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Key value for chemical safety assessment

Effects on fertility

Description of key information

2-generation study by gavage (OECD TG 416, GLP):
NOAEL (parental F0/F1) = 1000 mg/kg bw/day (no significant effect observed in the reproductive performance and systemic toxicity)
NOEL (offspring F1/F2) = 1000 mg/kg bw/day (no significant effect observed on the development of pups)

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose for cross-reference:
reference to same study
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
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 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.
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
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
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: 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.
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
Key result
Critical effects observed:
no
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: 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.
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
Key result
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Key result
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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

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.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 23 May 2008 to 15 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
the study was performed similarly to OECD guideline and following the principles of Good Labaratory Practice (but not audited by Quality assurance unit). This study was used as a preliminary test for a 2-generation study.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
only 2 dose levels, no microscopic examination was performed, used as a preliminary study to a multigeneration study (CIT study n° 34760 RSR) .
GLP compliance:
no
Remarks:
but follows the principles of Good Laboratory Practice.
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: approximately 10 weeks
- Weight at study initiation: mean body weight of 427 g (range: 413 g to 444 g) for the males and 266 g (range: 251 g to 292 g) for the females.
- Fasting period before study: no
- Housing: 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
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: from 23 May 2008 (arrival of the animals) to 27 July 2008 (necropsy of the last female)
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 acceptable in order to achieve the concentrations of 90 and 200 mg/mL. The resulting suspension was left under magnetic stirring until treatment of the animals.

VEHICLE
- Justification for use and choice of vehicle: Vehicle used in previous repeated (28-day) dose toxicity study (22314 TSR) in which stability, homogeneity and concentration of the test item in the vehicle were found satisfactory.
- Concentration in vehicle: 90 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
- Lot/batch no.: 126K0117 and 117K0127
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days.
- 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: individually
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No chemical analysis was performed.
Duration of treatment / exposure:
- In the males: 15 days before mating, during the mating period (up to 3 weeks), until sacrifice (i.e. at least 5 weeks in total).
- in the females: 15 days before mating, during the mating period (up to 3 weeks), during pregnancy, during lactation until day 4 post-partum inclusive.
Frequency of treatment:
Once a day, at approximately the same time each day, 7 days a week.
Dose / conc.:
0 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:
10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were selected on the basis of a 4-week toxicity study in the rat (CIT/Study No. 22314 TSR). In this study, dose-levels of 150, 450 and 1000 mg/kg/day resulted in no deaths or clinical signs, no relevant effects on body weight or food consumption, no toxicologically significant differences in hematology or blood biochemistry parameters and no effects were observed at microscopic examination. The NOEL was considered to be 1000 mg/kg/day.
- Rationale for animal assignment: according to a computerized stratification procedure, so that the average body weight of each group was similar.
Positive control:
not required.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day for mortality and morbidity including week-end and public holidays. Once a day for clinical signs.
- Cage side observations include routine examinations.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
> for males: the first day of treatment (day 1), then once a week until sacrifice;
> for 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 and days 1 and 5 post-partum.

FOOD CONSUMPTION:
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 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice.
During the pairing period, food consumption was not recorded for males or females.

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage, each morning during the mating period, until the females had mated.
Sperm parameters (parental animals):
Not examined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter size and number and sex of pups, stillbirths, live births, dead and cannibalized pups, postnatal mortality, presence of gross anomalies, clinical signs (daily), body weight (day 1 and day 5 post-partum, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Pups found dead or prematurely sacrificed were carefully examined for gross external abnormalities. Pups sacrificed on day 5 post-partum were discarded with no post-mortem examination. There was no preservation of tissues.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals , after the end of the mating period (after at least 5 weeks of treatment).
- Maternal animals: All surviving animals, on day 5 post-partum, or on day 25 post-coitum for females which had not delivered.

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all animals. 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 corpora lutea and implantation scars were also recorded for females sacrificed on day 5 post-partum.
The numbers of corpora lutea were recorded for females sacrificed on day 25 post-coitum due to no delivery. For these apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY / ORGAN WEIGHTS
All macroscopic lesions were preserved in 10% buffered formalin (except for the testes and epididymides which were preserved in Davidson’s fixative).
No microscopic examination was performed.
Postmortem examinations (offspring):
- Pups were sacrificed on day 5 post-partum.
- Pups found dead or prematurely sacrificed were carefully examined for gross external abnormalities. Pups sacrificed on day 5 post-partum were discarded with no post-mortem examination.
- There was no preservation of tissues.
Statistics:
Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by the Fisher exact probability test.
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,
- Mean litter size,
- Pup sex ratios
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A total of three animals treated at 1000 mg/kg/day showed relevant clinical signs: one male showed ptyalism from day 12 to day 26 of dosing, one female showed reflux at dosing on day 12 post-coitum only and another female displayed chromodacryorrhea on day 21 post-coitum and day 0 post-partum.
One male treated at 450 mg/kg/day showed reflux at dosing on day 9 of dosing.
It was considered that none of these represented signs of toxicity of the test item.
In addition, hairloss on the forelimbs was observed in one control female, and one male and one female treated at 1000 mg/kg/day. These signs are commonly observed in laboratory rats of this strain and are considered not to be related to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on males or females during the pre-mating phase.
During gestation, females treated at 1000 mg/kg/day gained more weight than the controls but the females treated at 1000 mg/kg/day had a mean of one pup more than the control females which explain the higher body weight gain.
Both female groups treated with Isostéarate d’oxyde de cérium et de fer gained more body weight than the controls during lactation, however four control females lost a small amount of weight during this period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on mean male or female food consumption.
The female group treated at 1000 mg/kg/day had slightly higher food consumption than the controls during lactation which corresponded with the higher body weight gain.
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:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
1 control female and 2 females at 450 mg/kg/day stayed for 1 week or more in diestrus and/or metestrus before mating; all females were pregnant. Other than these females, all estrous cycles were considered to be normal.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on mating; all females at all dose-levels mated and only one control male did not mate. The mean number of days of pairing before mating was similar to or lower than the control group.
There were two mated but non-pregnant females in the group treated 1000 mg/kg/day. Neither showed any macroscopic abnormality at post-mortem examination.
There were no effects on the mean numbers of corpora lutea, implantations or pups at any dose-level, nor on the duration of gestation or the extent of pre-implantation loss.
The mean post-implantation loss was slightly higher in the groups treated with the test item when compared with the controls, however the differences were not toxicologically significant. It was therefore considered that this did not represent an effect of treatment with the test item.
Sex ratio: The percentage of male pups was similar between all groups.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No relevant effects up to highest dose tested
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed at 1000 mg/kg/day. At 450 mg/kg/day, one pup was cold to the touch, showed generalized hematoma and was found dead on post-natal day 4. Another pup from the same litter appeared to have milk in the urogenital region. It was considered that there were no treatment-related clinical signs.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Pup survival was higher in the groups treated with the test substance than in the control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight on post-natal day 1 was similar between all groups, including the controls, but on post-natal day 5 the pups from the group treated at 1000 mg/kg/day had a lower, non-statistically significant, mean body weight than the controls (-7% in the males and -6% in the females). The mean body weight gain from post-natal days 1 to 5 was also lower at 1000 mg/kg/day (-15% in the males and -12% in the females) when compared with the controls. This may have some relationship to the fact that there was a mean of one pup more per female in this group because the individual values at 1000 mg/kg/day were within the control range, but a relationship to treatment with the test item cannot be excluded.
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
None of the pups found dead showed gross external abnormalities.
Histopathological findings:
not examined
Dose descriptor:
NOEL
Generation:
F1
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Reproductive effects observed:
no

Summary table of mating and fertility data

Dose level (mg/kg/d)

0

450

1000

Number of animals paired (M + F)

10 + 10

10 + 10

10 + 10

Number of males mated

9

10

10

Number of females mated

10

10

10

Mean number of days taken to mate

5.2

5.6

2.3

Number of pregnant females

10

10

8

 

Summary table of delivery data

 

Dose level (mg/kg/day)

0

450

1000

Number of pregnant females surviving delivery

Mean duration of gestation (days)

Mean number of corpora lutea

Mean number of implantations

Mean pre-implantation loss (%)

Mean number of pups delivered

Mean post-implantation loss (%)

10

22.0

16.2

13.7

16.1

12.3

10.7

10

21.9

15.0

14.3

5.6

12.4

13.5

8

22.0

16.6

15.9

4.2

13.5

14.4

 

Summary of pup body weight

 

Sex

Dose-level (mg/kg/day)

 

0

Male

450

 

1000

 

0

Female

450

 

1000

Body weight (g)

PND 1

PND 5

 

8.0

13.5

 

7.8

12.9

 

7.8

12.5

 

7.5

12.7

 

7.5

12.8

 

7.4

12.0

Body weight gain (g)

PND 1-5

 

+5.4

 

+5.2

 

+4.6

 

+5.1

 

+5.3

 

+4.5

PND = post-natal day

Conclusions:
Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.
Executive summary:

In a Reproduction/developmental toxicity screening test, the potential general and reproductive or developmental toxicity of Cerium and iron oxide isostearate were tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0, 450 or 1000 mg/kg/day in corn oil.

 

No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.

Pups showed no effects of treatment on survival.

Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg/day (-15% in the males and -12% in the females, not statistically significant). This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded. It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio. There were no treatment-related macroscopic abnormalities.

 

Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.

As this study was designed to choose the dose-levels which should be used in a 2-genarations study, it is however considered that 1000 mg/kg/day would be a suitable high dose-level for themultigeneration study.

 

This study is classified as acceptable. It is similar to OECD guideline on reproduction/developmental toxicity screening and is acceptable as a preliminary study to a multigeneration study.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from cerium and iron oxide isosterate, a substance that presents structural similarities with iron oxide isostearate, was used to cover this endpoint. See the Read-across justification document (Justification for analogue approach) attached in IUCLID Section 13.2 for the justification of the read-across.
See also the original letters from the French Competent Authorities requiring the read across to be done with Cerium and iron oxide isostearate substances, attached in Section 13.2 as well (French CA testing program July 2005, and French CA testing program Sep 2007).
Reason / purpose for cross-reference:
read-across source
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
Key result
Critical effects observed:
no
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
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOEL
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
Key result
Critical effects observed:
no
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
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Since no effects were seen in this study in the 2 generations, the No Observed Adverse Effect Level for cerium and iron oxide isosterate, an analogue of iron oxide isostearate, 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. Based on the stuctural similarities between target and sources substances, the same conclusions are assumed for iron oxide isostearate.
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, a structural analogue of 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 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, and weeks 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 for cerium and ison oxide isostearate was therefore considered to be 1000 mg/kg/day. Further, 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. Thus, no classification for reproductive toxicity is warranted based on the absence of relevant effect in this study, according to the criteria of UN/EU GHS

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, same results are assumed for iron oxide isostearate.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from cerium and iron oxide isosterate, a substance that presents structural similarities with iron oxide isostearate, was used to cover this endpoint. See the Read-across justification document (Justification for analogue approach) attached in IUCLID Section 13.2 for the justification of the read-across.
See also the original letters from the French Competent Authorities requiring the read across to be done with Cerium and iron oxide isostearate substances, attached in Section 13.2 as well (French CA testing program July 2005, and French CA testing program Sep 2007).
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOEL
Remarks:
general toxicity and reprotoxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No relevant effects up to highest dose tested
Critical effects observed:
no
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
In this reproduction/developmental toxicity screening test (OECD TG 421) performed as a dose-range finding study for a 2-generations study with cerium and iron oxide isostearate, an analogue of iron oxide isostearate, the NOEL for the general toxicity and reproductive performance of the parents was found to be = 1000 mg/kg bw/d due to absence of toxic effect observed in the measured parameters. The NOEL for the developmental toxicity was set at 450 mg/kg bw/d due to a slight effect on the body weight gain in pups.
Based on the stuctural similarities between target and sources substances, the same NOEL values are assumed for iron oxide isostearate.
Executive summary:

In a Reproduction/developmental toxicity screening test, the potential general and reproductive or developmental toxicity of Cerium and iron oxide isostearate, a structural analogue of iron oxide isostearate, was tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0, 450 or 1000 mg/kg/day in corn oil.

 

No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.

Pups showed no effects of treatment on survival.

Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg/day (-15% in the males and -12% in the females, not statistically significant). This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded. It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio.There were no treatment-related macroscopic abnormalities.

 

Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, same results are assumed for iron oxide isostearate.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
Studies performed with OECD guidelines, meeting REACH and CLP requirements
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The potential for toxicity to reproduction of the iron oxide isostearate was assessed by analogy with cerium and iron oxide isostearate (Active matter DPX10, CAS 753480-32-9) showing similar chemical structure and similar properties in accordance with the testing program established by the Competent Authorities in July 2005 and September 2007: see attached letters in section 13.2 and the Read-across justification document attached in this section as well.

The potential of cerium and iron oxide isostearate to induce effects on reproduction was assessed in a 2-generation reproduction study performed according to OECD guideline No 416 and in accordance with GLP (CIT report No.RSR). This study was scored as validity 1 according to Klimisch criteria and thus was considered as the Key study.

In this study, four groups of 25 male and 25 female Sprague-Dawley rats constituting the F0 parents received the test item Cerium and iron oxide isostearate or the vehicle only, 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, vehicle only), 150, 450 or 1000 mg/kg/day, under a constant dosage volume of 4 mL/kg/day.

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, 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. Another group of 25 males and 25 females received the vehicle alone under the same experimental conditions and acted as a control group.

The F0 and F1 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 of both F0 and F1 females were monitored during the last 3 weeks before mating and during the mating period, which lasted up to 13 days for F0 females and up to 16 days for F1 females. The females of both generations were allowed to litter and rear their progeny until weaning. The F1 and F2 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).

Furthermore, each animal of the F1 generation 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, and at 9 weeks of age, spontaneous locomotor activity was measured using automated infra-red sensor equipment.

After weaning of the pups, the males and females of both the F0 and F1 generation were sacrificed. Sperm analysis was performed on the first ten F0 and F1 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 of both generations, 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.

 

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.

  

In the 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.

There were no treatment-related effects on body weight or body weight gain and no test item treatment-related clinical signs were observed. There was a slight 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 of treatment with the test item on food consumption during the lactation 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 in F1 females, this variation was not considered to be of toxicological significance.

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 controlsand no treatment-related findings were observed at external examination of 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 was associated with increases in mean kidney and liver weights in F1 males. These changes were not observed in the females. 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 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 increase 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.

 

In the F2 generation pups, 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 at 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.

 

From the results observed in this study, it can be concluded that the No Observed Adverse Effect Level for reproductive performance and systemic toxicity of F0 and F1 generations was considered to be 1000 mg/kg/day.

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

Therefore, similar results and values for the no observed effect levels were assumed for iron oxide isostearate.

Further, in a Reproduction/developmental toxicity screening test, that was performed similarly to OECD GD 421 and following the principles of GLP (but not audited by Quality Assurance unit since this study was used as a preliminary test for a 2 -generatin study), the potential general and reproductive or developmental toxicity of Cerium and iron oxide isostearate,a structural analogue of iron oxide isostearate, was tested following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0, 450 or 1000 mg/kg/day in corn oil.

 

No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.

Pups showed no effects of treatment on survival.

Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg/day (-15% in the males and -12% in the females, not statistically significant). This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded. It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio.There were no treatment-related macroscopic abnormalities.

 

Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, same results are assumed for iron oxide isostearate.

