed.
Other effects:
no effects observed
Description (incidence and severity):
> Physical and reflex development during lactation (F1 pups):
All pups were positive for pinna unfolding and hair growth on post-natal day 5, tooth eruption on day 13 p.p. and auditory canal opening on day 17 p.p..
Not all pups were positive for eye opening on day 17 p.p., however the incidence was identical to the control group (one pup per group in groups 1 and 4).
The number of pups passing the surface righting and air righting tests was similar to or greater than in the control group at all dose-levels.
The number of pups failing the cliff avoidance test on day 11 p.p. was slightly higher at 450 and 1000 mg/kg/day than in the control group. In the absence of any effects on any of the other physical or reflex development tests, this difference was considered to be incidental.

> Auditory function (F1 pups) :
There was no treatment-related effect on auditory function.
All animals had a positive response to the auditory startle test. The latency and amplitude of the response was similar in all groups therefore it was concluded that no group showed impairment of hearing.

> Pupil constriction and locomotor activity (F1 pups)
There was no treatment-related effect on pupil constriction and locomotor activity.
All groups had similar numbers of horizontal and rearing movements during the 1-hour period as the controls.
All animals were positive for pupil constriction reflex at 4 weeks of age.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Remarks:
(offspring)
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effect observed on the development of pups

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item treatment-related clinical signs.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Pup mortality was not increased in the test item-treated groups;
Body weight and weight changes:
no effects observed
Description (incidence and severity):
the pups from the group treated at 150 mg/kg/day had a statistically significantly greater mean body weight gain between days 7 and 14 p.p. which resulted in higher mean body weights of both male and female pups at the end of lactation. There were no effects at 450 or 1000 mg/kg/day.
In the absence of any dose-related effect a relationship to the treatment was considered unlikely.
(see table 11)
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The test item administration in F1 parents did not induce any changes in organ weights in F2 pups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related findings at external examination of the pups.
Histopathological findings:
not examined
Description (incidence and severity):
As no macroscopic lesion was observed in F2 pups at external examination, no microscopic examination was performed.
Other effects:
no effects observed
Description (incidence and severity):
> Physical and reflex development (F2):
There were no treatment-related effects.
All pups were positive for pinna unfolding and hair growth on day 5 p.p. and tooth eruption on day 13 p.p..
Not all pups were positive for eye opening or auditory canal opening on day 17 p.p., however the incidence was nearly identical to the control group.
The number of pups passing the surface righting, cliff avoidance and air righting tests was similar to or greater than in the control group at all dose-levels.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOEL
Generation:
F2
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effect observed on the development of pups

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 3a: Body weight and body weight change (F0 generation)

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Premating

 

 

 

 

 

 

 

 

Days 1 - 71

+317

+331

+312

+331

+139

+144

+156**

+147

Days 1 - 127

+396

+416

+395

+416

/

/

/

/

Gestationa

 

 

 

 

 

 

 

 

GD 0 - 20

/

/

/

/

+144

+151

+143

+150

Lactation

 

 

 

 

 

 

 

 

LD 1 - 21

/

/

/

/

+16

+6

+18

+18

GD: gestation day, LD: lactation day, /: not applicable, a: only includes pregnant females with live fetuses.

Statistically significant **: p<0.01.

Table 3b: Body weight and body weight change (F1 generation)

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Premating

 

 

 

 

 

 

 

 

. Days 1 - 71

+462

+473

+477

+475

+222

+232

+226

+222

. Days 1 - 127

+566

+585

+594

+581

/

/

/

/

Gestation

 

 

 

 

 

 

 

 

. GD 0 - 20

/

/

/

/

+148

+161

+156

+166

Lactation

 

 

 

 

 

 

 

 

. LD 1 - 21

/

/

/

/

+19

+13

+17

+23

GD: gestation day, LD: lactation day, /: not applicable.

Table 4a : Estrous cycles (F0 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of cycles per female

4.7

4.8

4.8

4.7

Mean cycle length

(days)

4.0

4.0

4.1

4.1

Number of abnormally cycling females

1

2

2

2

An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.

Table 4b : Estrous cycles (F1 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of cycles per female

4.6

4.5

4.8

4.6

Mean cycle length

(days)

4.2

4.4

4.1

4.2

Number of abnormally cycling females

1

2

1

2

An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.

