Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 December 2009 - 25 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Silicic acid(H2Si2O5), barium salt(1:1), lead-doped
- Substance type: White powder
- Physical state: solid
- Molecular formula BaSi2O5:Pb
- Molecular weight 275.6 (based on theoretical value)
- Analytical purity: 100%
- Purity test date: 20-oct-2010
- Lot/batch No.: NP-800-11-232
- Expiration date of the lot/batch: 16 October 2010 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- pH (1% in water, indicative range) 9.5 – 9.6 (determined at NOTOX)
- Solubility in vehicle (1% Aq. Carboxymethyl cellulose): Not indicated
- Stability in vehicle (1% Aq. Carboxymethyl cellulose): See details on analytical verification of doses or concentrations.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
Animals were supplied by Charles River Deutschland, Sulzfeld, Germany instead of by Charles River Laboratories France, L'Arbresle Cedex, France. A different supplier is considered not to adversely affect the outcome of the study.

Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.

- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight range at start of treatment was 297-301 gr (males) or 195-198 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 – 22.2°C
- Humidity (%): 22 - 60%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests examinations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 09 december 2009 To: 25 January 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% Aqueous carboxymethyl cellulose
Details on exposure:
Method of formulation:Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level.

Storage conditions of formulations: At ambient temperature.

Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.


Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the oestrus cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After 14 days of unsuccessful pairing one female who had not shown evidence of mating was separated from her male.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase, according to a validated method (NOTOX project 492368). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Analyses were based on the presence of barium and lead in the test substance.

Concentrations of Group 2, 3 and 4 formulations were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

The formulation of Group 2 was homogeneous (i.e. coefficient of variation ≤ 10%). However, the coefficient of variation of the Group 4 formulation was outside the target range (i.e. 34%, based on individual accuracies of 72% to 177% for barium and 67 to 168% for lead). The level of accuracy appeared independent on the sampling position (i.e. top to bottom of the container). Additional analysis of the formulation of Group 4 yielded a mean accuracy of 109 and 99% for barium and lead respectively and a coefficient of variation of 17 and 3.9% for barium and lead respectively. Again, the level of accuracy appeared independent on the sampling position. It was considered that the relatively high coefficient of variation occurred due to the inherent variation of the analytical method applied since the results obtained on lead demonstrated that the formulation was homogenous. Therefore, it was concluded that formulations were prepared homogenously.

Initial analysis of Group 2 and 4 formulations after storage at room temperature for at least 6 hours yielded a relative difference of ≤ 10%. With the additional analysis of the Group 4 formulation a relative difference of -15% was obtained based on barium (and -3.5% based on lead). However, since the element barium is stable at room temperature and not volatile it was concluded that this lower value was obtained due to the inherent variation in the analytical method. Therefore, the formulations were concluded to be stable during storage at room temperature for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One Group 2 female and one Group 3 female were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100, 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the results a 10-Day range finding study, dose levels selected for the main study were 30, 100 and 300 mg/kg/day (see attachment).
No clinical signs (indicative of toxicity) occurred at 100 or 300 mg/kg/day. Therefore, clinical observations in the main study were conducted at least immediately after dosing.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily (early morning/late afternoon). Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule:at least immediately after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales.

BODY WEIGHT:
Yes - Time schedule for examinations:Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
Yes. Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes(average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils) Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time

CLINICAL CHEMISTRY:
Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Animals fasted: Yes - How many animals: 5 animals/sex/group
- Parameters checked in table were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity test (recording period: 1-hour for individual animals, using a computerised monitoring system; Pearson Technical Services, Suffolk, Great Britain).

During the motor activity test, males were caged individually and females were caged with their pups.

Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
Parameters examined in all male parental animals:
testis weight, epididymis weight.
In addition, for 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:
- Females which delivered: Lactation Days 5 or 7.
- Surviving females which failed to deliver: post-coitum Day 25 and 26 (females with evidence of mating) or 20 days after the last day of the mating period (females without evidence of mating).
- Males: Following completion of the mating period (a minimum of 28 days of dose administration).
- Spontaneous deaths: As soon as possible after death and always within 24 hours.
- Euthanized in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The number of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis <3>:
Identification marks (not processed) , Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Caecum, Pituitary gland, Cervix, Preputial gland, Clitoral gland, Prostate gland, Colon, Rectum, Coagulation gland, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides <1>, Seminal vesicles, (Eyes with optic nerve (if detectable) and Harderian gland) <1>, Skeletal muscle, (Skin), Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar-, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes <1>, Kidneys, Thymus, (Larynx), Thyroid including parathyroid (if detectable),(Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung (infused with formalin), Urinary bladder, Lymph nodes -mandibular, mesenteric-, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions

- All remaining animals and females which failed to deliver:<2>
Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes <1>, Epididymides <1>, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions, Identification marks(not processed)

<1> Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) and Milli-Ro water and transferred to formalin after fixation for at least 24 hours.
<2 > In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
<3> Recognizable fetuses were examined externally, sexed and preserved in 10% neutral-buffered formalin.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- Selected 5 animals/sex/group:
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate<1>, Liver, Seminal vesicles including coagulating glands<1>, Ovaries, Thyroid including parathyroid <1>, <1> weighed when fixed for at least 24 hours.

- All remaining males:
Epididymides and Testes

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4, and of selected females of Group 3 (due to high mortality incidence in Group 4).
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were killed in extremis.
- The reproductive organs* of all surviving animals that failed to mate, conceive, sire or deliver healthy pups:
Group 1: one male and female that failed to mate.
Group 2: two males and females (females had no offspring)
Group 3: one male of which his mated female was found dead, and one male and female (female had no offspring)
Group 4: two males and females (females had no offspring), three males of which the mated females were found dead/sacrificed.
- All gross lesions of all animals (all dose groups).

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

Inadvertently, a few tissues were not available for histopathology. Reasons for missing a few tissues included that these tissues were not discernable at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in the raw data and pathology report.Sufficient data was available for evaluation.

Inadvertently the uterus of one control female was not stained using the Salewski technique to detect any former implantation sites.This female was assumed to be non-pregnant based on absence of evidence of mating and macroscopically visible implantation sites or corpora lutea. Since this concerned only one control case, this did not adversely affect the overall interpretation of the study results.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-6.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
no
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)

The following additional methods of statistical analysis were used:
The number of implantation sites and corpora lutea was transformed by using 1/x, log x, x2 and √x to obtain a normal distribution. This was followed by ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

No statistical analysis was performed on histopathology findings.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Reproductive indices:
For each group, the following calculations were performed:

Percentage mated females: Number of females mated/Number of females paired x 100

Fertility index: Number of pregnant females/Number of females paired x 100

Conception index: Number of pregnant females/Number of females mated x 100

Gestation index: Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100

Viability index (%): Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY
At 300 mg/kg/day, one male was found dead after 21 days of treatment, and five females were found dead or killed in extremis towards the end of their pregnancy period (i.e. on Days 20 or 21 of the post coitum period). One other female at 300 mg/kg/day was killed in extremis on Day 8 of the post coitum period. At 100 mg/kg/day, a single female was killed in extremis on Day 21 of the post coitum period.
No mortality occurred at 30 mg/kg/day.

CLINICAL SIGNS
Lethargy, flat posture, pale appearance, ptosis, laboured or shallow respiration, piloerection and/or hypothermia were noted among most animals at 100 and 300 mg/kg/day that died spontaneously or were killed in extremis (see Mortality, above).

Incidental findings among surviving animals included salivation, alopecia, scales over several body parts and transient piloerection. These observations occurred within the range of background findings for rats of this age and strain, and were considered to be of no toxicological relevance at the observed incidence.No clinical signs were noted among control animals and males at 30 and 300 mg/kg/day.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all surviving selected animals.The variation in motor activity did not indicate a relation to treatment.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted up to 300 mg/kg/day.