Effects on developmental toxicity

Description of key information

Using a weight of evidence approach:

- 2-generation study by gavage (OECD 416, GLP):

NOEL (offspring F1/F2) = 1000 mg/kg bw/day (no significant effect observed on the development of pups)

- Teratogenicity studies from water soluble and insoluble iron compounds

NOEL (teratogenic effects) > 1000 mg/kg bw/day (no soft tissue and skeletal abnormalities induced)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
Pre and post natal informations from the 2-generations reproduction study
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
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 416
Deviations:
yes
Remarks:
yes 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
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 x21.5 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 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 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.
Details on mating procedure:
For both, F0 and F1 generations:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days had elapsed.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
Duration of treatment / exposure:
In F0 females:
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- during the full pregnancy
- during lactation until day 21 post-partum (p.p.) inclusive,
- females with no delivery were treated until the day prior to sacrifice.

In F0 males :
- 10 weeks before mating,
- during the mating period (up to 3 weeks),
- until sacrifice (after weaning of the pups).

> Constitution and treatment of the F1 generation : On day 22 p.p., one or two males and one or two females per litter (from as many litters as possible) were selected to obtain 25 animals/sex/group and therefore constitute the F1 generation. Day 22 p.p. was designated day 1 of the F1 generation.

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 tov 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).
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (vehicle)
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 (in both generations)
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 421 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.
Maternal examinations:
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: 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: 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. The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
In both F0 and F1generations:
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. 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.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
in both, F0 and F1generations:
> SACRIFICE
On completion of the treatment period, all surviving females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.
- 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.

> GROSS NECROPSY
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 were also recorded for mated females sacrificed on day 22 p.p..and prematurely sacrificed or found dead.
- 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.
- The tissues specified in Tissue Procedure Table 1 were preserved in 10% buffered formalin (except for the testes and epididymides which were preserved in Davidson’s fixative).

> HISTOPATHOLOGY / ORGAN WEIGHTS
in both, F0 and F1generations:
- Organ weights: The body weight of each animal was recorded before sacrifice at the end of the 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 (included in the section : Any other information on mat. and athods incluD. tables) 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.
- 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.

> OTHER:
F0 and F1 dams :
Females were allowed to litter normally and rear their progeny until weaning. Any sign of a difficult or prolonged parturition was recorded. The day of completed parturition was designated day 1 p.p.. The length of gestation was calculated.

F1 pups :
> Sexual development:
- 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 in the F1 generation:
- Auditory function: When the animals were 4 weeks old, they were tested for the acoustic startle response.
- Pupil constriction: When the animals were 4 weeks old, they were tested for the pupil constriction reflex.
- 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.
Ovaries and uterine content:
The ovaries and uterine content of F0 and F1 dams were examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
> 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. 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.
> Data calculations were performed for each group for : Pre-implantation loss, post-implantation loss, mating index: fertility index, gestation index, live birth index, viability index on day 4 p.p., lactation index on day 21 p.p..
> Data are expressed as group mean values ± standard deviation (body weight, food consumption,corpora lutea, implantations, fetuses, resorptions, pups, gestation length) or as proportions (pre-implantation loss, post-implantation loss, fetal observations, gestation index, live birth index, viability index). Whenever necessary, the experimental unit of comparison was the litter. Data of non-pregnant females are excluded from group mean calculations.
> 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.
Fetal examinations:
Postmortem examinations (offspring)
SACRIFICE
F1 and F2 pups
- Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital on day 4 p.p for pups not selected on day 4 p.p. or on day 22 p.p for pups not selected at weaning. F2 offspring were sacrificed on day 4 p.p. for pups not selected on day 4 post-partum or on day 22 p.p.
- Pups found dead or prematurely sacrificed: Moribund pups were sacrificed by an intraperitoneal injection of sodium pentobarbital.
- A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead, prematurely sacrifices pups, pups showing external abnormalities or clinical signs and on one randomly selected F1 and F2 pup/sex/litter sacrificed on day 22 p.p.. Special attention was paid to the reproductive organs.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as described in the foloowing paragraphes :

> External examinations: Yes
F1 and F2 pups :
The total litter size and numbers of pups of each sex were recorded as soon as possible after birth. The litters were observed daily in order to note the number of live, dead and cannibalized pups. Any gross malformation in pups was noted.
The following F1 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..
F2 pups: On day 4 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible four males and four females per litter in order to reduce the litter size-induced variability in the growth and development of the pups and thus increase the sensitivity of statistical analysis.

> Soft tissue examinations: Yes:
F1 and F2 pups:
* Gross necropsy: Internal examinations including the cervical, thoracic, and abdominal viscera. 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 / Organs weight
F1 and F2 pups
>The body weight of each pup selected for a macroscopic post-mortem examination was recorded before scheduled sacrifice on day 22 p.p..
> Brain (including medulla/pons cerebellar and cerebral cortex), spleen and thymus 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 macroscopic lesions and all pups with external abnormalities.

> Skeletal examinations: No
> Head examinations: organ weight measured

> Other examinations:
F1 and F2 pups:
- Clinical signs: The pups were observed daily for clinical signs.
- Body weight: The weight of each pup was recorded on days 1, 4, 7, 14 and 21 p.p..
- Physical development: The number of pups in each litter exhibiting the required characteristics of physical development was recorded at designated time-points:
. 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..
- Reflex development : The number of pups in each litter exhibiting the required characteristics of reflex development was recorded at designated time-points:
. surface righting reflex: on day 5 p.p.,
. cliff avoidance: on day 11 p.p.,
. air-righting reflex: on day 17 p.p..
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.
- Statistical analysis of auditory startle data was performed using the software SAS Enterprise Guide version 2.05.89 (SAS Release 8.02 TS Level 02M0, SAS Institute Inc). Animals whose data are not interpretable were excluded from subsequent statistical analyses. These statistical analyses were performed by sex.To compare the auditory startle response (categorical data using positive or negative values) between treatment groups, a Fisher’s exact test was used. If a global statistically significant difference was observed between treatment groups, multiple Fisher’s exact tests were performed to compare the auditory startle response between each test item-treated group and the control group.
- 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.
Indices:
> Reproductive indices in both F0 and F1 animals
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
Historical control data:
Not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
> F0 females : 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 animals:
There were no test item treatment-related clinical signs. A few signs of lesions (hairloss and nodosities) were recorded both in treated and controls male
and females animals. These findings are commonly observed 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).
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
> F0 females :
No premature death occurred during the study in F0 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 examin ation, 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..

> F1 females:
Some deaths occured but were relationship to the treatement was excluded :
- One female at 1000 mg/kg bw/d 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. On of these females had a fetus in the vagina but had not delivered any pups. The second one 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.
- One female at 450 mg/kgbw/d 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.
- One male in level dose group 150 mg/kg bw/d 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.
- 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. A second female of this group was prematurely sacrificed on lactation day 7 because of dead litter. There were no macroscopic or microscopic findings.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
> F0 dams (See table 2 in "Any information on results incl. table") :
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.

> F1 dams (See table 2 in "Any information on results incl. table"):
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 2 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.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
> F0 females:
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 females:
The female group treated at 1000 mg/kg/day showed a 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 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 females :
There were increases in the mean ovary weights (both sides) in high-dose females compared with controls:
+16*, +14, +17* for the left ovary, absolute weight at 150, 450 and 1000 mg/kg bw/d, respectively
+16, +11, +15* for the left ovary, relative weight at 150, 450 and 1000 mg/kg bw/d, respectively
+9, +12, +18* for the right ovary, absolute weight at 150, 450 and 1000 mg/kg bw/d, respectively
+9, +10, +16* for the right ovary, relative weight at 150, 450 and 1000 mg/kg bw/d, respectively
Statistically significant from controls: *: p<0.05.
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. See table 9 in "any infomation on results incl. Tables"
The test item administration in F0 parents did not induce any changes in organ weights in their pups.

> F1 females:
No significant effect of treatment were observed in organs weight of F1 females. When compared with controls, the test item administration in F1 parents was not associated with any changes in organ weights in their pups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
> F0 females:
Premature deaths
- 1000 mg/kg/day: in one 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 dams 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 females:
Premature death:
- 1000 mg/kg/day: In one found dead female (day 48), many white discolored foci in the kidneys, unilaterallydilated renal pelvis, dilated urinary bladder with red liquid content, and enlarged adrenals was observed. In 2 other 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 of them, all 17 fetuses remaining in the uterine horns were found dead, whereas in the other one, four fetuses were found dead and one fetus was still alive.
- 450 mg/kg/day: In the female found dead on day 10, the lungs were enlarged.
- 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 the other female sacrificed on lactation day 7 because of dead litter, there were no macroscopic findings.
Termianl 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.
Neuropathological findings:
no effects observed
Description (incidence and severity):
> F1 animals :
- 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.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
> F0 females :
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 tothe 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 females:
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 females. 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 (13.71 +/- 5.224 compared to 15.99 +/- 7.526 in control). 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:
There were no significant microscopic changes in the liver and the kidneys from high-dose females.

> F1 females:
Premature death:
- 1000 mg/kg/day: In the 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 the 2 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 onee of the female and minimal multifocal neutrophilic inflammation inthe other one. 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.
- Control: In one 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 other female sacrificed on lactation day 7 because of dead litter, there were no significant microscopic findings in the genital organs.

Terminal sacrifice:
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 females.
In the female (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 1 control females, 3 females from 150 mg/kg/day dose level group, 2 from the 450 mg/kg/day dose level group, and 2 from the 1000 mg/kg/day dose level group which were not pregnant, 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:
The mean value and standard deviation resulting from the quantitative evaluation of the primordial ovarian follicles of the right ovarry from control and high dose group females were 7.25 +/- 4.493 and 7.83 +/- 3.990, respectively and for corpora lutea : 9;80 +/- 2.626 and 11.92 +/- 5.436 in control and high dose group, respectively. 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.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
> F0 dams:
Estrous Cycles : no effects of treatment with the test item was observed on estrous cyclicity.
- The mean number of cycles per female were 4.7, 4.8, 4.8 and 4.7 for level groups 0, 150, 450 and 1000 mg/kg bw/day, respecitvely
- The mean Mean cycle length were 4, 4, 4.1 and 4.1 for level groups 0, 150, 450 and 1000 mg/kg bw/day, respecitvely
- The number of abnormally* cycling females were 1, 2, 2 and 2 for level groups 0, 150, 450 and 1000 mg/kg bw/day, respecitvely

> F1 dams: There were no effects of treatment with the test item on estrous cycles.
- The mean number of cycles per female were 4.6, 4.5, 4.8 and 4.6 for level groups 0, 150, 450 and 1000 mg/kg bw/day, respecitvely
- The mean Mean cycle length were 4.2, 4.4, 4.1 and 4.2 for level groups 0, 150, 450 and 1000 mg/kg bw/day, respecitvely
- The number of abnormally* cycling females were 1, 2, 1 and 2 for level groups 0, 150, 450 and 1000 mg/kg bw/day, respecitvely.

*An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
> F0 females
The mean number of implantation sites was similar in all groups as were the mean number of pups born and the post-implantation loss. Number of Post-implantation loss (calculated Manually) was 14.2, 14.2, 11,7 and 12.4 from dose-level group 0, 150, 450 and 1000 mg/kg bw day, respectively.

> F1 females:
There were no statistically significant differences, between control and treated groups, regarding mean numbers of implantations, delivered pups and post-implantation loss.

See also table 3 in "Any other other information on results incl. table):
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
> F1 pups:
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 of thefemale had no clinical signs during lactation yet 9/15 of the pups were dead or cannibalized on lactation day 1. The other 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. One female was pale and had piloerection on lactation day 1 and on the same day all the pups were found dead (seven pups) or cannibalized (five 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 2d female also had an entire dead litter on lactation day 1 but had no clinical signs. The last one lost 10/14 pups on lactation day 1 (nine were found dead and one was cannibalized) but had no clinical signs. The remaining four pups survived until weaning.
Two of these 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.

> F2 pups:
Pup mortality was not increased in the test item-treated groups..

See also table 3 in "Any other other information on results incl. table):
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
> F0 dams :
The mean duration of gestation was similar in all groups. The mean duration of gestation was 22, 21.8, 21.8 and 21.9 days in levels group 0, 150, 450 nad 1000 mg/kg bw/day, respectively.

> F1 dams :
The mean duration of gestation was identical in all groups.
See table 7 in section "any other information on results incl tables" for both F0 and F1 animals results.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
> F0 animals :
There were 1,1 and 2 non pregnant females at 150, 450 and 1000 mg/kg/day, respectively. No more than two non-pregnant females per group is considered to be within normal range. These females were sacrificed on day 25 p.c.. One female treated at 1000 mg/kg/day was pregnant but did not deliver.

> F1 animals :
There were 1, 4, 2 and 2 non pregnant females at 0, 150, 450 and 1000 mg/kg/day, respectively. 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.
See table 7 in section "any other information on results incl tables" for both F0 and F1 animals results.
Other effects:
no effects observed
Description (incidence and severity):
> F0 animals :
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.
The mean duration of gestation was similar in all groups.

> F1 animals:
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
See table 7 in section "any other information on results incl tables" for both F0 and F1 animals results.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No maternal toxicity observed
Remarks on result:
other:
Remarks:
For both F0 and F1 dams
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
> F1 pups:
There were no effects of treatment with the test item on mean pup body weight at day 1 and Day 21 or body weight gains at any dose-level. The test item administration in F0 parents did not induce any changes in organ weights in their pups.

> F2 pups:
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.
When compared with controls, the test item administration in F1 parents was not associated with any changes in organ weights in their pups.

See table 5 in "any other information on results incl. tables"
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Description (incidence and severity):
F1 pups:
Percentage of male pups at birth : The percentage of male pups was similar in all groups

F2 pups:
There was no effect of treatment on the mean percentage of male pups.

See also table 3 in "Any other other information on results incl. table".
Changes in litter size and weights:
not specified
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
> F1 pups:
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 .

> F2 pups:
Generally, the pups which were later cannibalized or found dead did not have clinical signs. There were no treatment-related findings at external examination of the pups

See also table 4 in "Any other other information on results incl. table".
External malformations:
no effects observed
Description (incidence and severity):
> F1 Pups :
- External examinations: 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.
- 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 1 dam given 450 mg/kg/day, had dehydration and coldness on days 6 to 8 p.p..

> F2 Pups:
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.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Description (incidence and severity):
As no macroscopic lesion was observed in F1 and F2 pups at external examination, no microscopic examination was performed.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed on pre and post natal development in F1 and F2 pups
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

> 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.

- Sexual development of F1 animals (see also table 6 in "Any other other information on results incl. table):

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.

>F2 pups:

- Physical and reflex development

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.

> Sperm analysis in F0 and F1 males : There were no effects of treatment with the test item on sperm parameters in F1 and F0 males (See table 8 in "Any other other information on results incl. table".

 

Table 2:Body weight and body weight change(F0/F1 dams)

F0 Dams

Dose-level (mg/kg bw/day

0

150

450

1000

Premating

Days 1 – 71

+139

 

+144

 

+156**

 

+147

 

Gestation dayaGD 0-20

+144

+151

+143

+150

Lactation

LD 1 - 21

+ 16

+ 6

+ 18

+ 18

F1 Dams

Dose-level (mg/kg bw/day

0

150

450

1000

Premating

Days 1 – 71

+222

 

+232

 

+226

 

+222

 

Gestation dayaGD 0-20

+148

+161

+156

+166

Lactation

LD 1 - 21

+ 19

+ 13

+ 17

+ 23

 GD: gestation day, LD: lactation day, a: only includes pregnant females with live fetuses, Statistically significant **: p<0.01.