Table 5a: Mating, fertility and parturition (F0 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Number of males + females paired

25 +25

25 + 25

25 + 25

25 + 25

Number of pairs mated

25

25

25

25

Mating index

100%

100%

100%

100%

Mean number of days taken to mate

2.0

2.9

3.4

3.0

Number of pregnant females

25

24

24

23

Fertility index

100%

96%

96%

92%

Number of females delivering live pups

25

24

24

22

Gestation index

100%

100%

100%

96%

Mean duration of gestation (days)

22.0

21.8

21.8

21.9

Table 5b: Mating, fertility and parturition (F1 generation)

Dose-level (mg/kg/day)

0

150

450

1000

Number of males paired

25

25

24

24

Number of males mated

25

23

24

24

Male fertility index

100%

92%

100%

100%

Number of females paired

25

25

24

24

Number of females mated

25

25

24

24

Mating index

100%

100%

100%

100%

Mean number of days taken to mate

3.0

4.3

2.7

2.8

Number of pregnant females

24

21

22

22

Fertility index

96%

84%

92%

92%

Number of females delivering live pups

23a

21

22

20a

Gestation index

96%

100%

100%

91%

Mean duration of gestation (days)

21.9

21.9

21.9

21.9

a: females sacrificed mid-parturition because of poor clinical condition.

Table 6a: Mating, fertility and parturition (F0 generation) Cont’

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of implantation sites

13.8

14.8

14.4

14.1

Mean number of pups born

11.8

12.7

12.7

12.3

Post-implantation loss (calculated manually)

14.2

14.2

11.7

12.4

Number of entire litters dead (no. of pups)

1 (14)

0

2 (24)

1 (14)

Number of dead pups: days 1 - 4

22

6**

38*

34*a

Number of dead pups: days 5 - 21

0

1

0

1

Number of litters with dead pups

6

4

6

11

% of male pups at birth

51.7%

46.7%

48.8%

50.0%

Statistically significant *: p<0.05, **: p<0.01.  a: does not include one pup which was cannibalized and could not be sexed.

Table 6b: Mating, fertility and parturition (F1 generation) Cont’

Dose-level (mg/kg/day)

0

150

450

1000

Mean number of implantation sites

12.1

14.4

13.1

14.4

Mean number of pups born

11.1

12.3

12.0

12.1

Post-implantation loss (calculated manually)

10.1

14.0

8.2

16.1

Number of entire litters dead

1

0

0

0

Number of dead pups: days 1 - 4

19

12

12

14

Number of dead pups: days 5 - 21

1

4

1

0

Number of litters with dead pups

11

9

5

8

% of male pups at birth

46.6

50.8

39.5

51.4

Table 7:Relevant changes in mean absolute and relative organ weights in treated F0 parents (% changes from controls)

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

150

450

1000

150

450

1000

Exam. animals / Num. of animals

25/25

25/25

25/25

25/25

23/25

24/25

Body weight

+3

-1

+3

0

+2

+2

- Kidney, left

 

 

 

 

 

 

  . absolute

+6*

+7*

+12**

+2

+6*

+3

  . relative

+3

+7*

+9**

+2

+3

+1

- Kidney, right

 

 

 

 

 

 

  . absolute

+3

+6*

+10**

+5

+5

+4

  . relative

0

+7**

+7**

+4*

+3

+2

- Liver

 

 

 

 

 

 

  . absolute

+10**

+9**

+17**

+2

+4

+1

  . relative

+7**

+10**

+14**

+2

+1

-1

- Ovary, left

 

 

 

 

 

 

  . absolute

 

 

 

+16*

+14

+17*

  . relative

 

 

 

+16

+11

+15*

- Ovary, right

 

 

 

 

 

 

  . absolute

 

 

 

+9

+12

+18**

  . relative

 

 

 

+9

+10

+16*

Statistically significant from controls: *: p<0.05, **: p<0.01.

Statistical significance determined for organ weights values and not percent changes.

Table 8:Relevant changes in mean absolute and relative organ weights in treated F1 parents (% changes from controls)

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

150

450

1000

150

450

1000

Exam. animals / Num. of animals

24/25

25/25

25/25

25/25

24/25

22/25

Body weight

+4

+5

+3

+4

+1

+2

- Kidney, left

 

 

 

 

 

 

  . absolute

+8*

+10**

+9**

+3

-2

+4

  . relative

+4

+5

+7*

-1

-3

+2

- Kidney, right

 

 

 

 

 

 

  . absolute

+7

+10**

+6*

+3

0

+5

  . relative

+3

+5

+4

-1

-2

+2

- Liver

 

 

 

 

 

 

  . absolute

+8*

+9*

+13**

+1

+2

0

  . relative

+4

+4

+10**

-3

+1

-2

Statistically significant from controls: *: p<0.05, **: p<0.01.

