[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In a study according OECD TG 422 performed with the read-across substance calcium carbonate (nano) no treatment-related effects were observed for reproduction, therefore, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 February 2010 to 02 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: approximately 12 weeks old
- Weight at study initiation: Males: 299 - 376 g; Females: 191 - 227 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.
- Diet: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used and was available ad libitum.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage and was available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55± 15%
- Air changes: at least fifteen air changes per hour
- Photoperiod: low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 02 March 2010 (first day of treatment) To: 18 April 2010 (final necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Distilled water. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd. (Harlan Laboratories Ltd. Project Number: 2974-0011). Results from the previous study showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at 4 ºC in the dark.
The treatment volume for each animal was 5 mL/kg.

Details on mating procedure:

- M/F ratio per cage: 1 male: 1 female basis within each dose group
- Length of cohabitation: up to fourteen days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing).
- After successful mating each pregnant female was caged individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken and analysed for concentration of Calcium carbonate (nano).

Due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of Calcium Carbonate (nano) in the test material formulations was determined using a gravimetric technique.

The results indicate that the prepared formulations were within ± 6% of the nominal concentration.

Duration of treatment / exposure:

Up to 48 consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Daily

Details on study schedule:

Chronological Sequence of Study:

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.

iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.

ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (Harlan Project Number: 2974-0011).
- Rationale for animal assignment: The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation, lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes - Food efficiency was calculated retrospectively for males throughout the study period and for females during the premating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: All animals in all dose groups and five selected males and females from each dose level, prior to termination.
- Battery of functions tested: sensory reactivity (grasp response, touch escape, vocalisation, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex, finger approach) grip strength, motor activity

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 post partum for females
- How many animals: five males and five females selected from each test and control group
- Parameters examined:
* Haemoglobin (Hb)
* Erythrocyte count (RBC)
* Haematocrit (Hct)
* Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
* Total leucocyte count (WBC)
* Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
* Platelet count (PLT)
* Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
* Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 post partum for females
- How many animals: five males and five females selected from each test and control group
- Parameters examined:
* Urea
* Inorganic phosphorus (P)
* Glucose
* Aspartate aminotransferase (ASAT)
* Total protein (Tot.Prot.)
* Alanine aminotransferase (ALAT)
* Albumin
* Alkaline phosphatase (AP)
* Albumin/Globulin (A/G) ratio (by calculation)
* Creatinine (Creat)
* Sodium (Na+)
* Total cholesterol (Chol)
* Potassium (K+)
* Total bilirubin (Bili)
* Chloride (Cl-)
* Bile acids (Bile)
* Calcium (Ca++)

Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Litter observations:

PARAMETERS EXAMINED
On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum. For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes: Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
* Adrenals
* Pituitary (post fixation)
* Brain
* Seminal vesicles
* Epididymides
* Spleen
* Heart
* Testes
* Kidneys
* Thymus
* Liver
* Thyroid (weighed post-fixation with Parathyroid)
* Ovaries
* Uterus (weighed with Cervix)
* Prostate

HISTOPATHOLOGY: Yes: Samples of the following tissues were removed from all animals and preserved:
* Adrenals
* Muscle (skeletal)
* Aorta (thoracic)
* Oesophagus
* Bone & bone marrow (femur including stifle joint)
* OVARIES
* Bone & bone marrow (sternum)
* Pancreas
* Brain (including cerebrum, cerebellum, medulla oblongata and pons)
* PITUITARY
* PROSTATE
* Caecum
* Rectum
* CERVIX
* Salivary glands (submaxillary)
* COAGULATION GLAND
* Sciatic nerve
* Colon
* SEMINAL VESICLES
* Duodenum
* Skin (hind limb)
* EPIDIDYMIDES
* Spinal cord (cervical, mid-thoracic and lumbar)
* Eyes
* Gross lesions
* Spleen
* Heart
* Stomach
* Ileum
* TESTES
* Jejunum
* Thymus
* Kidneys
* Thyroid/parathyroid
* Liver
* Trachea
* Lungs (with bronchi)
* Urinary bladder
* Lymph nodes (cervical and mesenteric)
* UTERUS
* MAMMARY TISSUE
* VAGINA

The tissues from five selected control and 1000 mg/kg bodyweight/day dose group animals, any animals dying during the study were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in capital letters from the remaining control and 1000 mg/kg bodyweight/day were also processed. In addition, sections of testes and epididymides from all control and 1500 mg/kg bodyweight/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS NECROPSY
The organs examined and weighed were identical to those listed in the parental animals section.