Table 3: Fertility parameters (F0 and F1 animals)

F0 Dams

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%

 

F1 Dams

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 (no. of pups)

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%

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

 

Table 4: Distribution of pup mortality (F1 and F1 pups)

F1 pups

Dose-level (mg/kg/day)

0

150

450

1000

Number of cannibalized pups

Number of cannibalized pups with clinical signs

2

0

3

3

7

0

11

1

Number of pups found dead

Number of found dead pups with clinical signs

20

0

4

0

31

1

24

0

Number of prematurely sacrificed pups

0

0

0

1a

F2 pups

Dose-level (mg/kg/day)

0

150

450

1000

Number of cannibalized pups

Number of cannibalized pups with clinical signs

5

3

6

0

4

0

5

1

Number of pups found dead

Number of found dead pups with clinical signs

15

1

10

1

8

0

9

1

Number of prematurely sacrificed pups

0

0

1a

0

a: pup 1 of female F0-1000 mg/kg bw/d: sacrificed after premature sacrifice of the day; female F1- 450 mg/kg bw/d: pup 8 because of dehydration, hypoactivity and coldness.

 

 

Table 5:Body weight during lactation (F1 and F2 pups)

F1 pups

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Body weight (g)

- D1

- D21

 

7.8

57.1

 

7.7

60.6

 

7.5

58.6

 

7.6

57.8

 

7.3

56.1

 

7.3

58.5

 

7.1

56.7

 

7.1

56.3

Body weight gain (g)

D1-21

+ 49.3

+ 52.9

+ 51.1

+ 50.2

+ 48.8

+ 51.2

+ 49.6

+ 49.2

F2 pups

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Body weight (g)

- D1

- D21

 

7.6

55.9

 

7.5

59.6

 

7.4

57.6

 

7.3

56.2

 

7.2

54.5

 

7.1

57.5

 

7.2

56.8

 

6.9

54.7

Body weight gain (g)

D7-14

D1-21

 

 

+ 17.4

+ 48.3

 

 

+19.2**

+ 52.1

 

 

+18.0

+ 50.2

 

 

+17.0

+ 48.9

 

 

+17.0

+ 47.3

 

 

+18.7*

+ 50.4

 

 

+17.9

+ 49.6

 

 

+16.8

+ 47.8

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

 

Table 6:Age at sexual maturity (F1 animals)

Sex

Male

Female

Dose-level

(mg/kg/day)

0

150

450

1000

0

150

450

1000

Mean age (d)

54.2

56.8

54.2

53.6

35.2

38.5

38.7

35.2

Body weight (g)

328

349

331

326

139

143

145

139

 

 


Table 7: Mating, fertility and parturition parameters (F0 and F1 animals)

F0 Dams

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

F1 Dams

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 8: Sperm analysis (F0 and F1 animals)

F0 males

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

F1 males

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

Conclusions:
In this study since no effect of the substance were observed at the tested dose levels, the No Observed Adverse Effect Level for maternal toxicity for both, F0 and F1 dams was found to be 1000 mg/kg bw/day.
Further, cerium and iron oxide isostearate did not delayed or impaired development in F1 or F2 pups and no external malformation or macroscopic findings were observed whatever the dose levels used. Therefore, the No Observed Effect Level (NOEL) for pre- 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 considered to be 1000 mg/kg/day. The NOAEL for maternal toxicity for both F0 and F1 dams was therefore found to be 1000 mg/kg bw/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 p and post natal developmental toxicity is warranted based on the absence of relevant effects in this study, according to the criteria of UN/EU GHS.

 

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

Endpoint:
developmental toxicity
Remarks:
Screening for reproductive/developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 23 May 2008 to 15 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was performed similarly to OECD guideline and following the principles of Good Labaratory Practice (but not audited by Quality ass urance unit). This study was used as a preliminary test for a 2-generation study and used 1 control and 2 level dose groups.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 421
Deviations:
yes
Remarks:
yes only 2 dose levels, no microscopic examination was performed, used as a preliminary study to a multigeneration study (CIT study n° 34760 RSR) .
GLP compliance:
no
Remarks:
But follows the principles of Good Laboratory Practice.
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: mean body weight of 427 g (range: 413 g to 444 g) for the males and 266 g (range: 251 g to 292 g) for the females.
- Fasting period before study: no
- Housing: 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
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: from 23 May 2008 (arrival of the animals) to 27 July 2008 (necropsy of the last
female)
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 acceptable in order to achieve the concentrations of 90 and 200 mg/mL. The resulting suspension was left under magnetic stirring until treatment of the animals. The dosage forms were administered by gavage using a plastic syringe fitted with a metal gavage tube, once a day, at approximately the same time. The quantity of dosage form administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 5 mL/kg/day was used.

VEHICLE
- Justification for use and choice of vehicle: Vehicle used in previous repeated (28-day) dose toxicity study (22314 TSR) in which stability, homogeneity and concentration of the test item in the vehicle were found satisfactory.
- Concentration in vehicle: 90 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
- Lot/batch no.: 126K0117 and 117K0127
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
As this study is a dose range finding study, no chemical analysis was performed.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 14 days
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Duration of treatment / exposure:
- in the females: 15 days before mating, during the mating period (up to 3 weeks), during pregnancy, during lactation until day 4 post-partum inclusive.
- In the males: 15 days before mating, during the mating period (up to 3 weeks), until sacrifice (i.e. at least 5 weeks in total).
Day 1 corresponds to the first day of treatment period.
Frequency of treatment:
Once a day, at approximately the same time each day, 7 days a week.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle given to control group
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were selected on the basis of a 4-week toxicity study in the rat (CIT/Study No. 22314 TSR). In this study, dose-levels of 150, 450 and 1000 mg/kg/day resulted in no deaths or clinical signs, no relevant effects on body weight or food consumption, no toxicologically significant differences in hematology or blood biochemistry parameters and no effects were observed at microscopic examination. The NOEL was considered to be 1000 mg/kg/day.
- Rationale for animal assignment: according to a computerized stratification procedure, so that the average body weight of each group was similar.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day during the treatment period including week-end and public holidays for mortality and signs of morbidity. From arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.
- Cage side observations include routine examinations.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
> for 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 and days 1 and 5 post-partum.
> for males: the first day of treatment (day 1), then once a week until sacrifice.

FOOD CONSUMPTION: Yes
> 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 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice.
> 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.
> During the pairing period, food consumption was not recorded for males or females.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
> SACRIFICE
- Maternal animals: All surviving animals, on day 5 post-partum, or on day 25 post-coitum for females which had not delivered.
- Male animals: All surviving animals, after the end of the mating period (after at least 5 weeks of treatment).

> GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all animals (females and males). 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 corpora lutea and implantation scars were also recorded for females sacrificed on day 5 postpartum. The numbers of corpora lutea were recorded for females sacrificed on day 25 post-coitum due to no delivery. For these apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

> HISTOPATHOLOGY / ORGAN WEIGHTS
All macroscopic lesions were preserved in 10% buffered formalin (except for the testes and epididymides which were preserved in Davidson’s fixative). No microscopic examination was performed.

OTHER:
- Parturition: Females were allowed to litter normally and rear their progeny until sacrifice of the pups on day 5 post-partum. Any sign of a difficult or prolonged parturition was recorded. The day of completed parturition was designated day 1 post-partum. The length of gestation was calculated.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Not specified
- Number of late resorptions: Not specified
- Other:
The numbers of corpora lutea and implantation scars were also recorded for females sacrificed on day 5 postpartum. The numbers of corpora lutea were recorded for females sacrificed on day 25 post-coitum due to no delivery. For these apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
The estrous cycle stage was determined from a fresh vaginal lavage, each morning during the mating period, until the females had mated.
Fetal examinations:
- Litter observations :
The following parameters were examined in F1 offspring: Total litter size and number and sex of pups, stillbirths, live births, dead and cannibalized pups, postnatal mortality, presence of gross anomalies, clinical signs (daily), body weight (day 1 and day 5 post-partum).
- External examinations: Yes
Pups found dead or prematurely sacrificed were carefully examined for gross external abnormalities. Pups sacrificed on day 5 post-partum were discarded with no post-mortem examination. There was no preservation of tissues.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Standardisation of litters on day 4 postpartum : no
Statistics:
Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
Indices:
> 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 5 post-partum,
- Mean litter size,
- Pup sex ratios
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 2 females treated at 1000 mg/kg/day showed relevant clinical signs: one female showed reflux at dosing on day 12 post-coitum only and another female displayed chromodacryorrhea on day 21 post-coitum and day 0 post-partum. It was considered that none of these represented signs of toxicity of the test item.
In addition, hairloss on the forelimbs was observed in one control female, and one female treated at 1000 mg/kg/day. These signs are commonly observed in laboratory rats of this strain and are considered not to be related to treatment with the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item females during the pre-mating phase.
During gestation, females treated at 1000 mg/kg/day gained more weight than the controls but the females treated at 1000 mg/kg/day had a mean of one pup more than the control females which explain the higher body weight gain.
Both female treated groups gained more body weight than the controls during lactation, however four control females lost a small amount of weight during this period.
See Table 1 in "any other information on results incl. table".
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on mean female food consumption. The female group treated at 1000 mg/kg/day had slightly higher food consumption than the controls during lactation which corresponded with the higher body weight gain.
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
One female from the group treated at 1000 mg/kg/day had enlarged and irregularly colored right papillary process of the liver with a firm consistency. Due to the low incidence of these findings, they were all considered to be unrelated to treatment with the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects on the mean numbers of corpora lutea, implantations or pups at any dose-level, nor on the duration of gestation or the extent of pre-implantation loss.
The mean post-implantation loss was slightly higher in the groups treated with the test item when compared with the controls, however the differences were not toxicologically significant. It was therefore considered that this did not represent an effect of treatment with the test item.
See Table 3 in "any other information on results incl. table".
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effect of the treatement (See Table 3 in "any other information on results incl. table").
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no effect of treatement on the duration of gestation (See Table 3 in "any other information on results incl. table").
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on mating; all females at all dose-levels mated and only one control male did not mate. The mean number of days of pairing before mating was similar to or lower than the control group.
One females in the control group, 3 females in the 450 mg/kg/day dose group stayed for 1 week or more in diestrus and/or metestrus before mating; all females were pregnant. Other than these females, all estrous cycles were considered to be normal.
There were two mated but non-pregnant females in the group treated 1000 mg/kg/day. Neither showed any macroscopic abnormality at post-mortem examination.
(See Table 2 in "any other information on results incl. table".
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No effect in dams
Abnormalities:
no effects observed
Fetal body weight changes:
not specified
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No effect of the treatment was observed. See Table 3 in "any other information on results incl. tables".
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of male pups was similar between all groups. See Table 3 in "any other information on results incl. tables".
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight on post-natal day 1 was similar between all groups, including the controls, but on post-natal day 5 the pups from the group treated at 1000 mg/kg/day had a lower, non-statistically significant, mean body weight than the controls (-7% in the males and -6% in the females). The mean body weight gain from post-natal days 1 to 5 was also lower at 1000 mg/kg/day (-15% in the males and -12% in the females) when compared with the controls.
This may have some relationship to the fact that there was a mean of one pup more per female in this group because the individual values at 1000 mg/kg/day were within the control range, but a relationship to treatment with the test item cannot be excluded.
See Table 4 in "any other information on results incl. tables".
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Pup survival was higher in the groups treated with Isostéarate d’oxyde de cérium et de fer than in the control group.
No clinical signs were observed at 1000 mg/kg/day. At 450 mg/kg/day, one pup was cold to the touch, showed generalized hematoma and was found dead on post-natal day 4. Another pup from the same litter appeared to have milk in the urogenital region.
It was considered that there were no treatment-related clinical signs. See Table 3 in "any other information on results incl. tables".

External malformations:
no effects observed
Description (incidence and severity):
None of the pups found dead showed gross external abnormalities.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Dose descriptor:
NOEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1:Body weight and body weight changein dams

Dose-level (mg/kg bw/day

0

450

1000

Pre-mating

Day 1

Day15

 

Days 1-15

 

 

266

289

 

+ 23

 

265

288

 

+ 23

 

267

290

 

+ 23

Gestation

Day 0 p.c.

Day 20 p.c.

 

Days 0-20 p.c.

 

300

453

 

+ 154

 

306

464

 

+ 158

 

299

459

 

+ 163

Lactation

Day 1 p.p.

Day 5 p.p.

 

Day 0-5 p.p.

 

363

367

 

+ 4

 

360

373

 

+ 13

 

349

369

 

+ 20*

p.c.:post-coitum,p.p.:post-partum,*: p<0.05.

 

 

 

Table 2: Mating and fertility data

Dose-level (mg/kg/day)

0

450

1000

Number of males + females paired

10 + 10

10 + 10

10 + 10

Pairs mated

10

10

10

Mating index (%)

100

100

100

Number of malesmated

9

10

10

Number of femalesmated

10

10

10

Mean number of days taken to mate

5.2

5.6

2.3

Number of pregnant females

10

10

8

Fertility index (%)

100

100

80

Females with live concepti

10

10

8

Gestation index

100

100

100

 

Table 3: Delivery data

Dose-level (mg/kg/day)

0

450

1000

Number of pregnant females surviving delivery

10

10

8

Mean duration of gestation (days)

22.0

21.9

22.0

With stillborn pups

N

%

 

0

0

 

0

0

 

0

0

With stillborn pups

N

%

 

0

0

 

0

0

 

0

0

With entire liveborn litter dying and/or missing, cannibalized, cled

Days 0-4

Day 0-5

 

 

0

0

 

 

0

0

 

 

0

0

Mean number ofcorpora lutea

16.2

15.0

16.6

Mean number of implantations

13.7

14.3

15.9

Mean pre-implantation loss (%)

16.1

5.6

4.2

Mean number of pups delivered

12.3

12.4

13.5

Mean post-implantation loss (%)(calculated manually)

10.7

13.5

14.4

Live birth index (%)

100

100

100

Pups surviving 4 days (N)

Viability index (%)

120

97.6

122

98.4

106

98.1

Pups surviving 5 days (N)

Lactation index (%)

120

100

122

100

106

100

Sex ratio – Male pups/total pups

Day 0 (%)

Day 5 5%)

 

47.6

47.5

 

48.4

49.2

 

48.1

49.1

 

Table 4:Body weight and body weight gainin pups

Sex

Male

Female

Dose-level (mg/kg bw/day

0

450

1000

0

450

1000

Body weight (g)

PND 1

PND 5

 

Body weight gain (g)

PND 1-5

 

 

8.0

13.5

 

 

+ 5.4

 

7.8

12.9

 

 

+ 5.2

 

7.8

12.5

 

 

+ 4.6

 

7.5

12.7

 

 

+ 5.1

 

7.5

12.8

 

 

+ 5.3

 

7.4

12.0

 

 

+ 4.5

PND: post-natal day.

Conclusions:
Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day. Pups showed no effects of treatment on survival and no gross external abnormalities. However the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.
Based on these results, the NOAEL for maternal toxicity is considered to be 1000 mg/kg bw/day and the NOEL for pre and post natal development is considered to be 450 mg/kg bw/day.
Executive summary:

In a Reproduction/developmental toxicity screening test, the potential general and reproductive or developmental toxicity of Cerium and iron oxide isostearate were evaluated following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0, 450 or 1000 mg/kg/day in corn oil.