A statistically significant lower absolute body weight of males at 300 mg/kg/day on Day 1 of the mating period, and lower body weight gain over Days 1-8 of the pre-mating period and over Day 8 of the pre-mating period to Day 1 of the mating period was observed. Given the temporary and slight nature of these changes, these were considered to be of no toxicological relevance.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

The (relative) food intake of females at 300 mg/kg/day during lactation appeared lower than controls, but the variation in food intake of these females (only two survived the lactation period) was similar to that observed among control females. The statistically significant higher relative food consumption of females at 300 mg/kg/day over Days 14-17 of the post-coitum period was of a temporary and slight nature. Therefore, these changes were considered to be of no toxicological relevance.

HAEMATOLOGY
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with the test substance.

The statistically significant lower activated partial thromboplastin time (APTT) level of females at 300 mg/kg/day was not considered to represent a change of toxicological relevance since values remained within the range considered normal for rats of this age and strain, and the opposite effect would be expected in case of target organ toxicity.

CLINICAL BIOCHEMISTRY
A statistically significant higher cholesterol and bilirubin value was noted for males at 300 mg/kg/day. One of the two surviving females at 300 mg/kg/day showed a notably high bile acid and inorganic phosphate level. The other surviving female showed a notablyhigher alkaline phosphatase activity (ALP).

The notably higher aspartate aminotransferase activity (ASAT) of one male at 300 mg/kg/day was confined to a single animal of this dose group, and was therefore considered not to be of toxicological relevance. The statistically significant lower creatinine level of males at 30 and 100 mg/kg/day occurred inthe absence of a dose-related trend, and means remained within the range considered normal for rats of this age and strain. No toxicological relevance was therefore ascribed to this change.

MACROSCOPIC EXAMINATION
At 300 mg/kg/day, most animals that were euthanized in extremis or died before their scheduled necropsies showed an enlarged liver and kidneys, autolysis, a gray-white focus on the left lateral lobe of the liver, several reddish foci on the glandular mucosa of the stomach and/or many dark red foci on the lungs. Two surviving males at 300 mg/kg/day showed red/yellowish foci in the stomach.

At 30 and 100 mg/kg/day, no toxicologically relevant macroscopic abnormalities were noted. No macroscopic abnormalities were noted for the single female that was euthanized in extremis at 100 mg/kg/day. Yellowish nodules on the epididymides (uni- or bilateral) were recorded for several males of the control group and at 30 and 100 mg/kg/day. Although the incidence of this finding was slightly higher than would be expected for rats of this age and strain, the incidence did not show a dose-related trend. Therefore, no toxicological relevance was ascribed to this observation.Incidental findings among control and treated animals included alopecia, reduced size of the testes, epididymides and/or seminal vesicles, agenesis of the testes, tan discoloration of the clitoral gland, uterus containing fluid, pelvic dilation of the kidneys and an enlarged mandibular lymph node with reddish discoloration. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. As such, these macroscopic findings were considered to be of no toxicological relevance.

ORGAN WEIGHTS
No toxicologically relevant changes in organ weight and organ to body weights were recorded at any dose level.

Statistically significant increases in absolute thyroid weights and relative kidney weights of females at 100 mg/kg/day occurred in the absence of a dose-related trend and means remained within the range considered normal for rats of this age and strain. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

MICROSCOPIC EXAMINATION
There were no microscopic findings which could be attributed to treatment with the test item.

A total of 9 males were recorded with sperm granuloma of the epididymides - three in the control group, three at 30 mg/kg/day, two at 100 mg/kg/day and one at 300 mg/kg/day. Whilst there was no relationship to treatment, the incidence of this finding was somewhat higher than usually encountered in this strain of rat. One control male had severe unilateral seminiferous epithelial degeneration of the testis with corresponding unilateral very severe oligospermia of the epididymis which may have contributed to the suspected infertility in this animal. In all other animals suspected of infertility there were no microscopic findings which could explain their lack of reproductive performance.