Statistics:

Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Reproductive indices:

MATING PERFORMANCE AND FERTILITY: The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices: For each group the following were calculated:

Mating Index (%) = (No. of animals mated/No. of animals paired) x 100
Pregnancy Index (%) = (No. of pregnant females/No. of animals mated) x 100

GESTATION AND PARTURITION DATA: The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length: Calculated as the no. of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index: The following was calculated for each group:

Parturition Index (%) = (No. of females delivering live offspring/No. of pregnant females) x 100

Offspring viability indices:

LITTER RESPONSES: The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss = ([No. of corpora lutea - no. of implantation sites]/No. of Corpora Lutea) x100
Post–implantation loss = ([No. of implantation sites - Total no. of offspring born]/No. of implantation sites) x 100

ii) Live Birth and Viability Indices: The following indices were calculated for each litter as follows:

Live Birth Index (%) = (No. of offspring alive on Day 1/No. of offspring born) x 100
Viability Index (%) = (No. of offspring alive on Day 4/No. of offspring alive on Day 1) x 100

iii) Sex Ratio (% males): Sex ratio was calculated for each litter value on Days 1 and 4 post partum:

(Number of male offspring/Total number of offspring) x 100

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths that were considered to be related to test material toxicity.
One male treated with 1000 mg/kg bodyweight/day was killed in extremis on Day 39. Histopathological examinations of this animal revealed the cause of death to be due to a misplaced gavage with perforation leading to necrotizing inflammation around the trachea, oesophagus, lungs and thymus. This was therefore considered to be unrelated to test material toxicity.

CLINICAL SIGNS (PARENTAL ANIMALS)
There were no toxicologically significant changes detected.
Episodes of generalised fur loss were evident in three females treated with 1000 mg/kg bodyweight/day and two females treated with 100 mg/kg bodyweight/day. One female treated with 300 mg/kg bodyweight/day had a missing upper front tooth between Days 31 and 35. These incidences in isolation were considered not to be of toxicological significance. Two control females also had fur loss between Day 32 and Day 45. One male treated with 300 mg/kg bodyweight/day had an open wound from Day 27 onwards, followed by scab formation and fur loss from Day 28. Observations of this nature are commonly observed in group housed animals and are not considered to be related to treatment.
The male that was killed in extremis on Day 39 had noisy respiration on Days 36 and 39 and pilo erection, a decreased respiration rate, lethargy and hunched posture prior to termination.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment related effects detected in bodyweight development.
Statistical analysis of the data did not reveal any significant intergroup differences.

FOOD CONSUMPTION
No adverse effect on food consumption was detected for males during the treatment period, or for females during the pre-mating, gestation or lactation phases of the study.

FOOD EFFICIENCY
Food efficiency (the ratio of bodyweight gain to dietary intake) was not affected for males throughout the treatment period, or for females during the pre-mating phase.

REPRODUCTIVE FUNCTION: No treatment-related effects were detected on fertility for treated animals when compared to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating: No treatment-related effects were detected in mating performance. With the exception of one control pair, which mated six days following pairing, all paired animals mated within the first four days of pairing.
Gestation length: No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22 to 23½ days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically significant effects were detected in the organ weights measured.
Males treated with 100 mg/kg bodyweight/day showed a statistically significant reduction in spleen weight both absolute and relative to terminal bodyweight. Females treated with 300 mg/kg bodyweight/day showed a statistically significant increase in relative brain weight. In the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no toxicologically significant macroscopic abnormalities detected in terminal kill animals.
Three males treated with 300 mg/kg bodyweight/day had red lungs at necropsy. A further male from this treatment group had pale lungs and dark cervical lymph nodes. One male treated with 100 mg/kg bodyweight/day also had dark cervical lymph nodes and hydronephrosis in the right kidney. In the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance. One female treated with 1000 mg/kg bodyweight/day, two females treated with 100 mg/kg bodyweight/day and two control females showed fur loss at necropsy. Observations of this nature are commonly observed following lactation and in conjunction with the observation also being present in control females the intergroup differences were considered unrelated to treatment.
The male that was killed in extremis on Day 39 showed thickening in the stomach, white fluid in the thoracic cavity, dark kidneys, red lungs and flaccid testes.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment related microscopic abnormalities detected in terminal kill animals.
All findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age or findings associated with the status post partum.
The cause of death in the male that was killed in extremis was considered to be due to a misplaced gavage with perforation leading to necrotizing inflammation around the trachea, oesophagus, lungs and thymus. This was therefore considered to be unrelated to test material toxicity.

HAEMATOLOGY
No toxicologically significant effects were detected.
Males treated with 1000 mg/kg bodyweight/day showed a statistically significant reduction in mean corpuscular haemoglobin and mean corpuscular volume. All individual values were within the normal ranges for rats of the strain and age used and in isolation were considered not to be of toxicological importance.

CLINICAL CHEMISTRY
No toxicologically significant effects were detected.
Males treated with 1000 mg/kg bodyweight/day showed a statistically significant reduction in total protein and a statistically significant increase in chloride concentration. Males from all treatment groups also showed statistically significant reductions in phosphorus. All individual values were within the normal ranges for rats of the strain and age used and in isolation were considered not to be of toxicological importance.