 

No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.

Pups showed no effects of treatment on survival.

Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg/day (-15% in the males and -12% in the females, not statistically significant). This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded. It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio. There were no treatment-related macroscopic abnormalities in pups.

 

Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.

As this study was designed to choose the dose-levels which should be used in a 2-genarations study, it is however considered that 1000 mg/kg/day would be a suitable high dose-level for themultigeneration study.

 

This study is classified as acceptable. It is similar to OECD guideline on reproduction/developmental toxicity screening and is acceptable as a preliminary study to a multigeneration study.

Endpoint:
developmental toxicity
Remarks:
Teratogenicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Publicly available FDA research laboratory study where no study guideline is mentioned but the method used is comparable to an OECD 414. However, methods and discussion of the resuts are reported briefly.160 mg/kg bw/day (nominal)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Groups each of approximately 24 pregnant albino CD-1 mice were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. On day 17, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.
GLP compliance:
no
Remarks:
The study was performed before implementation of GLP requirements.
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: gang-houses in disposable plastic cages
- Diet (e.g. ad libitum): free acces to food
- Water (e.g. ad libitum): free acces to fresh tap water.
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): daily controlled, from 74 to 81 °F
- Humidity (%): daily controlled, from 64 to 78 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: To: not specified
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Administration of the test substance as a water suspension (10 ml/kg bw)
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Cohoused. Virgin adult female albino CD-l outbred mice were mated with young adult males. Observation of the vaginal sperm plug was considered Day 0 of gestation.
One male was not permitted to impregnate more than one female per group.
Duration of treatment / exposure:
From day 6 to day 15 of gestation
Frequency of treatment:
daily
Duration of test:
17 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control : vehicle (water)
Dose / conc.:
1.6 mg/kg bw/day (nominal)
Dose / conc.:
7.4 mg/kg bw/day (nominal)
Dose / conc.:
34.5 mg/kg bw/day (nominal)
Dose / conc.:
160 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 to 28 mice/group
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 22 to 28 ( see table 1 in "Any other information on materials and methods incl. tables") pregnant albino CD-1 mice were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. One group of mice was treated with the vehicle, at a level equivalent to the group receiving the highest dose, for the negative control group and one group was treated with 150 mg/kg bw Aspirin as a positive control group. Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. All animals were observed daily for appearance, behavior, food consumption and weight.
On day 17, all dams were subjected to caesarean section and the number of implantation sites, resorption sites, number of corpora lutea, number of live and dead foetuses and sex were recorded. The body weight of the live pups was recorded. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external abnormalities. One-third of the foetuses of each litter were examined for visceral abnormalities using the Wilson technique. Colored photographs were taken of all grossly abnormal fetuses. The remaining two-thirds were cleared in potassium hydroxide (KOH) , stained with alizarin red S dye and examined for skeletal defects.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily - All animals were observed daily for appearance and behavior with particular attention to food consumption and weight.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 11, 15, and 17 ofgestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: yes
- Sacrifice on gestation day # 17
- Organs examined: uterus

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations sites: Yes
- Number of resorptions: Yes
- Other : number of abortion, number of live litters/fetuses, number of dead fetuses, average fetus weight, sex ratio
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: Yes: No specified
Statistics:
not specified
Indices:
not specified
Historical control data:
not specified
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
The authors indicated that the administration of up to 160 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on maternal survival.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect of the treatment with the test substance was observed on the body weight gain of the dams as compared to the control group (see Table 5 in "Any other information on results incl. tables).
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Details on results:
The authors concluded that Ferrous sulfate showed no maternal toxicity at dose levels up to 160 mg/kg bw in mice.
Number of abortions:
no effects observed
Description (incidence and severity):
No abortion occured in the treated dams whatever the dose level used as compared to controls (see Table 2 in "Any other information on results incl. tables).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the number of implantation sites whatever the dose used. Total number of implantation sites were 207, 212, 216, 237 and 215 for the dose level groups 0 (vehicle), 1.6, 7.4, 34.5 and 160 mg/kg bw/d, respectiveley. (see Table 2 in "Any other information on results incl. tables).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more sites resorbed were : 6, 7, 11, 8 and 7 in the dose level groups 0 (vehicle), 1.6, 7.4, 34.5 and 160 mg/kg bw/d, respectiveley.
The number of dams with all sites resorbed were : 0, 1, 0, 0 and 0 in the dose level groups 0 (vehicle), 1.6, 7.4, 34.5 and 160 mg/kg bw/d, respectiveley. (see Table 2 in "Any other information on results incl. tables).
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more dead fetuses was 0, 0, 2, 4 and 2 in the dose level groups 0 (vehicle), 1.6, 7.4, 34.5 and 160 mg/kg bw/d, respectiveley.
They were no dam with all dead fetuses whatever the dose level group. (see Table 2 in "Any other information on results incl. tables).
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the number of dams surviving at term (see Table 1 in "Any other information on results incl. tables).
Other effects:
no effects observed
Description (incidence and severity):
Further, there were no effect of the treatment on the total number of corpora lutea (see Table 2 in "Any other information on results incl. tables).
Details on maternal toxic effects:
The authors concluded that Ferrous sulfate showed no maternal toxicity or teratogenic effect at dose levels up to 160 mg/kg bw in mice.
Key result
Dose descriptor:
NOEL
Effect level:
>= 160 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effect
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the average fetus weight (average fetus weight (in g) were 0.94, 0.88, 0.94, 0.92 and 0.95 in the dose level groups 0 (vehicle), 1.6, 7.4, 34.5 and 160 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no apparent reduction in number of live fetuses. The total number of live fetuses were 198, 191, 198, 223 and 199 in the dose level groups 0 (vehicle), 1.6, 7.4, 34.5 and 160 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No apparent change in sex ratio was observed whatever the dose level group considered: the sex ratio were 0.87, 0.97, 1.06, 1.10 and 0.92 in the dose level groups 0 (vehicle), 1.6, 7.4, 34.5 and 160 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No grossly abnormal fetuses were observed in the study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in skeletal tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 3 in "Any other information on results incl. tables).
Visceral malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in soft tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 4 in "Any other information on results incl. tables).
Details on embryotoxic / teratogenic effects:
The authors concluded that Ferrous sulfate showed no teratogenic effect at dose levels up to 160 mg/kg bw in mice.
Key result
Dose descriptor:
NOEL
Effect level:
>= 160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Number of mated and pregnant dams /group

 

 

Total

Surviving at term

Material

Dose (mg/kg)**

Mated

Pregnant

Total

Pregnant1

Sham (vehicle)

0.0

22

20

22

20

Aspirin*

150.0

22

18

22

18

 

 

 

Ferrous sulfate

1.6

28

19

28

19

7.4

25

20

25

20

34.5

25

20

25

20

160.0

27

21

27

21

1: includes all dams examined at term, * positive control, ** administered as a water suspension (10 ml/kg bw)

 

Table 2: Reproduction data

 

Negative control

Positive control (Aspirin)

 

Ferrous sulfate

Dose (mg/kg) :

0.0

 

150 mg/kg

1.6

7.4

34.5

160.0

Pregnancies

Total No.

Died or aborted (before Day 17)

To term (on day 17)

 

20

0

20

 

18

0

18

 

 

19

0

19

 

20

0

20

 

20

0

20

 

21

0

21

Corpora Lutea

Total No.

Average/dam mated

 

226

10.8

 

227

10.3

 

225

8.04

 

243

9.72

 

269

10.8

 

242

8.96

Live litters

Total No.*

 

20

 

18

 

18

 

20

 

20

 

21

Implant Sites

Total No.

Average/dam mated*

 

207

10.4

 

204

11.3

 

212

11.2

 

216

10.8

 

237

11.9

 

215

10.2

Resorptions

Total No.*

Dams with 1 or more sites resorbed

Dams with all sites resorbed

Per cent partial resorptions

Per cent complete resorptions

 

9

6

--

30.0

--

 

20

9

--

50.0

--

 

21

7

1

36.8

5.26

 

16

11

--

55.0

--

 

10

8

--

40.0

--

 

14

7

--

33.3

--

Live fetuses

Total No.

Average/dam*

Sex ratio (M/F)

 

198

9.90

0.87

 

183

10.2

1.01

 

191

10.1

0.97

 

198

9.90

1.06

 

223

11.2

1.10

 

199

9.48

0.92

Dead fetuses

Total No.*

Dams with 1 or more dead

Dams with all dead

Per cent partial dead

Per cent all dead

 

--

--

--

--

--

 

1

1

--

5.56

--

 

--

--

--

--

--

 

2

2

--

10.0

--

 

4

3

--

15.0

--

 

2

2

--

9.52

--

Average fetus weight (g)

0.94

0.83

0.88

0.94

0.92

0.95

*Includes only those dams examined at term

 

Table 3: Summary of skeletal findings*

 

Negative control

Positive control (Aspirin)

 

Ferrous sulfate

Dose (mg/kg) :

0.0

 

150 mg/kg

1.6

7.4

34.5

160.0

Live fetuses examined (at term)

140/20

130/18

133/18

140/20

155/20

139/21

Sternebrae

Incomplete oss.

Scrambled

Bipartite

Fused

Extra

Missing

Other

 

19/9

 

 

 

 

10/3

 

34/10

 

1/1

 

 

22/9

 

9/6

 

 

 

 

15/8

 

48/13

 

 

 

 

27/9

 

17/9

 

 

 

 

13/6

 

10/5

 

 

 

 

11/3

Ribs

Incomplete oss.

Fuse/split

Wavy

Less than 12

Less than 13

Other

 

 

 

 

 

21/9

 

 

13/4

 

1/1

48/15

 

 

 

1/1

 

28/10

 

1/1

 

1/1

 

16/8

 

 

 

 

 

31/13

 

 

 

 

 

21/10

Vertebrae

Incomplete oss.

Scrambled

Fused

Extra ctrs. oss.

Scoliosis

Tail defects

Other

 

4/2

 

6/3

2/1

6/1

 

7/4

 

4/3

 

5/3

 

5/3

Skull

Incomplete closure

Missing

Craniostosis

Other

 

 

 

1/1

 

 

 

1/1

Extremities

Incomplete oss.

Missing

Extra

 

1/1

 

1/1

 

5/3

 

5/4

 

2/1

 

1/1

Miscellaneous

Hyoid, missing

Hyoid, reduced

 

24/9

14/9

 

24/11

21/11

 

26/10

16/10

 

34/11

16/9

 

36/15

14/8

 

24/10

11/7

*Numerator = Number of fetuses affected; Denominator = Number of litters affected

 

Table 4: Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam number

Number of pups

Description

Aspirin (Positive control)

150.0

23347

1

Gastroschisis

 

 

Ferrous sulfate

 

1.6

23484

23496

1

1

Gastroschisis

Gastroschisis

160.0

23593

1

Gastroschisis

 

Table 5: Average body weight of pregnant dams

 

 

Day

Material

Dose Level (mg/kg)**

0

6

11

15

17**

 

 

Average body weight (g)

Sham (vehicle)

0.0

29

32

35

42

49 (20)

Aspirin*

150.0

32

35

38

45

51 (18)

 

 

Ferrous sulfate

1.6

33

35

39

46

52 (19)

7.4

33

35

39

47

53 (20)

34.5

32

34

39

47

52 (20)

160.0

30

33

36

44

48 (21)

* Positive control, ** Number of surviving dams in parentheses

Conclusions:
The authors concluded that Ferrous sulfate showed no maternal toxicity or teratogenic effect at dose levels up to 160 mg/kg bw in mice. In the experimental conditions of the study, the NOEL for maternal toxicity and teratogenic effect can be established for both, at >= 160 mg/kg bw/d (highest dose tested).
Executive summary:

Groups each of approximately 24 pregnant albino CD-1 mice were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. On day 17, all dams were subjected to caesarean section and the number of implantation sites, corpora lutea, resorption sites and live and dead fetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities

The authors concluded that the administration of up to 160 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Endpoint:
developmental toxicity
Remarks:
Teratogenicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Publicly available FDA research laboratory study where no study guideline is mentioned but the method used is comparable to an OECD 414. However, methods and discussion of the resuts are reported briefly.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Groups each of approximately 24 pregnant albino rats (wistar derived stock) were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormalities. All foetuses were examined for the presence of external congenital abnormalities. One-third of thefoetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.
GLP compliance:
no
Remarks:
The study was performed before implementation of GLP requirements.
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: From 209 to 218 g at day 0 of the study
- Fasting period before study: not specified
- Housing: individually housed in mesh bottom cages
- Diet (e.g. ad libitum): free acces to food
- Water (e.g. ad libitum): free acces to fresh tap water.
- Acclimation period: not specified
ENVIRONMENTAL CONDITIONS
- Temperature (°C): daily controlled, from 74 to 81 °F
- Humidity (%): daily controlled, from 64 to 78 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified
IN-LIFE DATES: not specified
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Administration of the test substance as a water suspension (10 ml/kg bw)
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Cohoused. Virgin adult female albino rat (Wistar derived stock) were mated with young adult males. Observation of the vaginal sperm plug was considered Day 0 of gestation.
One male was not permitted to impregnate more than one female per group.
Duration of treatment / exposure:
From day 6 through day 15 of gestation.
Frequency of treatment:
daily
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control: vehicle (water)
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
9.3 mg/kg bw/day (nominal)
Dose / conc.:
43.1 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
The authors said that dose levels were up to 160 mg/kg bw/day.
No. of animals per sex per dose:
21 to 25 rat/group
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 21 to 25 ( see table 1 in "Any other information on materials and methods incl. tables") pregnant albino rats (Wistar derived stock) were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. One group of rats was treated with the vehicle, at a level equivalent to the group receiving the highest dose, for the negative control group and one group was treated with 250 mg/kg bw Aspirin as a positive control group. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. All animals were observed daily for appearance, behavior, food consumption and weight.
On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites, number of corpora lutea, number of live and dead foetuses and sex were recorded. The body weight of the live pups was recorded. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined grossly for the presence of external abnormalities. One-third of the foetuses of each litter were examined for visceral abnormalities using the Wilson technique. Colored photographs were taken of all grossly abnormal fetuses. The remaining two-thirds were cleared in potassium hydroxide (KOH) , stained with alizarin red S dye and examined for skeletal defects.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily - All animals were observed daily for appearance and behavior with particular attention to food consumption and weight.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 11, 15, and 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gai n data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: yes
- Sacrifice on gestation day # 20
- Organs examined: uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations sites: Yes
- Number of resorptions: Yes
- Other : number of abortion, number of live litters/fetuses, number of dead fetuses, average fetus weight, sex ratio
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: Yes: No specified
Statistics:
not specified
Indices:
not specified
Historical control data:
not specified
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
The authors indicated that the administration of up to 200 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on maternal survival.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect of the treatment with the test substance was observed on the body weight gain of the dams as compared to the control group (see Table 5 in "Any other information on results incl. tables).
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Details on results:
The authors concluded that Ferrous sulfate induced no clearly discernible maternal toxicity at dose levels up to 200 mg/kg bw in rats.
Number of abortions:
no effects observed
Description (incidence and severity):
No abortion occured in the treated dams whatever the dose level used as compared to controls (seeTable 2 in "Any other information on results incl. tables).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the number of implantation sites whatever the dose used. Total number of implantation sites were 218, 240, 222, 231 and 212 for the dose level groups 0 (vehicle), 2.0, 9.3, 43.1 and 200 mg/kg bw/d, respectiveley. (see Table 2 in "Any other inf ormation on results incl. tables).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more sites resorbed were : 3, 2, 3, 0 and 0 in the dose level groups 0 (vehicle), 2.0, 9.3, 43.1 and 200 mg/kg bw/d, respectiveley.
The number of dams with all sites resorbed were : 0, 0, 0, 0 and 0 in the dose level groups 0 (vehicle), 2.0, 9.3, 43.1 and 200 mg/kg bw/d, respectiveley. (see Table 2 in "Any other information on results incl. tables).
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more dead fetuses or with all dead fetuses was 0 in control and all dose levels groups (see Table 2 in "Any other information on results incl. tables).
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the number of dams surviving at term (see Table 1 in "Any other information on results incl. tables).
Other effects:
no effects observed
Description (incidence and severity):
Further, there were no effect of the treatment on the total number of corpora lutea (see Table 2 in "Any other information on results incl. tables).
Details on maternal toxic effects:
The authors concluded that Ferrous sulfate showed no maternal toxicity or teratogenic effect at dose levels up to 200 mg/kg bw in rats.
Key result
Dose descriptor:
NOEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effect
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the average fetus weight (average fetus weight (in g) were 3.82, 3.97, 3.95, 3.81 and 3.94 in the dose level groups 0 (vehicle), 2.0, 9.3, 43.1 and 200 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no apparent reduction in number of live fetuses. The total number of live fetuses were 215, 238, 216, 231 and 212 in the dose level groups 0 (vehicle), 2.0, 9.3, 43.1 and 200 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No apparent change in sex ratio was observed whatever the dose level group considered: the sex ratio were 0.94, 0.92, 0.95, 0.89 and 0.91 in the dose level groups 0 (vehicle) 2.0, 9.3, 43.1 and 200 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
One grossly abnormal fetus was reported in the negative control (vehicle) group and one also in the positive (aspirin) control group. But none were reported in the treated groups with the test substance.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in skeletal tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 3 in "Any other information on results incl. tables).
Visceral malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in soft tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 4 in "Any other information on results incl. tables).
Details on embryotoxic / teratogenic effects:
The authors concluded that Ferrous sulfate showed no teratogenic effect at dose levels up to 200 mg/kg bw in rats.
Key result
Dose descriptor:
NOEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Number of mated and pregnant dams /group