Statistical significance determined for organ weights values and not percent changes.

Table 9a : Sperm analysis (F0 generation)

Dose-level (mg/kg/day)

0

1000

Mean number of epididymal sperm (106/cauda)

171

173

% of motile sperm

96

93

% of morphologically normal sperm

96

96

Mean number of sperm heads (106/g testis)

125

125

 

Table 9b : Sperm analysis (F1 generation)

Dose-level (mg/kg/day)

0

1000

Mean number of epididymal sperm (106/cauda)

177

191

% of motile sperm

90

99

% of morphologically normal sperm

83

92

Mean number of sperm heads (106/g testis)

122

121

Table 10: Body weight and body weight change in F1 generation during lactation (pups) 

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Body weight (g)

 

 

 

 

 

 

 

. Day 1

7.8

7.7

7.5

7.6

7.3

7.3

7.1

7.1

. Day 21

57.1

60.6

58.6

57.8

56.1

58.5

56.7

56.3

Body weight gain (g)

 

 

 

 

 

 

 

. Days 1 - 21

+49.3

+52.9

+51.1

+50.2

+48.8

+51.2

+49.6

+49.2

Applicant's summary and conclusion

Conclusions:
Since no effects were seen in this study in the 2 generations, the No Observed Adverse Effect Level for the F0 and F1 parents for systemic toxicity and reproductive performances were considered to be 1000 mg/kg bw/day. Further, the test substance did not cause delayed or impaired development in the F1 or F2 generations following treatment of the parents. Therefore, the No Observed Effect Level (NOEL) for peri- and post-natal development of F1 and F2 generations was considered to be 1000 mg/kg/day.
Executive summary:

In a 2-generation reproduction study scored as validity 1 according to Klimisch criteria (CIT report No. 34760 RSR, OECD guideline 416, GLP), four groups of 25 male and 25 female Sprague-Dawley rats received the test item Cerium and iron oxide isostearate, daily for 10 weeks prior to mating, during mating, gestation and lactation until weaning of the pups. The test item was administered as a suspension in corn oil, by oral gavage, at dose-levels of 0 (control), 150, 450 or 1000 mg/kg/day. A constant dosage volume of 4 mL/kg/day was used.

The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once a week. Body weight and food consumption were recorded weekly. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days. The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development were assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening and, surface righting, cliff avoidance and air righting reflexes).

After weaning of the pups, the males and females of the F0 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs of the control and high-dose group animals and on all macroscopic lesions.

 

F1 generation

After weaning (day 22post-partum) of the progeny of the F0 generation, one or two males and one or two females per litter were selected to constitute the F1 generation of four groups of 25 male and 25 female rats. Three groups received the test item, Cerium and iron oxide isostearate, and the fourth group (control) received the vehicle only (corn oil) daily for 10 weeks prior to mating and, during mating, gestation and lactation until weaning of the pups. The test item was administered as a suspension in corn oil, by oral gavage, at dose-levels of 150, 450 or 1000 mg/kg/day under a constant dosage‑volume of 4 mL/kg/day.

 

The animals were checked at least twice daily for mortality or morbidity and at least once daily for clinical signs. A detailed clinical examination was performed once a week. Body weight and food consumption were recorded weekly. Each animal was assessed for sexual maturity (balanopreputial separation or vaginal opening) and the day of age and body weight was recorded on the day each animal was positive. At 4 weeks of age, the animals were assessed for auditory function (acoustic startle response) and pupil constriction, andweeks of age, spontaneous locomotor activity was measured using an automated infra-red sensor equipment. The estrous cycles were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 16 days.

The females were allowed to litter and rear their progeny until weaning. The pups were regularly weighed throughout the lactation period and observed daily for clinical signs. Physical and reflex development was assessed (pinna unfolding, hair growth, tooth eruption, auditory canal opening, eye opening and, surface righting, cliff avoidance and air righting reflexes).

At weaning of the pups, the males and females of the F1 generation were sacrificed. Sperm analysis was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed, including counting the number of implantation sites in females, and designated organs were weighed. A microscopic examination was performed on the reproductive organs of the control and high-dose groups and on all macroscopic lesions.

 

In addition, pups of both the F0 and F1 animals were submitted for a macroscopic examination. One randomly selected pup/sex/litter for F1 and F2 litters, all pups found dead or prematurely sacrificed and any pups showing external abnormalities or clinical signs were weighed and then submitted for a macroscopic examination of the principal thoracic and abdominal organs with special attention paid to the reproductive organs.