NEUROBEHAVIOUR
- Behavioural Assessments: Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
- Functional Performance Tests: There were no treatment related changes in functional performance.
- Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically significant effects observed at any dose level.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

All females from control, 100, 300 and 1000 mg/kg bodyweight/day dose groups gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

VIABILITY (OFFSPRING)
No significant differences were detected for corpora lutea and implantation counts for treated animals when compared to controls. Litter sizes and viability for treated groups were also comparable to controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING)
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak, pale and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test material toxicity.
No treatment-related effects were detected for surface righting reflex for offspring from treated animals when compared to offspring from control females.
Statistical analysis of the data did not reveal any significant intergroup differences.

BODY WEIGHT (OFFSPRING)
There were no differences in litter weights or mean offspring bodyweights between control and treated animals.
Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decendent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No statistically significant treatment-related effects at the high dose

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

No treatment-related effects were observed for reproduction, therefore, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to Annex IX, 8.7.3 of REGULATION (EC) No 1907/2006 OF THE EUROPEAN PARLIAMENT (REACH), an extended one-generation reproductive toxicity study having regard to the likely route of human exposure, shall be proposed for substances within the tonnage band 100 - 1000 tpa, if the 90-day study or other available studies on reproduction indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.
As discussed in more detail below, there is no evidence of substance-related effects with regard to reproductive toxicity as demonstrated in the available OECD TG 422 study, OECD TG 414 studies and the Repeated Dose 90-DayToxicity studies in rats.
A 28 day repeated dose oral toxicity study combined with a reproduction/ developmental toxicity screening test was performed in the rat in accordance with OECD Guideline 422 (Dunster, 2010). The read-across substance and component of the test substance Calcium carbonate (nano) was administered by gavage to rats (Wistar, 10 animals/sex/group) for up to forty-eight consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. There were no treatment related effects observed on mating, fertility or gestation length at any dose level. The offspring litter size, viability, growth and development were all comparable to controls and no adverse effects were noted. Since no treatment-related effects were observed for reproduction, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day. A prenatal developmental toxicity study was performed to evaluate the developmental effects of moderate dietary calcium increases in rats fed nutritionally adequate diets (Shackelford et al, 1993). The test method used was not reported; however, it is considered to be similar to OECD Guideline 414. Female rats (CR CD/VAF) were given 0.50 (control), 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate in AIN-76A diets for 6 weeks before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed. No dose-related changes were found in maternal clinical findings, the average number of implantations, resorptions and viable foetuses, or foetal length or weight. There were no statistically significant increases as compared with the control group in the litter incidence regarding specific external, visceral or skeletal variations of the foetuses. Therefore, dietary calcium was neither fetotoxic nor teratogenic at the concentrations used. Since no adverse effects were noted at the highest dose level tested (1.25% Ca in diet), the NOAEL for teratogenic and maternal toxic effects in rats is in excess of 1.25% Ca, equivalent to approximately 1963 - 2188 mg/kg bw/day of calcium carbonate. A number of supporting studies are reported in the literature where calcium has been administered to pregnant women (Bogden et al, 1995 and Belizan et al, 1997). No teratogenic or reproductive effects were noted, indicating that calcium supplementation up to doses of 2 g/day are considered to be non-toxic. Based on the lack of effect upon reproduction or offspring development observed in these studies at dose levels well in excess of normal human exposure doses, calcium carbonate is not a significant risk to the reproductive process and further studies are unlikely to show any significant effects. Furthermore, calcium is an essential element to all life forms and ecosystems and occurs naturally within the environment. Humans are widely exposed to naturally occurring calcium carbonate, e.g. via drinking water on a day to day basis. Calcium is the fifth most abundant element in the human body (European Commission Scientific Committee on Food, 2003). The calcium content of the human body is 25 to 30 g at birth (0.8% of the body weight) and between 900 and 1300 g in adult men (up to 1.7% of body weight). Over 99% of the total calcium of the body is located in the bones, where it accounts for 39% of the total body bone mineral content and in the teeth, mostly as hydroxyapatite. Calcium carbonate is also a food additive approved by the Council Directive 95/2/EC on food additives (the substance has the acronym E 170). Ingested calcium and carbonate ions are actively regulated by the body. Calcium must be ingested with the diet in sufficient amounts to allow for calcium deposition during bone growth and modelling and to compensate for obligatory intestinal, faecal and dermal losses during the life-time. Calcium deficiency can result from low dietary intake, low absorption or excessive losses. A decrease in ionised calcium in the extracellular fluid stimulates the secretion of parathyroid hormone to mobilise calcium from bone and maintain the pre-set serum calcium level. Parathyroid hormone also increases the intracellular calcium concentration in many types of cells which in turn sets off a large number of reactions involving the permeability of the plasma membrane, signalling pathways, including activation and deactivation of enzymes, cyclic-nucleotide formation and break-down, cytoskeletal rearrangement, and gene transcription. The pathophysiologic changes and disorders resulting from this include hypertension and arteriosclerosis, Alzheimer’s disease, muscular dystrophy, diabetes mellitus and malignancies. The European Commission Scientific Committee on Food has produced a report on the Tolerable Upper Intake Level of Calcium (2003). The report describes the results obtained in a number of studies on calcium salts and concludes that the derivation of an upper intake level (UL) for calcium can be based on the evidence of large placebo controlled intervention studies for preventive purposes with supplemental calcium carbonate of up to 2000 mg calcium in addition to the calcium intake from the diet (>400 mg/day). These studies have been conducted in more than 3000 pregnant women and no adverse effects have been reported. There are no data to suggest an increased susceptibility for lactating women. Therefore, the UL of 2500 mg calcium per day also applies to pregnant and lactating women, as well as adults.
In conclusion, an additional extended one-generation reproductive toxicity study in the rat is unlikely to provide any further evidence of reproductive toxicity as the existing studies have demonstrated a lack of effect at dose levels well in excess of expected human exposure.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reproductive effects observed:

not specified

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:

OECD 422 (read-across)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was performed with the read-across substance calcium carbonate (nano) according to OECD Guideline 422 and GLP with concentrations of the read-across substance calcium carbonate (nano) of 0, 100, 300 and 1000 mg/kg bw/day (Dunster, 2010).

There were no unscheduled deaths that were considered to be related to test material toxicity. One male treated with 1000 mg/kg bodyweight/day was killed in extremis on Day 39. Histopathological examinations of this animal revealed the cause of death to be due to a misplaced gavage with perforation leading to necrotizing inflammation around the trachea, oesophagus, lungs and thymus. This was therefore considered to be unrelated to test material toxicity.

There were no toxicologically significant changes detected.

Episodes of generalised fur loss were evident in three females treated with 1000 mg/kg bw/day and two females treated with 100 mg/kg bodyweight/day. One female treated with 300 mg/kg bw/day had a missing upper front tooth between Days 31 and 35. These incidences in isolation were considered not to be of toxicological significance. Two control females also had fur loss between Day 32 and Day 45. One male treated with 300 mg/kg bw/day had an open wound from Day 27 onwards, followed by scab formation and fur loss from Day 28. Observations of this nature are commonly observed in group housed animals and are not considered to be related to treatment.

The male that was killed in extremis on Day 39 had noisy respiration on Days 36 and 39 and pilo erection, a decreased respiration rate, lethargy and hunched posture prior to termination.

There were no treatment related effects detected in bodyweight development. Statistical analysis of the data did not reveal any significant intergroup differences. No adverse effect on food consumption was detected for males during the treatment period, or for females during the pre-mating, gestation or lactation phases of the study. Food efficiency (the ratio of bodyweight gain to dietary intake) was not affected for males throughout the treatment period, or for females during the pre-mating phase.

No treatment-related effects were detected on fertility for treated animals when compared to controls. No treatment-related effects were detected in mating performance. With the exception of one control pair, which mated six days following pairing, all paired animals mated within the first four days of pairing.No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22 to 23½ days.

No toxicologically significant effects were detected in the organ weights measured. Males treated with 100 mg/kg bodyweight/day showed a statistically significant reduction in spleen weight both absolute and relative to terminal bodyweight. Females treated with 300 mg/kg bodyweight/day showed a statistically significant increase in relative brain weight. In the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological significance.

There were no toxicologically significant macroscopic abnormalities detected in terminal kill animals. Three males treated with 300 mg/kg bodyweight/day had red lungs at necropsy. A further male from this treatment group had pale lungs and dark cervical lymph nodes. One male treated with 100 mg/kg bodyweight/day also had dark cervical lymph nodes and hydronephrosis in the right kidney. In the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance. One female treated with 1000 mg/kg bodyweight/day, two females treated with 100 mg/kg bodyweight/day and two control females showed fur loss at necropsy. Observations of this nature are commonly observed following lactation and in conjunction with the observation also being present in control females the intergroup differences were considered unrelated to treatment.

The male that was killed in extremis on Day 39 showed thickening in the stomach, white fluid in the thoracic cavity, dark kidneys, red lungs and flaccid testes.

There were no treatment related microscopic abnormalities detected in terminal kill animals.

All findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age or findings associated with the status post-partum. The cause of death in the male that was killed in extremis was considered to be due to a misplaced gavage with perforation leading to necrotizing inflammation around the trachea, oesophagus, lungs and thymus. This was therefore considered to be unrelated to test material toxicity.

No toxicologically significant effects were detected. Males treated with 1000 mg/kg bodyweight/day showed a statistically significant reduction in mean corpuscular haemoglobin and mean corpuscular volume. All individual values were within the normal ranges for rats of the strain and age used and in isolation were considered not to be of toxicological importance.