 

 

Total

Surviving at term

Material

Dose (mg/kg)**

Mated

Pregnant

Total

Pregnant1

Sham (vehicle)

0.0

25

19

25

19

Aspirin*

250.0

21

21

21

21

 

 

Ferrous sulfate

2.0

25

20

25

20

9.3

21

20

21

20

43.1

24

20

24

20

200.0

22

20

22

20

1: includes all dams examined at term, * positive control, ** administered as a water suspension

 

Table 2: Reproduction data

 

Negative control

Positive control (Aspirin)

 

Ferrous sulfate

Dose (mg/kg) :

0.0

 

250 mg/kg

2.0

9.3

43.1

200.0

Pregnancies

Total No.

Died or aborted (before Day 20)

To term (on day 20)

 

19

0

19

 

21

0

21

 

20

0

20

 

20

0

20

 

20

0

20

 

20

0

20

Corpora Lutea

Total No.

Average/dam mated

 

242

9.68

 

241

11.5

 

256

10.2

 

243

11.6

 

252

10.5

 

232

10.6

Live litters

Total No.*

 

19

 

19

 

20

 

20

 

20

 

20

Implant Sites

Total No.

Average/dam mated*

 

218

11.5

 

212

10.1

 

240

12.0

 

222

11.1

 

231

11.6

 

212

10.6

Resorptions

Total No.*

Dams with 1 or more sites resorbed

Dams with all sites resorbed

Per cent partial resorptions

Per cent complete resorptions

 

3

3

--

15.8

--

 

48

12

2

57.1

9.52

 

2

2

--

10.0

--

 

6

3

--

15.0

--

 

--

--

--

--

--

 

--

--

--

--

--

Live fetuses

Total No.

Average/dam*

Sex ratio (M/F)

 

215

11.3

0.94

 

163

8.58

0.99

 

238

11.9

0.92

 

216

10.8

0.95

 

231

11.6

0.89

 

212

10.6

0.91

Dead fetuses

Total No.*

Dams with 1 or more dead

Dams with all dead

Per cent partial dead

Per cent all dead

 

--

--

--

--

--

 

1

1

--

4.76

--

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

Average fetus weight (g)

3.82

2.64

3.97

3.95

3.81

3.94

*Includes only those dams examined at term

 

Table 3: Summary of skeletal findings*

 

Negative control

Positive control (Aspirin)

 

Ferrous sulfate

Dose (mg/kg) :

0.0

 

250 mg/kg

2.0

9.3

43.1

200.0

Live fetuses examined (at term)

151/19

114/19

166/20

151/20

159/20

146/20

Sternebrae

Incomplete oss.

Scrambled

Bipartite

Fused

Extra

Missing

Other

 

28/12

 

1/1

 

 

2/2

 

73/18

 

12/7

 

 

72/19

 

23/11

 

 

 

 

1/1

 

22/12

 

 

 

 

4/1

 

14/8

 

1/1

 

 

9/5

 

22/11

 

1/1

 

 

2/1

Ribs

Incomplete oss.

Fuse/split

Wavy

Less than 12

Less than 13

Other

 

 

 

30/9

 

17/11

7/4

45/15

1/1

79/18

 

1/1

 

25/11

 

6/3

 

21/9

 

2/1

 

4/3

 

29/12

 

1/1

 

16/9

Vertebrae

Incomplete oss.

Scrambled

Fused

Extra ctrs. oss.

Scoliosis

Tail defects

Other

 

4/4

 

54/6

 

1/1

1/1

 

9/5

 

10/4

 

7/3

 

3/3

Skull

Incomplete closure

Missing

Craniostosis

Other

 

32/8

 

44/14

 

45/15

 

34/11

 

30/11

 

26/12

Extremities

Incomplete oss.

Missing

Extra

 

 

 

3/316

 

 

 

 

 

 

 

 

Miscellaneous

Hyoid, missing

Hyoid, reduced

 

15/8

28/12

 

38/16

7/3

 

21/13

26/13

 

13/4

11/9

 

13/7

23/10

 

9/6

24/12

*Numerator = Number of fetuses affected; Denominator = Number of litters affected

 

Table 4: Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam number

Number of pups

Description

Negative control

0.0

43323

1

Encephalomeningocele;

Anopia; Micromeglia

Aspirin (Positive control)

250.0

43336

43345

43346

1

1

1

Gastroschisis

Gastroschisis

Encephalomeningocele

 

 

Ferrous sulfate

2.0

43498

4

Gastroschisis

9.3

43530

1

Hydrocephalus

200.0

43591

1

Gastroschisis

 Table 5: Average body weight of pregnant dams

 

 

Day

Material

Dose Level (mg/kg)**

0

6

11

15

20**

 

 

Average body weight (g)

Sham (vehicle)

0.0

215

235

251

275

344 (19)

Aspirin*

250.0

211

228

241

261

309 (21)

 

 

Ferrous sulfate

2.0

218

234

254

281

356 (20)

9.3

209

237

253

278

347 (20)

43.1

218

231

249

273

338 (20)

200.0

210

231

247

267

336 (20)

* Positive control, ** Number of surviving dams in parentheses

Conclusions:
The authors concluded that Ferrous sulfate showed no maternal toxicity or teratogenic effect at dose levels up to 200 mg/kg bw in rats. In the experimental conditions of the study, the NOEL for maternal toxicity and teratogenic effect can be established for both, at >= 200 mg/kg bw/d (highest dose tested).
Executive summary:

Groups each of approximately 21 to 25 pregnant albino rats (wistar derived stock) were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining  two-thirds for skeletal abnormalities.

The authors concluded that the administration of up to 200 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Endpoint:
developmental toxicity
Remarks:
Teratogenicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Publicly available FDA research laboratory study where no study guideline is mentioned but the method used is comparable to an OECD 414. However, methods and discussion of the resuts are reported briefly.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Groups each of approximately 24 pregnant albino CD-1 mice were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. On day 17, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.
GLP compliance:
no
Remarks:
The study was performed before implementation of GLP requirements.
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: From 28 to 32 at day 0 of the pregnancy
- Fasting period before study: not specified
- Housing: gang-housed in disposable plastic cages
- Diet (e.g. ad libitum): free acces to food
- Water (e.g. ad libitum): free acces to fresh tap water.
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): daily controlled, from 77 to 82 °F
- Humidity (%): daily controlled, from 50 to 72 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified
IN-LIFE DATES: From: To: not specified
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Administration of the test substance as a water suspension (10 ml/kg bw)
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Cohoused. Virgin adult female albino CD-l outbred mice were mated with young adult males. Observation of the vaginal sperm plug was considered Day 0 of gestation. One male was not permitted to impregnate more than one female per group.
Duration of treatment / exposure:
From day 6 through day 15 of gestation.
Frequency of treatment:
daily
Duration of test:
17 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control: vehicle (water)
Dose / conc.:
16 mg/kg bw/day (nominal)
Dose / conc.:
74.3 mg/kg bw/day (nominal)
Dose / conc.:
345 mg/kg bw/day (nominal)
Dose / conc.:
1 600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 to 26 mice/group
Control animals:
yes, concurrent vehicle
not specified
Details on study design:
Groups of 22 to 26 (see table 1 in "Any other information on materials and methods incl. tables") pregnant albino CD-1 mice were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. One group of mice was treated with the vehicle, at a level equivalent to the group receiving the highest dose, for the negative control group and one group was treated with 150 mg/kg bw Aspirin as a positive control group. Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. All animals were observed daily for appearance, behavior, food consumption and weight.
On day 17, all dams were subjected to caesarean section and the number of implantation sites, resorption sites, number of corpora lutea, number of live and dead foetuses and sex were recorded. The body weight of the live pups was recorded. The urinogenital tract of each dam was examined in detail for anatomical normality. All foetuses were examined for the presence of external abnormalities. One-third of the foetuses of each litter were examined for visceral abnormalities using the Wilson technique. Colored photographs were taken of all grossly abnormal fetuses. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily - All animals were observed daily for appearance and behavior with particular attention to food consumption and weight.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 11, 15, and 17 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: yes
- Sacrifice on gestation day # 17
- Organs examined: uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations sites: Yes
- Number of resorptions: Yes
- Other : number of abortion, number of live litters/fetuses, number of dead fetuses, average fetus weight, sex ratio
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: Yes: No specified
Statistics:
not specified
Indices:
not specified
Historical control data:
not specified
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
The authors indicated that the administration of up to 1600 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on maternal survival.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect of the treatment with the test substance was observed on the body weight gain of the dams as compared to the control group (see Table 5 in "Any other information on results incl. tables).
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Details on results:
The authors concluded that Ferric sodium pyrophosphate had no clearly discernible maternal toxicity at dose levels up to 1600 mg/kg bw in mice.
Number of abortions:
no effects observed
Description (incidence and severity):
No abortion occured in the treated dams whatever the dose level used as compared to controls (see Table 2 in "Any other information on results incl. tables).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the average number of implantation sites/dam whatever the dose used. The average number of implantation sites/dam were 12.2, 11.3, 11.8, 10.8 and 12 for the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley. (see Table 2 in "Any other information on results incl. tables).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more sites resorbed were : 5, 7, 7, 7 and 10 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley.
The number of dams with all sites resorbed were : 0, 0, 1, 0 and 0 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley. (see Table 2 in "Any other information on
results incl. tables).
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more dead fetuses was 4, 0, 3, 1 and 4 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley. The number of dams with all dead fetuses was 4, 0, 3, 1 and 3 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the number of dams surviving at term (see Table 2 in "Any other information on results incl. tables).
Other effects:
no effects observed
Description (incidence and severity):
Further, there were no effect of the treatment on the total number of corpora lutea (see Table 2 in "Any other information on results incl. tables).

Details on maternal toxic effects:
The authors concluded that Ferric sodium pyrophosphate showed no maternal toxicity or teratogenic effect at dose levels up to 1600 mg/kg bw in mice.
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effect
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the average fetus weight (average fetus weight (in g) were 0.92, 0.95, 0.90, 0.92 and 0.94 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no apparent reduction in number of live fetuses. The average number of live fetuses/dam were 11.6, 10.7, 10.9, 10.3 and 11.0 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No apparent abnormal change in sex ratio was observed whatever the dose level group considered: the sex ratio were 0.91, 0.92, 0.84, 1.12 and 1.20 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No grossly abnormal fetuses were observed in the study.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in skeletal tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 3 in "Any other information on results incl. tables).
Visceral malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in soft tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 4 in "Any other information on results incl. tables).
Details on embryotoxic / teratogenic effects:
The authors concluded that Ferric sodium pyrophosphate showed no teratogenic effect at dose levels up to 1600 mg/kg bw in mice.
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Number of mated and pregnant dams /group

 

 

Total

Surviving at term

Material

Dose (mg/kg)**

Mated

Pregnant

Total

Pregnant1

Sham (vehicle)

0.0

26

25

26

25

Aspirin*

150.0

26

24

26

24

 

 

Ferric sodium pyrophosphate

16.0

23

19

23

19

74.3

22

21

22

21

345.0

24

21

24

21

1600.0

24

20

24

20

1: includes all dams examined at term, * positive control, ** administered as a water suspension (10 ml/kg bw)

 

Table 2: Reproduction data

 

Negative control

Positive control (Aspirin)

 

Ferric sodium pyrophosphate

Dose (mg/kg) :

0.0

 

150 mg/kg

16.0

74.3

345.0

1600.0

Pregnancies

Total No.

Died or aborted (before Day 17)

To term (on day 17)

 

25

0

25

 

24

0

24

 

 

19

0

19

 

21

0

21

 

21

0

21

 

20

0

20

Corpora Lutea

Total No.

Average/dam mated

 

327

12.6

 

294

11.3

 

237

10.3

 

265

12.0

 

249

10.4

 

253

10.5

Live litters

Total No.*

 

25

 

24

 

19

 

20

 

21

 

20

Implant Sites

Total No.

Average/dam mated*

 

304

12.2

 

276

11.5

 

214

11.3

 

248

11.8

 

227

10.8

 

239

12.0

Resorptions

Total No.*

Dams with 1 or more sites resorbed

Dams with all sites resorbed

Per cent partial resorptions

Per cent complete resorptions

 

9

5

--

20.0

--

 

19

12

--

50.0

--

 

11

7

--

36.8

--

 

17

7

1

33.3

4.76

 

10

7

--

33.3

--

 

15

10

--

50.0

--

Live fetuses

Total No.

Average/dam*

Sex ratio (M/F)

 

291

11.6

0.91

 

253

10.5

0.89

 

203

10.7

0.92

 

228

10.9

0.84

 

216

10.3

1.12

 

220

11.0

1.20

Dead fetuses

Total No.*

Dams with 1 or more dead

Dams with all dead

Per cent partial dead

Per cent all dead

 

4

4

--

16.0

--

 

4

4

--

16.7

--

 

--

--

--

--

--

 

3

3

--

14.3

--

 

1

1

--

4.76

--

 

4

3

--

15.0

--

Average fetus weight (g)

0.92

0.93

0.95

0.90

0.92

0.94

*Includes only those dams examined at term

 

Table 3: Summary of skeletal findings*

 

Negative control

Positive control (Aspirin)

 

Ferric sodium pyrophosphate

Dose (mg/kg) :

0.0

 

150 mg/kg

16.0

74.3

345.0

1600.0

Live fetuses examined (at term)

201/25

175/24

141/19

159/20

151/21

153/20

Sternebrae

Incomplete oss.