The spermatogenic staging profiles were normal for all males evaluated in the control and 300 mg/kg groups, respectively.

The remainder of recorded microscopic findings were within the range of background pathology encountered in Wistar rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality and associated clinical signs at 100 and 300 mg/kg/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters (mating, fertility and conception indices, precoital time, and the number of corpora lutea and implantation sites) were noted up to 300 mg/kg/day.

DEVELOPMENTAL DATA
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

- Gestation:
At 300 mg/kg/day, the gestation index was noticeably lower than controls (25% vs. 100% for the control group), due to premature death of 6 of the 8 pregnant females in this group. The absolute number of living pups at first litter check was also considerably lower at 300 mg/kg/day compared to controls (21 pups vs. 106 pups for the control group), since only two litters with live pups were available for analysis. Despite the difference in absolute number, the mean number of pups per litter was similar between the 300 mg/kg/day and control groups (10.5 and 11.8 pups per litter, respectively).
Duration of gestation was unaffected by treatment.

- Parturition and maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

- Mortality
A single pup of female no. 42 (control group) and a single pup of female no. 63 (100 mg/kg/day) was found missing during lactation. The incidence remained within the range considered normal for pups of this age and therefore no toxicological relevance was attributed to these missing pups.

- Clinical signs
Incidental clinical symptoms were noted only for pups in the control group and consisted of small size and absence of milk in the stomach. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age. Furthermore, because these findings were isolated to only control animals, they were considered to be of no toxicological relevance.

- Body weight
Mean body weight of male and female pups at 300 mg/kg/day on Day 4 of lactation appeared slightly lower than controls. The change was however slight (within the range considered normal for pups of this age and within the range encountered for pups among other dose groups), and no evidence of deficiencies in maternal care, or a high incidence of insufficient milk in the stomach of the pups was observed. Therefore, no toxicological relevance was ascribed to these slightly lower pup body weights.

- Macroscopy
A single control pup was noted as small at macroscopic examination. As this was a control pup this finding was not considered to be toxicologically relevant. No macroscopic findings were noted in any other pup, including pups examined from females at 300 mg/kg/day that died or were euthanized in extremis.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Silicic acid (H2Si2O5), barium salt (1:1), lead-doped was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for a total of 28 days). The females were exposed for 2 weeks prior to mating, during mating, during postmating, and at least 4 days of lactation (for 41-47 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results:
A single male and six females at 300 mg/kg/day and a single female at 100 mg/kg/day were either found dead or euthanized in extremis. Females were mostly found dead or sacrificed towards the end of their pregnancy period. Among most of these animals clinical signs indicative of ill-health were noted. A cause of death could however not be established based on in-life data, macroscopy or histopathology. The single death case at 100 mg/kg/day was also
considered to be related to treatment since the time of death (towards end of pregnancy) and the nature of clinical signs observed prior to its death corresponded to that noted among most deaths at 300 mg/kg/day.

Any macroscopic abnormalities observed among surviving animals at 300 mg/kg/day were not associated with treatment-related histopathological changes and were therefore considered to be of no toxicological relevance.

Apart from mortality and associated clinical signs at 100 and 300 mg/kg/day, no toxicologicallyrelevant changes were recorded for any of the parameters (functional observations, body weight, food consumption, clinical laboratory investigations, organ weights, macroscopy and microscopic examination) investigated in this study, including at 30 mg/kg/day.

Reproductive/Developmental results:
No reproductive/developmental toxicity was observed at any dose level.

Although only two females at 300 mg/kg/day survived to deliver live offspring due to the high mortality incidence, it could be concluded from the available data that no treatment-related effect on reproductive/developmental parameters occurred at 300 mg/kg/day. The lower gestation index and number of living pups at first litter check at 300 mg/kg/day were ascribed to parental mortality, and were therefore not indicative of primary developmental toxicity.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were
derived:
Parental NOAEL: 30 mg/kg/day
Reproductive NOAEL: 300 mg/kg/day
Developmental NOAEL: 300 mg/kg/day