No toxicologically significant effects were detected. Males treated with 1000 mg/kg bodyweight/day showed a statistically significant reduction in total protein and a statistically significant increase in chloride concentration. Males from all treatment groups also showed statistically significant reductions in phosphorus. All individual values were within the normal ranges for rats of the strain and age used and in isolation were considered not to be of toxicological importance.

Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls. There were no treatment related changes in functional performance.There were no treatment-related changes in sensory reactivity.

All females from control, 100, 300 and 1000 mg/kg bodyweight/day dose groups gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

No significant differences were detected for corpora lutea and implantation counts for treated animals when compared to controls. Litter sizes and viability for treated groups were also comparable to controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak, pale and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test material toxicity. No treatment-related effects were detected for surface righting reflex for offspring from treated animals when compared to offspring from control females. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no differences in litter weights or mean offspring bodyweights between control and treated animals. Statistical analysis of the data did not reveal any significant intergroup differences. Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.

The NOAEL for maternal toxicity and reproductive toxicity was considered to be 1000 mg/kg bw/day in rats.

Effects on developmental toxicity

Description of key information

A study according OECD TG 414 in rats is available with the read-across substance calcium carbonate (nano). No treatment-related effects were observed for reproduction/ developmental toxicity, therefore, a NOEL for reproductive/ developmental toxicity was considered to be 1000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

Second species

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
REACH Annex IX Column 2 Section 8.7.2 states as follows: “The study shall be initially performed on one species. A decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data.”
Based on the outcome of the first test with rats and all other relevant available data, a study on a second species, i. e. non-rodent species, is considered not necessary, as discussed in more detail below. There is no evidence of substance-related effects with regard to reproductive and developmental toxicity as demonstrated in the available Screening on Reproduction/Developmental Toxicity Study (OECD 422) and the Prenatal Developmental Toxicity studies, which are available for the read-across substances (OECD 414).
A 28 day repeated dose oral toxicity study combined with a reproduction/ developmental toxicity screening test was performed in the rat in accordance with OECD Guideline 422 (Dunster, 2010). The read-across substance and component of the test substance Calcium carbonate (nano) was administered by gavage to rats (Wistar, 10 animals/sex/group) for up to forty-eight consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. There were no treatment related effects observed on mating, fertility or gestation length at any dose level. The offspring litter size, viability, growth and development were all comparable to controls and no adverse effects were noted. Since no treatment-related effects were observed for reproduction, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.
A prenatal developmental toxicity study was performed to evaluate the developmental effects of moderate dietary calcium increases in rats fed nutritionally adequate diets (Shackelford et al, 1993). The test method used was not reported; however, it is considered to be similar to OECD Guideline 414. Female rats (CR CD/VAF) were given 0.50 (control), 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate in AIN-76A diets for 6 weeks before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed. No dose-related changes were found in maternal clinical findings, the average number of implantations, resorptions and viable foetuses, or foetal length or weight. There were no statistically significant increases as compared with the control group in the litter incidence regarding specific external, visceral or skeletal variations of the foetuses. Therefore, dietary calcium was neither fetotoxic nor teratogenic at the concentrations used. Since no adverse effects were noted at the highest dose level tested (1.25% Ca in diet), the NOAEL for teratogenic and maternal toxic effects in rats is in excess of 1.25% Ca, equivalent to approximately 1963 - 2188 mg/kg bw/day of calcium carbonate. A number of supporting studies are reported in the literature where calcium has been administered to pregnant women (Bogden et al, 1995 and Belizan et al, 1997). No teratogenic or reproductive effects were noted, indicating that calcium supplementation up to doses of 2 g/day are considered to be non-toxic. Based on the lack of effect upon reproduction or offspring development observed in these studies at dose levels well in excess of normal human exposure doses, calcium carbonate is not a significant risk to the reproductive process and further studies are unlikely to show any significant effects.
In conclusion, based on the available study results, adverse effects were observed neither on fertility parameters in male and female animals nor on development of the offspring. Therefore, according to Column 2, Section 8.7.2, Annex IX of REACH Regulation a study on developmental toxicity and teratogenicity in a second species is not considered scientifically justified, also in regards to animal welfare reasons.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Species:

rabbit

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 February 2010 to 02 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: approximately 12 weeks old
- Weight at study initiation: Males: 299 - 376 g; Females: 191 - 227 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.
- Diet: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used and was available ad libitum.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage and was available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55± 15%
- Air changes: at least fifteen air changes per hour
- Photoperiod: low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 02 March 2010 (first day of treatment) To: 18 April 2010 (final necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Distilled water. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd. (Harlan Laboratories Ltd. Project Number: 2974-0011). Results from the previous study showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at 4 ºC in the dark.
The treatment volume for each animal was 5 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken and analysed for concentration of Calcium carbonate (nano).

Due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of Calcium Carbonate (nano) in the test material formulations was determined using a gravimetric technique.