Scrambled

Bipartite

Fused

Extra

Missing

Other

 

38/14

 

 

 

 

11/7

 

54/18

 

1/1

 

 

24/11

 

38/16

 

3/1

 

 

12/4

 

22/9

 

2/1

 

 

21/4

 

42/13

 

1/1

 

 

13/6

 

25/8

 

1/1

 

 

9/4

Ribs

Incomplete oss.

Fuse/split

Wavy

Less than 12

Less than 13

Other

 

 

 

 

 

45/14

 

 

 

 

 

60/17

 

 

 

 

 

48/15

 

 

 

1/1

 

24/12

 

 

1/1

 

 

45/15

 

 

 

 

 

32/15

Vertebrae

Incomplete oss.

Scrambled

Fused

Extra ctrs. oss.

Scoliosis

Tail defects

Other

 

2/2

 

4/3

 

 

 

4/4

 

10/2

 

2/2

1/1

 

1/1

Skull

Incomplete closure

Missing

Craniostosis

Other

 

 

 

 

 

 

 

 

Extremities

Incomplete oss.

Missing

Extra

 

2/2

 

5/3

 

2/2

 

8/1

 

1/1

 

 

Miscellaneous

Hyoid, missing

Hyoid, reduced

 

28/13

14/11

 

37/13

16/11

 

20/9

12/7

 

38/9

11/9

 

26/12

16/9

 

23/10

12/6

*Numerator = Number of fetuses affected; Denominator = Number of litters affected

 

Table 4: Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam number

Number of pups

Description

Aspirin (Positive control)

150.0

25442

25446

1

1

Encephalomeningocele

Cleft palate; Exophthalmos;

Umbilical hernia

Ferric sodium pyrophosphate

74.3

25615

1

Umbilical hernia

 

Table 5: Average body weight of pregnant dams

 

 

Day

Material

Dose Level (mg/kg)**

0

6

11

15

17**

 

 

Average body weight (g)

Sham (vehicle)

0.0

31

34

38

46

53 (25)

Aspirin*

150.0

30

33

37

45

52 (24)

 

 

Ferric sodium pyrophosphate

16.0

29

33

37

45

51 (19)

74.3

31

34

40

46

51 (21)

345.0

32

35

38

46

52 (21)

1600.0

28

31

35

42

48 (20)

* Positive control, ** Number of surviving dams in parentheses

Conclusions:
The authors concluded that Ferric sodium pyrophosphate had no maternal toxicity or teratogenic effect at dose levels up to 1600 mg/kg bw in mice. In the experimental conditions of the study, the NOEL for maternal toxicity and teratogenic effect can be established for both, at >= 1600 mg/kg bw/d (highest dose tested).
Executive summary:

Groups each of approximately 24 pregnant albino CD-1 mice were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. On day 17, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities

The authors concluded that the administration of up to 1600 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Endpoint:
developmental toxicity
Remarks:
Teratogenicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Publicly available FDA research laboratory study where no study guideline is mentioned but the method used is comparable to an OECD 414. However, methods and discussion of the resuts are reported briefly.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Groups each of approximately 24 pregnant albino rat (Wistar derived stock) were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.
GLP compliance:
no
Remarks:
The study was performed before implementation of GLP requirements.
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: not specified
- Age at study initiation: not specified
- Weight at study initiation: From 210 to 221 g at day 0 of the study
- Fasting period before study: not specified
- Housing: individually housed in mesh bottom cages
- Diet (e.g. ad libitum): free acces to food
- Water (e.g. ad libitum): free acces to fresh tap water.
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): daily controlled, from 74 to 81 °F
- Humidity (%): daily controlled, from 50 to 71 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified
IN-LIFE DATES: not specified
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Administration of the test substance as a water suspension (10 ml/kg bw).
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Cohoused. Virgin adult female albino rat (Wistar derived stock) were mated with young adult males. Observation of the vaginal sperm plug was considered Day 0 of gestation. One male was not permitted to impregnate more than one female per group.
Duration of treatment / exposure:
From day 6 through day 15 of gestation.
Frequency of treatment:
daily
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control: vehicle (water)
Dose / conc.:
16 mg/kg bw/day (nominal)
Dose / conc.:
74.3 mg/kg bw/day (nominal)
Dose / conc.:
345 mg/kg bw/day (nominal)
Dose / conc.:
1 600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 to 29 rat/group
Control animals:
not specified
Details on study design:
Groups of 22 to 29 (see table 1 in "Any other information on materials and methods incl. tables") pregnant albino rats (Wistar derived stock) were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. One group of rats was treated with the vehicle, at a level equivalent to the group receiving the highest dose, for the negative control group and one group was treated with 250 mg/kg bw Aspirin as a positive control group. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. All animals were observed daily for appearance, behavior, food consumption and weight.
On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites, number of corpora lutea, number of live and dead foetuses and sex were recorded. The body weight of the live pups was recorded. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined grossly for the presence of external abnormalities. One third of the foetuses of each litter were examined for visceral abnormalities using the Wilson technique. Colored photographs were taken of all grossly abnormal fetuses. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily - All animals were observed daily for appearance and behavior with particular attention to food consumption and weight.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 11, 15, and 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: yes
- Sacrifice on gestation day # 20
- Organs examined: uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations sites: Yes
- Number of resorptions: Yes
- Other : number of abortion, number of live litters/fetuses, number of dead fetuses, average fetus weight, sex ratio
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: Yes: No specified
Statistics:
not specified
Indices:
not specified
Historical control data:
not specified
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
The authors indicated that the administration of up to 1600 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on maternal survival.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect of the treatment with the test substance was observed on the body weight gain of the dams as compared to the control group (see Table 5 in "Any other information on results incl. tables).
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Details on results:
The authors concluded that Ferric sodium pyrophosphate induced no clearly discernible maternal toxicity in pregnant rats at dose levels up to 1600 mg/kg bw.
Number of abortions:
no effects observed
Description (incidence and severity):
No abortion occured in the treated dams whatever the dose level used as compared to controls (seeTable 2 in "Any other information on results incl. tables).
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the number of implantation sites whatever the dose used. The average number of implantation sites/dam were 10.2, 10.8, 12.4, 11.6 and 11.9 for the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley. (see Table 2 in "Any other information on results incl. tables).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more sites resorbed were : 1, 0, 0, 0 and 0 in the dose level groups 0 (vehicle),16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley.
The number of dams with all sites resorbed were : 0, 0, 0, 0 and 0 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley. (see Table 2 in "Any other information on results incl. tables).
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of dams with 1 or more dead fetuses or with all dead fetuses was 0 in control and all dose levels groups (see Table 2 in "Any other information on results incl. tables).
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the number of dams surviving at term (see Table 1 in "Any other information on results incl. tables).
Other effects:
no effects observed
Description (incidence and severity):
Further, there were no effect of the treatment on the total number of corpora lutea (see Table 2 in "Any other information on results incl. tables).
Details on maternal toxic effects:
The authors concluded that Ferric sodium pyrophosphate had no maternal toxicity or teratogenic effect at dose levels up to 1600 mg/kg bw in rats.
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no effect
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The treatment with the test substance had no effect on the average fetus weight (average fetus weight (in g) were 3.95, 4.11, 4.12, 3.90 and 4.11 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no apparent reduction in number of live fetuses. The average number of live fetuses/dam were 10.1, 10.8, 12.4, 11.6 and 11.9 in the dose level groups 0 (vehicle), 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No apparent change in sex ratio was observed whatever the dose level group considered: the sex ratio were 0.94, 1.16, 1.03, 1.17 and 1.07 in the dose level groups 0 (vehicle) 16.0, 74.3, 345.0 and 1600 mg/kg bw/d, respectiveley (see Table 2 in "Any other information on results incl. tables).
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The authors reported that none grossly abnormal fetus were observed in control and treated groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in skeletal tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 3 in "Any other information on results incl. tables).
Visceral malformations:
no effects observed
Description (incidence and severity):
The number of abnormalities seen in soft tissues of the test groups did not differ from the number occurring spontaneously in the negative (sham treated) controls (see Table 4 in "Any other information on results incl. tables).
Details on embryotoxic / teratogenic effects:
The authors concluded that Ferric sodium pyrophosphate had no maternal toxicity or teratogenic effect at dose levels up to 1600 mg/kg bw in rats.
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Number of mated and pregnant dams /group

 

 

Total

Surviving at term

Material

Dose (mg/kg)**

Mated

Pregnant

Total

Pregnant1

Sham (vehicle)

0.0

25

19

25

19

Aspirin*

250.0

24

19

24

19

 

 

Ferric sodium pyrophosphate

16.0

29

21

29

21

74.3

22

20

22

20

345.0

24

20

24

20

1600.0

22

20

22

20

1: includes all dams examined at term, * positive control, ** administered as a water suspension

Table 2: Reproduction data

 

Negative control

Positive control (Aspirin)

 

Ferric sodium pyrophosphate

Dose (mg/kg) :

0.0

 

250 mg/kg

16.0

74.3

345.0

1600.0

Pregnancies

Total No.

Died or aborted (before Day 20)

To term (on day 20)

 

19

0

19

 

19

0

19

 

21

0

21

 

20

0

20

 

20

0

20

 

20

0

20

Corpora Lutea

Total No.

Average/dam mated

 

225

9.00

 

222

9.25

 

262

9.03

 

268

12.2

 

251

10.5

 

257

11.7

Live litters

Total No.*

 

19

 

17

 

21

 

20

 

20

 

20

Implant Sites

Total No.

Average/dam mated*

 

194

10.2

 

202

10.6

 

227

10.8

 

247

12.4

 

232

11.6

 

238

11.9

Resorptions

Total No.*

Dams with 1 or more sites resorbed

Dams with all sites resorbed

Per cent partial resorptions

Per cent complete resorptions

 

2

1

--

5.26

--

 

27

5

2

26.3

10.5

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

Live fetuses

Total No.

Average/dam*

Sex ratio (M/F)

 

192

10.1

0.94

 

175

9.21

0.94

 

227

10.8

1.16

 

247

12.4

1.03

 

232

11.6

1.17

 

238

11.9

1.07

Dead fetuses

Total No.*

Dams with 1 or more dead

Dams with all dead

Per cent partial dead

Per cent all dead

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

 

--

--

--

--

--

Average fetus weight (g)

3.95

2.49

4.11

4.12

3.90

4.11

*Includes only those dams examined at term

 

 

Table 3: Summary of skeletal findings*

 

Negative control

Positive control (Aspirin)

 

Ferric sodium pyrophosphate

Dose (mg/kg) :

0.0

 

250 mg/kg

16.0

74.3

345.0

1600.0

Live fetuses examined (at term)

135/19

123/17

157/21

174/20

160/20

166/20

Sternebrae

Incomplete oss.

Scrambled

Bipartite

Fused

Extra

Missing

Other

 

43/14

 

 

 

1/1

26/9

 

66/16

 

13/7

 

 

91/16

 

36/12

 

 

 

 

11/7

 

42/12

 

 

 

 

10/5

 

32/13

 

 

 

 

 

 

44/10

 

 

 

 

 

Ribs

Incomplete oss.

Fuse/split

Wavy

Less than 12

Less than 13

Other

 

 

 

19/10

 

3/1

 

10/2

14/5

40/13

2/1

95/16

 

 

 

28/10

 

 

1/1

9/6

 

7/4

 

 

 

2/2

 

 

 

9/6

Vertebrae

Incomplete oss.

Scrambled

Fused

Extra ctrs. oss.

Scoliosis

Tail defects

Other

 

9/2

 

70/17

 

1/1

 

 

4/3

 

6/3

 

4/3

 

13/6

Skull

Incomplete closure

Missing

Craniostosis

Other

 

17/8

 

56/17

5/3

 

22/11

 

10/7

 

20/10

 

24/10

Extremities

Incomplete oss.

Missing

Extra

 

3/1

 

8/3

 

 

 

 

 

 

 

 

Miscellaneous

Hyoid, missing

Hyoid, reduced

 

19/8

15/9

 

76/17

7/6

 

24/8

10/7

 

24/10

24/10

 

31/11

20/12

 

11/5

33/11

*Numerator = Number of fetuses affected; Denominator = Number of litters affected

 

Table 4: Summary of soft tissue abnormalities

Material

Dose level (mg/kg)

Dam number

Number of pups

Description

Aspirin (Positive control)

250.0

45439

45442

45452

1

1

1

Hydrocephalus

Gastroschisis

Encephalomyelocele; Umbilical hernia

Ferric sodium pyrophosphate

1600.0

45676

1

Hydrocephalus

 

Table 5: Average body weight of pregnant dams

 

 

Day

Material

Dose Level (mg/kg)**

0

6

11

15

20**

 

 

Average body weight (g)

Sham (vehicle)

0.0

211

226

246

265

326 (19)

Aspirin*

250.0

217

234

241

258

307 (19)

 

 

Ferric sodium pyrophosphate

16.0

212

230

248

268

330 (21)

74.3

221

238

258

282

357 (20)

345.0

211

229

244

266

332 (20)

1600.0

210

230

248

271

343 (20)

* Positive control, ** Number of surviving dams in parentheses

Conclusions:
The authors concluded that Ferric sodium pyrophosphate showed no maternal toxicity or teratogenic effect at dose levels up to 1600 mg/kg bw in rats. In the experimental conditions of the study, the NOEL for maternal toxicity and teratogenic effect can be established for both, at >= 1600 mg/kg bw/d (highest dose tested).
Executive summary:

Groups each of 22 to 29 pregnant albino rat (Wistar derived stock) were dosed by oral intubation with the test substance from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining  two-thirds for skeletal abnormalities.

The authors concluded that the administration of up to 1600 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of the study was to determine the effects in pregnant rats of the cation Fe3+ on the toxicity and teratogenicity of mercury or cadmium-trisodium nitrilotriacetate (Na3 NTA) mixtures and to compare the effects of a natural chelator, trisodium citrate dihydrate with those of Na3NTA on the induction of methyl mercury or cadmium toxicity and teratogenicity.
In this study, ferric trichloride (FeCl3) was administered alone or in combination with other chemicals to compare the effects of the different chemicals on the maternal toxicity (body weight gain, food and water consumption, hematology, uterine content and histopathology) and on the development of the fetuses and the incidence of skeletal and visceral malformations.
Groups of 20 pregnant female rats were treated via drinking water with either 0 (controls) or 7.8 mg/kg/day of ferric trichloride alone or in combination with the other substances from day 6 to day 15.
Thus, this study allows some conclusions to be drawn on the effects of FeCl3, a soluble form of iron, on maternal toxicity and on the developmental and teratogenicity on the rat pups.



GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Charles River CD
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: not specified
- Weight at study initiation: about 250 g
- Fasting period before study: not specified
- Housing: individually in stainless steel cages randomly arranged on the racks
- Diet (e.g. ad libitum): purified diet, at libitum
- Water (e.g. ad libitum): distilled and deionizd drinking water at libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 1 °C
- Humidity (%): 50 +/- 5 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12hr light-dark cycle

IN-LIFE DATES: From: To: not specified
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration was checked. The intended level was 7 mg/kg bw/d. The ingested level was found to be 7.8 mg/kg bw/d
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: No data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no details in the publication

After an orientation period, daily vaginal smears were made and a male rat was placed with each female just preceding the second estrus
Duration of treatment / exposure:
gestation day 6 to 15
Frequency of treatment:
continuous in drinking water
Duration of test:
16 days
Dose / conc.:
7.8 mg/kg bw/day (actual dose received)
Remarks:
ingested levels of Ferric trichloride
No. of animals per sex per dose:
20 females per group
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT:
- Time schedule for examinations: every 3 days

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption : measured daily

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 21
- Histopathological analysis of at least liver and kidneys in dams.

OTHER:
- Standard hemogram was done on blood obtained from the dams during laparotomies
- Metal content in tissues (from sections of liver, kidney and brain in 5 dams/group) using atomic absorption spectrophotometry.

The authors said that the procedures described in their previous study (Nolen et al., 1972) were followed to determine the effects of these materials on reproduction and embryogeny.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of pregnant females
- Number of corpora lutea
- Number of implantation
- Number of litters
- Average number of resorptions
- Number of live fetuses
Fetal examinations:
- Weights of fetuses
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Metal contents in whole fetuses from 5 rats per group were frozen and assayed for metals using atomic absorption spectrophotometry.
Statistics:
The continuous data, such as the numbers of fetuses per litter, weights, feed consumption, etc., were analyzed according to dams or litters using the analysis of variance (Snedecor, 1946) and partitioned by the method described by Scheffe (1952). The discrete data, such as the number of dead or deformed fetuses and the incidence of certain prominent soft-tissue defects and skeletal variations were analyzed by chi-square analysis (Snedecor, 1946). In addition, the total incidences of soft-tissue defects and skeletal variations were tested by the Wilcoxon Rank Sum Test (Siegal, 1956).
Historical control data:
No data
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No mortality was reported in the animal group treated with FeCl3.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
While the administration of the test materials in the drinking water caused the rats to change slighty their water consuption, no significant effect of FeCl3 was observed on the body weight gain of the treated dams as compared to controls (see table 1 below in section "Any information on results including tables").
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant effect of FeCl3 was observed on the daily feed consumption of the dams as compared to controls (see table 1 below in section "Any information on results including tables").
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No significant effect of FeCl3 was observed on the daily water consumption of the treated dams as compared to controls (see table 1 below in section "Any information on results including tables").
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
The authors concluded that there were no significant difference in the numbers of white blood cells, red blood cells or the amount of haemoglobin caused by the treatments in the FeCl3 group as compared to the control animals.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
While some lesions were reported in liver and kidney in the animal groups treated with the other chemicals tested in the study, no lesion in these organs were reported by the authors regarding dams treated with FeCl3. The authors also reported that no pathology was seen in the other organs attributable to the experimental materials.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No significant difference in the iron content in maternal liver and kidney was observed between the control and the FeCl3 treated groups (see table 5 below in section "Any information on results including tables").
Number of abortions:
no effects observed
Description (incidence and severity):
The authors said that the number of pregnant females varied within the groups of treatment but there were no significant differences in the average number of either corpora lutea or implantations. No significant difference were noted in the number of litters between the controls and FeCl3 treated groups (17 versus 16, respectively) (see table 2 below in section "Any information on results including tables".
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant difference were noted in the average number of resorptions between the controls and FeCl3 treated groups (0.9 versus 0.5, respectively) (see table 2 below in section "Any information on results including tables".
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
The authors said that there were few dead fetuses, and the number was not significantly affected by the treatments.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No significant difference was noted in the number of pregnant between the controls and FeCl3 treated groups (17 versus 16, respectively) (see table 2 below in section "Any information on results including tables".
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No maternal toxicity effect.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No significant difference was noted in the weight of male and females fetuses between the controls and FeCl3 treated groups (5.4 for males and 5.0 for females versus 5.3 for male and 5.0 for female, respectively) (see table 2 below in section "Any information on results including tables".
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No significant difference were noted in the average number of live fetuses between the controls and FeCl3 treated groups (12.1 versus 12.6, respectively) (see table 2 below in section "Any information on results including tables".
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No significant difference were noted in the average number of live fetuses and in the weight of male and females fetuses between the controls and FeCl3 treated groups (12.1 versus 12.6, respectively) (see table 2 below in section "Any information on results including tables".
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
no effects observed
Description (incidence and severity):
No significant effect of FeCl3 was observed on the percentage of defective fetuses/litter and frequency of skeletal abnormalities as compared to controls. The precentage of % of defective fetuses/litter in control and FeCl3 treated group were 23 % and 25.6 %, respectively (see table 4 below in section "Any information on results including tables".
Visceral malformations:
no effects observed
Description (incidence and severity):
No significant effect of FeCl3 was observed on the percentage of defective tissues and frequency of specific soft tissue abnormalities as compared to controls (see table 3 below in section "Any information on results including tables". The precentage of % of defective fetuses/litter in control and FeCl3 treated group were 32.5 % and 27.6 %, respectively.
Other effects:
no effects observed
Description (incidence and severity):
No significant difference in the iron content in the fetuses was observed between the control and the FeCl3 treated groups (see table 5 below in section "Any information on results including tables").
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed on pups
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no


Table 1: Average gain in body weight, feed consumption and water consumption of rat during pregnancy

Analysed parameters

Control (distilled water)

FeCl3 (7.8 mg/kg bw/d)

Gain in body weight (g)

Days 0-6

31

29

Days 6-15

60

57

Days16-21

86

70

Daily feed consumption (g)

Days 6-15

22

21

Days16-21

22

21

Daily water consumption (g)

Days 6-15

36

32

Days16-21

37

37

 

No significant effect of FeCl3 was observed in dams on body weight gain, the daily feed consumptions and the daily water consumption as compared to controls.

 

Table 2: Effect of ferric trichloride (FeCl3) on embryo and fetuses in rats

Analysed parameters

Control (distilled water)

FeCl3 (7.8 mg/kg bw/d)

 Number of litters

 17

 16

 Average number of resorptions

 0.9

 0.5

 Average number of live fetuses

 12.1

 12.6

 Weight of fetuses (males)

 5.4

 5.3

 Weight of fetuses (females)

 5.0

 5.0

 

No significant effect of FeCl3 was observed on the number of litters, average number of resorptions, average number of live fetuses and weight pups as compared to controls.

 

Table 3: Effect of ferric trichloride on fetal soft tissue abnormalities in rats

Analysed parameters

Control (distilled water)

FeCl3 (7.8 mg/kg bw/d)

Number of fetuses examined  

 

 132

 128

% defective fetuses/litter

 32.5

 27.6

Number of fetuses with some specific soft tissue abnormalities

 Hydronephrosis

 7

 16

 Hydroureter

 40

 35

 Bladder defects

 6

 4

 Cleft palate

 0

 0

 Undescended testicles

 2

 

No significant effect of FeCl3 was observed on the percentage of defective fetuses/litter and frequency of specific soft tissue abnormalities as compared to controls.

 

 

Table 4: Effect of ferric trichloride on fetal skeletal abnormalities and variations in rats

Analysed parameters

Control (distilled water)

FeCl3 (7.8 mg/kg bw/d)

Number of fetuses examined  

 

68

67

% defective fetuses/litter

23

25.6

Number of fetuses with some specific skeletal variations

 Hypoplastic sternebrae

 7

16

 Missing 5th sternebrae

 0

 0

 Hypoplastic 10th thoracic vertebra

 1

 2

 

No significant effect of FeCl3 was observed on the percentage of defective fetuses/litter and frequency of skeletal abnormalities as compared to controls.


Table 5: Iron concentrations in maternal livers and kidney in dams and in fetus (ppm in wet tissue +/-SD)

Analysed parameters

Control (distilled water)

FeCl3 (7.8 mg/kg bw/d)

Liver

53.8 +/- 11.8

58.0 +/- 14.9

Kidney

80.3 +/- 17.8

67.3 +/- 9.5

Fetus

44.6 +/- 27.4

37.8 +/- 15.7

 

No significant difference between iron content in maternal liver and kidney and in fetuses was observed between the control and FeCl3 groups.

Conclusions:
In this study, performed according to a method that meets generally accepted scientific principles for teratogenicity study and which was performed before GLP implementation, no maternal toxic effect in dams or developmental and teratogenic effect in the fetuses were observed after administration via drinking water of 7.8 mg/kg bw/day of ferric trichloride. Under the experimental conditions of this study, the NOEL of FeCl3 for maternal toxicity and for developmental and teratogenicity for the pups was therefore set as > 7,8 mg/kg bw day.
Executive summary:

The objective of the study was to determine the effects in pregnant rats of the cation Fe3+ on the toxicity and teratogenicity of mercury or cadmium-trisodium nitrilotriacetate (Na3 NTA) mixtures. The study was performed before GLP implementation according to a method that meets generally accepted scientific principles for teratogenicity study.

In this study, groups of 20 pregnant rats were treated via the drinking water with 0 (vehicle, distilled water), 7,8 mg/kg bw/day ferric trichloride (FeCl3) alone or in combination with other chemicals from day 6 to day 15 of organogenesis. Then, the dams were sacrificed and examined at day 21.

Body weight gains, food and water consumptions, hematology, histopathology, metal content in liver, kidneys and brain, number of pregnancies, number of litters and live fetuses as well as uterine content (number of corpora lutea, number of implantation sites, average number of resorptions) were analysed in dams.

Pups were examined for fetus weights, soft tissue and skeletal abnormalities. Metal contents was also measured in whole fetuses.

No mortality was reported in the animal group treated with FeCl3. No significant effect of FeCl3 was observed in dams on body weight gain, the daily feed consumptions and the daily water consumption as compared to controls. The authors claimed that no significant difference in the numbers of white blood cells, red blood cells or the amount of haemoglobin caused by the treatments in the FeCl3 group were observed as compared to the control animals. In addition, no lesion in these organs were reported by the authors regarding dams treated with FeCl3.

There were no significant differences in the average number of either corpora lutea or implantations and no significant difference were noted in the number of litters between the controls and FeCl3 treated groups. The number of dead fetuses was not significantly affected by the treatment. FeCl3 had no significant effect on the average number of resorptions, average number of live fetuses and weight pups as compared to controls. Further, no significant effect of this substance was also observed on percentage of defective fetuses/litter or frequency of specific soft tissue abnormalities or skeletal abnormalities as compared to controls.

No significant difference between iron content in maternal liver and kidney and in fetuses was observed between the control and FeCl3 groups.

According to these results, the NOEL of FeCl3 for maternal toxicity and for developmental and teratogenicity was therefore set as > 7,8 mg/kg bw day.

Endpoint:
developmental toxicity
Remarks:
Pre and post natal informations from the 2-generations reproduction study with an structural analogue
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from cerium and iron oxide isosterate, a substance that presents structural similarities with iron oxide isostearate, was used to cover this endpoint. See the Read-across justification document (Justification for analogue approach) attached in IUCLID Section 13.2 for the justification of the read-across.
See also the original letters from the French Competent Authorities requiring the read across to be done with Cerium and iron oxide isostearate substances, attached in Section 13.2 as well (French CA testing program July 2005, and French CA testing program Sep 2007).
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No maternal toxicity observed
Remarks on result:
other:
Remarks:
For both, F0 and F1 dams
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed on pre and post natal development in F1 and F2 pups and no external abnormalities
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
In a 2-generation reproduction study (OECD TG 416, K1) performed with cerium and iron oxide isostearate, a structural analogue of iron oxide isostearate, no effect on maternal toxicity was observed in F0 and F1 dams. Further, no effect was observed on pre and post natal development and no external malformation or macroscopic findings were observed in F1 et F2 pups whatever the dose levels used. Therefore, the No Observed Adverse Effect Level for maternal toxicity for both, F0 and F1 dams was found to be 1000 mg/kg bw/day and the No Observed Effect Level (NOEL) for pre- and post-natal development of F1 and F2 generations was considered to be 1000 mg/kg/day in this study.
Therefore, the same is assumed for iron oxide isostearate.
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, a structural analogue of 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 considered to be 1000 mg/kg/day. The NOAEL for maternal toxicity for both F0 and F1 dams was therefore found to be 1000 mg/kg bw/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/da

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

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, same results and conclusions are expected for iron oxide isostearate.

 

 

Endpoint:
developmental toxicity
Remarks:
Screening for reproductive/developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from cerium and iron oxide isosterate, a substance that presents structural similarities with iron oxide isostearate, was used to cover this endpoint. See the Read-across justification document (Justification for analogue approach) attached in IUCLID Section 13.2 for the justification of the read-across.
See also the original letters from the French Competent Authorities requiring the read across to be done with Cerium and iron oxide isostearate substances, attached in Section 13.2 as well (French CA testing program July 2005, and French CA testing program Sep 2007)
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No relevant effects up to highest dose tested
Abnormalities:
no effects observed
Dose descriptor:
NOEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
In a screening for reproductive /developmental toxity study (OECD TG 421, not GLP, K2) performed with cerium and iron oxide isostearate, a structural analogue of iron oxide isostearate, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day. Pups showed no effects of treatment on survival and no gross external abnormalities. However the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.
Based on the results of this study, the NOEL for maternal toxicity was considered to be 1000 mg/kg bw/day and the NOEL for pre and post natal development was considered to be 450 mg/kg bw/day.
Therefore, the same results is assumed for iron oxide isostearate.
Executive summary:

In a Reproduction/developmental toxicity screening test, the potential for reproductive or developmental toxicity of Cerium and iron oxide isostearate, a structural analogue of iron oxide isostearate, was evaluated following daily oral administration by gavage to 10-week old Sprague-Dawley rats (10/sex) from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0, 450 or 1000 mg/kg/day in corn oil. The study was performed similarly to an OECD 421 and following the principle of GLP (but not audited by Quality assurance unit) and was therefore quoted K2 acccording to Klimisch criteria. This study was used as a preliminary study for a 2 -generation study.

No unscheduled deaths or treatment-related clinical signs occurred during the study. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.

Pups showed no effects of treatment on survival.

Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg/day (-15% in the males and -12% in the females, not statistically significant). This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded. It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio.There were no treatment-related macroscopic abnormalities in pups.

 

Based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5.

As this study was designed to choose the dose-levels which should be used in a 2-generations study, it is however considered that 1000 mg/kg/day would be a suitable high dose-level for the multigeneration study.

This study was classified as acceptable. It is similar to OECD guideline on reproduction/developmental toxicity screening and is acceptable as a preliminary study to a multigeneration study.

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, similar results are expected for iron oxide isostearate.

 

.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
other: rat and mouse
Quality of whole database:
Several studies performed either with OECD guidelines and GLP or which are comparable to OECD guidelines with acceptable restrictions.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

To assess the potential developmental and teratogenic effects of the substance iron oxide isostearate, a weight of evidence approach was followed.

First, all data from the substance and its structural analogues (see Read-across justification document (Justification for analogue approach) attached in IUCLID Section 13.2) were considered. Thus, the 2 generation reproduction study (OECD 416) and the screening for reproductive/developmental toxicitystudy (OECD 421), performed with the structural analogue cerium and iron oxide isostearate, were considered suitable to assess the pre and post natal developmental effects of iron oxide isostearate.