 

Results

 

F0 generation

There were no found dead animals. A total of four females (one at 1000 mg/kg/day, two at 450 mg/kg/day and one in controls) were prematurely sacrificed during lactation, and microscopic examination findings excluded a relationship to the test item.

No test item treatment-related clinical signs were observed and there were no treatment-related effects on body weight or body weight gain. There was a statistically significant increase in mean food consumption in males and females treated at 1000 mg/kg/day during the pre-mating period and mid-gestation and in females treated at 450 mg/kg/day at the beginning of the pre-mating period.

There were no effects on estrous cyclicity, mating or fertility parameters at any dose-level, however pup mortality was statistically significantly higher at 450 and 1000 mg/kg/day. One female at 1000 mg/kg/day and one female at 450 mg/kg/day had clinical signs which could indicate poor maternal nesting or nursing behavior. Few of the dead or cannibalized pups from the other litters had clinical signs. Since the majority of the found dead pups were concentrated in a few litters, it is considered unlikely that their deaths were related directly to test item treatment and that it is more probably related to poor maternal nesting/nursing behavior. The presence of one dead litter in the control group suggests that the dead litters were incidental to treatment with the test item.

No effects on spermatogenesis were detected after analysis of epididymidal sperm motility and morphology and count and, after analyis of testicular sperm count. At histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle. At all dose-levels, there were a few organ weight changes which were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent for the sexes.

There were no test item‑related microscopic findings in the genital organs of F0 parents.

There was a statistically significant increase in the mean ovary weight in F0 females treated at 1000 mg/kg/day, correlating with an increase in the mean number ofcorpora lutea. This finding was not considered to be toxicologically significant based on the direction of the change. In this group, mean number of primordial follicles was slightly decreased. Since such variation was not found F1 females, this variation was not considered to be of toxicological significance.

 

F1 generation

The F1 generation showed no effects of treatment while pups; there were no test item treatment‑related clinical signs, no effects on body weight and no differences in physical or reflex development when compared with the controls.There were no treatment-related findings at external examination of the F1 pups.

 

There were no test item treatment-related mortalities.

As for the F0 generation, the males treated at 450 or 1000 mg/kg/day and the females treated at 1000 mg/kg/day had statistically significantly increased food consumption which correlated with a tendency towards a non statistically significant increase in body weight gains. The females treated at 150 or 450 mg/kg/day also had slightly increased food consumption during lactation.

There were no effects on estrous cyclicity or mating.

Pup mortality was not higher in test item-treated groups than in the controls.

No effects on spermatogenesis were detected after analysis of epididymidal sperm motility, morphology or count or testicular sperm count. At histopathological examination of the testes, there were no qualitative changes in tubule development through the different stages of the spermatogenic cycle.

The test item administration at all dose-levels induced statistically significant dose-related increases in mean kidney and liver weights in F1 males. On microscopic examiniation, the increase in mean liver weight was correlated with minimal centrilobular hypertrophy in males at 1000 mg/kg/day. There were no microscopic correlates in the kidneys. There were no effects on organ weights and no relevant microscopic findings in the liver and kidneys in F1 females.

There were no test item-related macroscopic findings in F1 parents. There were no test item‑related microscopic findings in the genital organs from F1 parents.

There were increases in the mean number ofcorpora luteain high-dose F1 females. This finding was not considered to be toxicologically significant based on the direction of the change. There were no significant differences in the mean number of primordial follicles between control and high‑dose F1 females.

F2 generation

There were no test item treatment-related clinical signs and no effects on physical or reflex development.

The pups from the groups treated at 150 mg/kg/day had statistically significantly higher mean body weight gains mid-lactation but there were no effects at 450 or 1000 mg/kg/day. Therefore a relationship to the treatment with the test-item was considered unlikely.

There were no test item-related changes in organ weights or macroscopic findings in the pups.

The No Observed Adverse Effect Level for reproductive performance and systemic toxicity of F0 and F1 generations was therefore considered to be 1000 mg/kg/day.

Cerium and iron oxide isostearate did not cause delayed or impaired development in the F1 or F2 generations following treatment of the parents.

Therefore, the No Observed Effect Level (NOEL) for peri- and post-natal development of F1 and F2 generations was considered to be 1000 mg/kg/day.

 

No classification for reproductive toxicity is warranted based on the absence of relevant effects in this study, according to the criteria of Annex VI Directive 67/548/EEC or UN/EU GHS.

 

This study is classified as acceptable. It satisfies the OECD 416 guideline requirements onTwo-generation reproduction toxicity testing.