The results indicate that the prepared formulations were within ± 6% of the nominal concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male: 1 female basis within each dose group
- Length of cohabitation: up to fourteen days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing).
- After successful mating each pregnant female was caged individually during the period of gestation and lactation.

Duration of treatment / exposure:

Up to 48 consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Daily

Duration of test:

Up to 48 consecutive days

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (Harlan Project Number: 2974-0011).
- Rationale for animal assignment: The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays, except during parturition where applicable. All observations were recorded.
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation, lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes - Food efficiency was calculated retrospectively for females during the premating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: All animals in all dose groups and five selected females from each dose level, prior to termination.
- Battery of functions tested: sensory reactivity (grasp response, touch escape, vocalisation, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex, finger approach) grip strength, motor activity

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 4 post partum for females
- How many animals: five females selected from each test and control group
- Parameters examined:
* Haemoglobin (Hb)
* Erythrocyte count (RBC)
* Haematocrit (Hct)
* Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
* Total leucocyte count (WBC)
* Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
* Platelet count (PLT)
* Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
* Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 4 post partum for females
- How many animals: five females selected from each test and control group
- Parameters examined:
* Urea
* Inorganic phosphorus (P)
* Glucose
* Aspartate aminotransferase (ASAT)
* Total protein (Tot.Prot.)
* Alanine aminotransferase (ALAT)
* Albumin
* Alkaline phosphatase (AP)
* Albumin/Globulin (A/G) ratio (by calculation)
* Creatinine (Creat)
* Sodium (Na+)
* Total cholesterol (Chol)
* Potassium (K+)
* Total bilirubin (Bili)
* Chloride (Cl-)
* Bile acids (Bile)
* Calcium (Ca++)

Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Statistics:

Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
There were no unscheduled deaths that were considered to be related to test material toxicity.

CLINICAL SIGNS
There were no toxicologically significant changes detected.
Episodes of generalised fur loss were evident in three females treated with 1000 mg/kg bodyweight/day and two females treated with 100 mg/kg bodyweight/day. One female treated with 300 mg/kg bodyweight/day had a missing upper front tooth between Days 31 and 35. These incidences in isolation were considered not to be of toxicological significance. Two control females also had fur loss between Day 32 and Day 45. One male treated with 300 mg/kg bodyweight/day had an open wound from Day 27 onwards, followed by scab formation and fur loss from Day 28. Observations of this nature are commonly observed in group housed animals and are not considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment related effects detected in bodyweight development.
Statistical analysis of the data did not reveal any significant intergroup differences.

FOOD CONSUMPTION
No adverse effect on food consumption was detected for females during the pre-mating, gestation or lactation phases of the study.

FOOD EFFICIENCY
Food efficiency (the ratio of bodyweight gain to dietary intake) was not affected for females during the pre-mating phase.

REPRODUCTIVE FUNCTION: No treatment-related effects were detected on fertility for treated animals when compared to controls.

REPRODUCTIVE PERFORMANCE
Mating: No treatment-related effects were detected in mating performance. With the exception of one control pair, which mated six days following pairing, all paired animals mated within the first four days of pairing.
Gestation length: No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22 to 23½ days.

ORGAN WEIGHTS
No toxicologically significant effects were detected in the organ weights measured.
Females treated with 300 mg/kg bodyweight/day showed a statistically significant increase in relative brain weight. In the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological significance.

GROSS PATHOLOGY
There were no toxicologically significant macroscopic abnormalities detected in terminal kill animals.
One female treated with 1000 mg/kg bodyweight/day, two females treated with 100 mg/kg bodyweight/day and two control females showed fur loss at necropsy. Observations of this nature are commonly observed following lactation and in conjunction with the observation also being present in control females the intergroup differences were considered unrelated to treatment.

HISTOPATHOLOGY
There were no treatment related microscopic abnormalities detected in terminal kill animals.
All findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age or findings associated with the status post partum.

HAEMATOLOGY
No toxicologically significant effects were detected.

CLINICAL CHEMISTRY
No toxicologically significant effects were detected.

NEUROBEHAVIOUR
- Behavioural Assessments: Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
- Functional Performance Tests: There were no treatment related changes in functional performance.
- Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
No significant differences were detected for corpora lutea and implantation counts for treated animals when compared to controls. Litter sizes and viability for treated groups were also comparable to controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING)
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak, pale and physical injury were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test material toxicity.
No treatment-related effects were detected for surface righting reflex for offspring from treated animals when compared to offspring from control females.
Statistical analysis of the data did not reveal any significant intergroup differences.

BODY WEIGHT (OFFSPRING)
There were no differences in litter weights or mean offspring bodyweights between control and treated animals.
Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decendent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

No treatment-related effects were observed for reproduction/ developmental toxicity, therefore, a NOEL for reproductive/ developmental toxicity was considered to be 1000 mg/kg bw/day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Minor deviations from the guideline which do not affect the outcome of the study.