To assess the potential teratogenic effects of the substance, as no reliable teratogenic study was found on the substance or its analogues, studies on the metallic and fatty acid moieties were considered. Indeed, as for many other metal compounds, the metal cation in the compound is generally considered as responsible for the observed toxicity, and that the severity of the effect (if any) is, in most of the case, related to the extent of exposure to the metal cation. The more toxic metal cation is dissolved into the medium under consideration (e.g., bodily fluids or environmental media), the more bioavailable/bioaccessible the metal will be. Note that other properties of the substance (e.g., particle size and particle size distribution) as well as the characteristics of the receiving compartment (e.g., pH) may also affect bioavailability/bioaccessibility. As no reliable study was identified for nano iron compounds, reliable teratogenicity studies, done with water soluble (iron trichloride, solubility > 700 g/l; ferrous sulfate, solubility > 200 g/l) and water insoluble (ferric sodium pyrophosphate) iron compounds, were thus considered in this strategy of weight of evidence, as a worst case, for the metallic moiety of the substance. Indeed, considering the physico-chemical properties of iron oxide isostearate and the absence of toxicity observed in all the 16 studies performed on the substance or its analogues, the absorption, the distribution and the accumulation of iron oxide isostearate is expected to be really limited by oral, dermal and inhalation routes of exposure (see Toxicokinetics, metabolism and distribution assessment in the IUCLID section 7.1). Therefore, using data on soluble and insoluble forms of iron oxide is thus considered as a worst case approach in this assessment. For the fatty acid moiety, EFSA concluded in 2017 that fatty acids with longer chains (> C12), which are substances that can be used as food additive, are not expected to have safety concern at a quite high level (up to 10 % as seen in a subchronic toxicity study) (see EFSA Scientific opinion: Re-evaluation of fatty acids (E 570) as a food additive, adopted April 4, 2017).

First, in a 2-generation reproduction study scored as validity 1 according to Klimisch criteria (OECD guideline 416, GLP – Spézia F. 2011), four groups of 25 male and 25 female Sprague-Dawley rats received the test item Cerium and iron oxide isostearate, a structural analogue of 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 in the 2 generations.

The animals of the 2 generations were checked at least twice daily for mortality/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. The F0 and F1 females were allowed to litter and rear their progeny until weaning.

Reproductive indices (pre-implantation loss, post-implantation loss, mating index, fertility index, gestation index, number of corpora lutea, number of implantations, and number of pups delivered) were evaluated in both F0 and F1 animals. Offspring viability indices (live birth index, viability index on day 4 post-partum, lactation index on day 21 post-partum, mean litter size, pup sex ratios were also measured in both generations.

After weaning of the F1 and F2 pups, the males and females of the F0 and F1 generation were sacrificed. Sperm analysis (epididymal sperm motility, morphology and count, testicular sperm count, testicular staging) was performed on the first ten males of the control and high-dose groups (groups 1 and 4). A complete macroscopic examination was performed in females, including counting the number of implantation sites 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 in both F0 and F1 parents.

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. The 4 groups of animals received the test item daily under the concentrations and timing described above.

The F1 and F2 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). Further, each F1 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, and at 8 weeks of age, spontaneous locomotor activity was measured.

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.

There were no test item treatment-related mortalities in F0 and 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 F0 (at 1000 mg/kg/day) at and F1 males (at 450 or 1000 mg/kg/day) and F0 and F1 females (at 1000 mg/kg/day) treated at during the pre-mating period and mid-gestation, which correlated with a tendency towards a non statistically significant increase in body weight gains, and in females treated at 450 mg/kg/day at the beginning of the pre-mating period. 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. No effects on spermatogenesis were detected after analysis of epididymidal sperm motility and morphology and count and, after analysis 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 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 highdose F1 females.

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

The F1 generation showed no effects of treatment while pups. There were no test item treatmentrelated clinical signs, no effects on body weight and no differences in physical or reflex development when compared with the controls. Further, there was no treatment-related effect on sexual development, i.e. no treatment-related effects on balanopreputial separation and no test item treatment-related effects on vaginal opening at any dose-level.

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 at external examination in the pups. As no macroscopic lesion was observed in F1 and F2 pups at external examination, no microscopic examination was performed.

As no effect were seen in this study, the No Observed Adverse Effect Level for reproductive performance and systemic toxicity for F0 and F1 generations was considered to be 1000 mg/kg/day. The NOAEL for maternal toxicity for both F0 and F1 dams was therefore considered to be 1000 mg/kg bw/day.

Cerium and iron oxide isostearate did not cause delayed or impaired development in the F1 or F2 generations following treatment of the parents. Further, no test item-related changes in organ weights or macroscopic findings at external examination was found in the pups. 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.

Thus, no classification for pre and post developmental toxicity is warranted based on the absence of relevant effects in this study, according to the criteria of UN/EU GHS.

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, same results and conclusions are assumed for iron oxide isostearate.

Further, in a reproduction/developmental toxicity screening study, performed as a dose-range finding study for the 2-generation study by using similar experimental conditions to an OECD 421 and following the principle of GLP (but not audited by Quality assurance unit and thus, quoted K2 acccording to Klimisch criteria), the potential effects of the same structural analogue cerium and iron oxide isostearate was evaluated (Davies R. 2010,).

 In this study, the rats (10/sex/group) received the test substance daily by gavage from 2 weeks before mating, through mating and, for the females, through gestation until day 5 post partum, at the dose levels of 0, 450 or 1000 mg/kg/day in corn oil. The animals were observed at least twice a day during the treatment period for mortality/morbidity and once a day for clinical signs. The body weights and food consumption were measured once a week until sacrifice for males and once a week until mated and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum in females. The estrous cycle stage was determined from a fresh vaginal lavage, each morning during the mating period, until the females had mated.

Females were allowed to litter normally and rear their progeny until sacrifice of the pups on day 5 post-partum. The numbers of corpora lutea and implantation scars were recorded for females sacrificed on day 5 postpartum or on day 25 post-coitum when delivery did not occurred. For these apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique. The following reproductive and offspring viability 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 live birth index, viability index on day 5 post-partum, mean litter size and pup sex ratios

F1 offspring: were examined for total litter size and number, sex ratio, stillbirths, live births, dead and cannibalized pups, postnatal mortality, presence of gross anomalies, clinical signs (daily), body weight (day 1 and day 5 post-partum). A complete macroscopic post-mortem examination was performed on all animals (females and males). Special attention was paid to the reproductive organs. But no microscopic examination was performed.

No unscheduled deaths or treatment-related clinical signs occurred during the study in the parents. There were no effects of the treatment on body weight, body weight gain or food consumption at any dose level. There were no relevant differences from controls for pairing, mating, fertility and delivery parameters.

Pups showed no effects of treatment on survival. Mean pup body weight gain was lower for males and females from the group treated at 1000 mg/kg/day but the difference was not statistically significant when compared to controls animals. This may be related to the slightly higher number of pups per litter in this group but a relationship to treatment cannot be excluded. It was considered that the test item did not have any effects on pup development in utero, pup survival, clinical signs or sex ratio. There were no treatment-related macroscopic abnormalities in pups.

Overall, based on the experimental conditions of this study, it was considered that the test item did not affect the adult animals after treatment at 450 or 1000 mg/kg/day, however the pups of the animals treated at 1000 mg/kg/day did have a lower mean body weight gain from post-natal days 1 to 5 with no clear conclusion on the cause. However, this effect was not observed in the 2 generations reproduction study reported above.

In a teratogenicity study performed with a method comparable to an OECD guideline no 414, groups of 22 to 28 pregnant albino CD-1 mice were dosed by oral intubation with ferrous sulfate (water soluble) at dose levels of 0 (control, vehicle), 1,6, 7,4, 34, 5 and 160 mg/kg bw/day from day 6 through day 15 of gestation (D.E Bailey, 1974). Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. On day 17, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.

The authors observed that the administration of up to 160 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal and fetal survival, on pups body weight or on sex ratio. No grossly abnormal fetuses were observed in the study and the number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

In the experimental conditions of the study, the NOEL for maternal toxicity and teratogenic effect in mouse can be established for both, at >= 160 mg/kg bw/d (highest dose tested).

Ferrous sulfate was further tested in rats using similar experimental conditions described above (D.E Bailey, 1974). Groups of 21 to 25 pregnant albino rats (wistar derived stock) were dosed by oral intubation with the test substance at dose levels of of 0 (control, vehicle), 2, 9.3, 43.1 and 200 mg/kg bw/day from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormalities. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.

In this study, again, the administration of up to 200 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival on pup body weight or on sex ratio. No grossly abnormal fetuses were seen in the group receiving the test substance. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

In the experimental conditions of the study, the NOEL for maternal toxicity and teratogenic effect in rats can be established for both, at >= 200 mg/kg bw/d (highest dose tested).

The same laboratory tested then the potential teratogenicity effects of ferric sodium pyrophosphate (an insoluble form of iron) with the same method, which is comparable to an OECD guideline no 414 (D.E Bailey, 1975). Groups of 22 to 26 pregnant albino CD-1 mice were dosed by oral intubation with the test substance at dose levels of 0 (control, vehicle), 16, 74.3, 345 and 1600 mg/kg bw/day from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation. On day 17, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormality. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.

The authors concluded that the administration of up to 1600 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal and fetal survival, on pups body weight or on sex ratio. No grossly abnormal fetuses were observed in the study and the number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Under these experimental conditions, it can be concluded that the NOEL for maternal toxicity and teratogenic effect in mice can be established for both, at >= 1600 mg/kg bw/d (highest dose tested) ferric sodium pyrophosphate.

Ferric sodium pyrophosphate was also tested in rats using the same experimental conditions described above (D.E Bailey, 1975). Groups of 22 to 29 pregnant albino rats (wistar derived stock) were dosed by oral intubation with the test substance at dose levels of of 0 (control, vehicle), 16, 74.3, 345 and 1600 mg/kg bw/day from day 6 through day 15 of gestation. Body weights were recorded on days 0, 6, 11, 15 and 20 of gestation. On day 20, all dams were subjected to caesarean section and the number of implantation sites, resorption sites and live and dead foetuses recorded. The body weight of the live pups was also taken. The urinogenital tract of each dam was examined in detail for abnormalities. All foetuses were examined for the presence of external congenital abnormalities. One-third of the foetuses were examined for visceral abnormalities and the remaining two-thirds for skeletal abnormalities.

The administration of up to 1600 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival on pups body weight or on sex ratio. No grossly abnormal fetuses were observed in the study and the number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Similarly, under the experimental conditions of this study, the NOEL for maternal toxicity and teratogenic effect in rats can be established for both, at >= 1600 mg/kg bw/d (highest dose tested).

 

In a study, that was performed before GLP implementation according to a method that meets generally accepted scientific principles for teratogenicity study, the water-soluble ferric trichloride (FeCl3) was administered alone or in combination with other chemicals to study the impact of Fe3+ on the toxicity and teratogenicity of mercury or cadmium compounds and chelators (Nolen et al, 1972). Groups of 20 pregnant female rats were treated via drinking water with either 0 (control, vehicle) or 7.8 mg/kg/day of ferric trichloride alone or in combination with the other substances from day 6 to day 15 of organogenesis. Then, the dams were sacrificed and examined at day 21. Body weight gains, food and water consumptions, hematology, histopathology, metal content in liver, kidneys and brain, number of pregnancies, uterine content, number of litters and live fetuses as well as uterine content (number of corpora lutea, number of implantation sites, and average number of resorptions) were analysed in dams. Pups were examined for development, fetus weights and the incidence of soft tissue and skeletal abnormalities. Metal contents was also measured in whole fetuses.

No mortality was reported in the animal group treated with FeCl3. No significant effect of FeCl3 was observed in dams on body weight gain, daily feed consumptions and daily water consumption as compared to controls. The authors indicated that no significant difference in the numbers of white blood cells, red blood cells or the amount of hemoglobin caused by the treatments in the FeCl3 group were observed as compared to the control animals. In addition, no lesions in organs were reported by the authors regarding dams treated with FeCl3.

There were no significant difference in the average number of either corpora lutea or implantations and no significant difference were noted in the number of litters between the controls and FeCl3 treated groups. The number of dead fetuses was not significantly affected by the treatment. FeCl3 had no significant effect on the average number of resorptions, average number of live fetuses and weight pups as compared to controls. Further, no significant effect of this substance was also observed on percentage of defective fetuses/litter or frequency of specific soft tissue abnormalities or skeletal abnormalities as compared to controls.

No significant difference between iron content in maternal liver and kidney and in fetuses was observed between the control and FeCl3 groups.

According to these results, the NOEL of FeCl3 for maternal toxicity and for developmental and teratogenicity was therefore set as > 7,8 mg/kg bw day.

Further, iron oxide isostearate and its analogues were found to have no toxicological effect in all the 16 studies performed according to OECD guidelines and in compliance with GLP: no systemic effect were found in acute oral and dermal toxicity studies, no skin/eye irritation and no skin sensitization was observed and no systemic and reproductive/developmental effect and no alterations in organs were seen in repeated dose toxicity studies (OECD 407 and 421) and in a reproduction study (OECD 416) performed by oral route at dose levels up to 1000 mg/kg bw/day in which rats were treated with the substance for up to 18/19 weeks. No genotoxic or clastogenic effects were observed in absence and presence of metabolic activation in 5 in vitro studies (OCD 471, 473 and 476).

Taking into consideration all the studies performed on the substance and its structural analogues as well as the studies performed on the metallic moiety of the substance, no systemic toxicity was found, more particularly no effect on the reproductive organs and function and no pre and post natal toxicity and no teratogenic effects were evidenced. This weight of evidence approach is considered sufficient to assess the developmental toxicity/teratogenicity potential of iron oxide isostearate and to conclude that no classification is warranted according to the criteria of UN/EU GHS based on the absence of relevant effect in these studies. Therefore, no further testing is required.

 

References :

- D.E Bailey, 1974. Teratologic evaluation of FDA 71-64 (ferrous sulphate) in mice and rats, studies no 2123 (19) for mice and 2143 (19) for rat. Food and Drug Research Laboratories, Inc., Waverly, N.Y., United States of America. Also reported in JECFA 1993 (IRON - WHO Food Additives – Serie 18 – IPCS INCHEM).

- D.E Bailey, 1975. Teratologic evaluation of FDA 73-82 (ferric sodium pyrophosphate) in mice and rat, studies no 2123 (6) mice and 2143 (6) rat. Food and Drug Research Laboratories, Inc., Waverly, N.Y., United States of America. Also reported in JECFA 1993 (IRON - WHO Food Additives – Serie 18 – IPCS INCHEM).

- Davies R. 2010, study report no 34759 RSR, Reproduction/developmental toxicity screening test by oral route (gavage) in rats, CIT – France.

- Nolen GA et al, 1972. Effects of trisodium nitrilotriacetate, trisodium citrate and a trisodium nitrilotriacetate-ferric chloride mixture on cadmium and methyl mercury toxicity and teratogenesis in rats. Toxicol Appl Pharmacol 23(2):238-250.

- Spézia F. 2011, study report no 34760 RSR, Two-generation reproduction toxicity study by oral route (gavage) in rats, CIT – France.

Justification for classification or non-classification

Taking into consideration all the studies performed on the substance and its structural analogues as well as the studies performed on the metallic moiety of the substance, no systemic toxicity was found, more particularly no effect on the reproductive organs and function and no pre and post natal toxicity and no teratogenic effects were evidenced. This weight of evidence approach is considered sufficient to assess the developmental toxicity/teratogenicity potential of iron oxide isostearate and to conclude that no classification is warranted according to the criteria of UN/EU GHS based on the absence of relevant effect in these studies.

Additional information