Principles of method if other than guideline:

This study was designed to evaluate the developmental effects of moderate dietary calcium increases in rats fed nutritionally adequate diets. Female Charles River CD/VAF Plus rats were given 0.50 (control), 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate in AIN-76A diets for 6 weeks before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: CD/VAF Plus Charles River

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Wilmington, MA, USA)
- Age at study initiation: Females - 52 days old; Males - 44 days old
- Weight at study initiation: Females - approximately 217 ± 1 g
- Fasting period before study: None
- Housing: Stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 27 °C
- Humidity (%): 25 - 72 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Once
- Mixing appropriate amounts with (Type of food): AIN-76A Diet
- Storage temperature of food: -1 to -6 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of calcium analysed using the methods of the Association of Official Analytical Chemists

Details on mating procedure:

Cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: No data - at least overnight for one night
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Females: 6 weeks prior to mating, through mating to day 20 of gestation

Frequency of treatment:

Rats allowed access to feed ad libitum

Duration of test:

Approximately 62 days

Dose / conc.:

0.75 other: %

Remarks:

nominal in diet

Dose / conc.:

1 other: %

Remarks:

nominal in diet

Dose / conc.:

1.25 other: %

Remarks:

nominal in diet

No. of animals per sex per dose:

Females: Pre-mating - 69/dose; Pregnant rats - 45-48/dose
Males: Received test material only during the mating period

Control animals:

other: The group receiving 0.5% calcium were used as the control group

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Every 3 days for the 6 weeks before mating and during gestation, but not during mating (females only)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes (females only)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Major organs (no further details reported)

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: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

The incidence of clinical parameters was analysed by comparing treatment groups with the control group by using Fisher's exact test.
Data on feed consumption were analysed using analysis of variance (ANOVA) and a protected least significant difference (LSD) test (two-tailed).
Weight gains were analysed using analysis of covariance (ANOCOVA) after adjusting for the initial body weight, and an LSD test (two-tailed).
Trend analysis was performed on feed consumption.
The number of corpora lutea, the average number of implantations, the average number of viable foetuses for each litter and the average number of male or female foetuses for each litter were analysed by ANOVA and a LSD test (one-tailed).
Data on implantation efficiency, early deaths, late deaths, and total resorptions (early and late deaths) were analysed by the Freeman-Tukey arcsine transformation for binomial proportions. The transformed data for each litter were then analysed by ANOVA and a LSD test (one-tailed), comparing the control group with experimental groups.
Fisher's exact test was used to analyse data on litters with runts and litters totally resorbed, comparing the controls group with treatment groups.
Foetal body weights and crown-rump lengths were analysed by a nested ANOVA and a LSD (one-tailed).
Data on the specific litter incidence of sternebral, skeletal and visceral variations were analysed using Fisher's exact test.
Proportions of litters with foetuses showing external variations were analysed using Fisher's exact test.
The average number of foetuses with variations per litter was analysed using the Freeman-Tukey arcsine transformation and then an ANOVA and a protected LSD test (one-tailed).
Litters that had foetuses with one or more sternebral, skeletal and visceral variations were analysed with Fisher's exact test.
Trend analysis was performed on the litter data for external variations using the Cox exact one-tailed test for unadjusted positive trend.

Historical control data:

No historical control data were provided

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical findings included alopecia, exudate around the eyes, exudate around the nose, bent teeth, lesion, lump in the left flank or leg and mammary lump. The number of animals with alopecia was significantly increased in animals fed 1.00 % calcium (8.7 vs 0 % in the control) during the 6 weeks before mating and in the groups fed 0.75 and 1.00 % calcium (10 and 16.3 % respectively vs 0 % in the control) during gestation.
One non-pregnant animal was observed to have diarrhoea on day 4 after assignment to the 1.25 % calcium diet.
One pregnant female fed 1.00 % dietary calcium developed bleeding from the vagina on days 15 and 16 of gestation. The incidence of diarrhoea and bleeding from the vagina was not considered to be related to the dietary calcium.
Three females died during the study, but the deaths were not attributable to excess calcium in the diet.
Values for mean daily feed consumption per female given excess dietary calcium for 6 weeks before mating and during gestation are shown in Table 1. The 0.75, 1.00 and 1.25 % groups ate slightly more than the controls during most of the study, but not all the increases were statistically significant.
Mean values for body weight gain for animals given excess dietary calcium for 6 weeks before mating and gestation are given in Table 2. Although some differences in weight gain between control and treated groups were significant, there was no consistent pattern of increase or decease in the body weight gain.
Maternal reproductive parameters are listed in Table 3. There were no adverse reproductive effects associated with ingestion of excess dietary calcium. Significant increases were observed in implantation efficiency at 1.25 % dietary calcium and in the average number of viable female foetuses at 0.75 and 1.25 % dietary calcium. These increases were not dose related and were not considered to be caused by dietary calcium.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1.25 other: % w/w calcium

Basis for effect level:

other: highest dose tested

Remarks on result:

other: This result corresponds to a NOAEL for maternal toxic effects of calcium carbonate in rats of between 1963 and 2188 mg/kg bw/d.

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no adverse effects to the foetuses caused by feeding excess dietary calcium to the dams (Table 3). Foetal body weights and crown-rump lengths of male and female animals given excess dietary calcium were similar to those of the control groups. Feeding excess dietary calcium had no effect on the number of litters with male or female runts. The incidences of specific external variations are shown in Table 4. In the control group, one foetus did not possess a mouth (astomia) and a lower jaw (agnathia). At the 1.25 % dietary calcium level, one foetus had multiple anomalies: agnathia, cleft palate, protruding tongue, beak-like snout, malformed head, open eye on the right side, oedema of the trunk, omphalocele, exposed heart and lungs (diaphragmatic cleft), club foot on the left front paw, scoliosis, imperforate anus and kinked tail. The occurrence of this foetus with multiple anomalies was not thought to be related to feeding the rats excess dietary calcium before and during pregnancy. The data in table 4 were analysed by trend analysis and Fisher's exact test. The Cox exact trend test showed no significant positive trends in the litter incidence of individual or combined variations. When analysed by Fisher's exact test, there were no significant differences between treated and control groups in the litter incidences of these same parameters. Under the conditions of the study, there were no significant increases in the litter incidence of specific or combined external variations. The numbers of foetuses with sternebral ossification deficiencies and the litter incidences are summarised in Table 5. A comparison of the proportion of litters with foetuses having sternebral variations showed no significant differences among the groups given excess calcium and the control group. The increase in the number of litters with foetuses with bipartite sternebrae in the 1.25 % group was not significant (p=0.0854). The incidences of sternebral variations are presented in Table 6. The variations counted are based on the classification of the types and not on the individual sternebrae affected. The average number of foetuses per litter with one or more sternebral variations increased in the 0.75, 1.00 and 1.25 % groups, but the differences were not significant. There was no significant increase in the average number of foetuses per litter with at least two or three sternebral variations in the groups fed excess dietary calcium. Statistical analysis of the proportion of litters that had foetuses with variations (at least one, two or three) showed no significant increase between the excess calcium and the control groups. The incidences of specific skeletal variations, excluding sternebrae, were not dose related and there were no significant differences among any of the values (Table 7). The average number of sacral and caudal vertebrae was not affected by dietary calcium (data not shown). The average number of foetuses per litter with at least one skeletal variation was lower in the 0.75, 1.00 and 1.25 % dietary calcium groups than in the control (Table 8). However, the ANOVA of the Freeman-Tukey arcsine-transformed data showed no differences in the average number of foetuses with variations (p>0.10). The proportion of litters that had foetuses with variations (at least one, two or three) showed no dose-related or significant differences between any of the values obtained in this study. There was no dose related effect on the incidence of any other type of soft-tissue variation (Table 9). The proportion of litters with hydrocephalus was greater in the control group than in the treatment groups, but the differences were not significant. The incidence of visceral problems by foetus and by litter is shown in Table 10. The average number of foetuses with at least one, two or three soft-tissue variations was similar in all groups. Although the average number of foetuses with at least one variation was lower in the 1.00 and 1.25 % groups, ANOVA did not indicate a significant decrease compared with controls. Although there was a dose-dependent decrease in the proportion of litters with foetuses with at least one variation. Fisher's exact test (two-tailed) did not show any significant difference. Thus the proportion of litters with foetuses with at least one two or three soft-tissue variations was similar in all groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1.25 other: % w/w calcium

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Remarks on result:

other: This result corresponds to a NOAEL for teratogenic effects of calcium carbonate in rats of between 1963 and 2188 mg/kg bw/d.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Mean daily feed consumption (g/animal/day) of rats given excess dietary calcium.

Concentration of Calcium in the Diet (%)
Days	0.5	0.75	1.00	1.25
	Non-pregnant rats 6 weeks before mating
	(69)	(69)	(69)	(69)
0-6	15.2 ± 0.4	15.7 ± 0.3	15.8 ± 0.3	16.4 ± 0.4
6-12	17.4 ± 0.3	17.7 ± 0.3	17.8 ± 0.3	18.2 ± 0.3
12-18	17.0 ± 0.3	17.6 ± 0.3	18.1 ± 0.3^b	18.4 ± 0.3^c
18-24	17.9 ± 0.3	18.1 ± 0.3	18.8 ± 0.3^a	18.9 ± 0.3^b
24-30	17.6 ± 0.3	17.9 ± 0.3	18.8 ± 0.3^b	18.6 ± 0.3^a
30-36	17.8 ± 0.3	17.7 ± 0.3	18.6 ± 0.3^d	18.4 ± 0.2
36-42	17.8 ± 0.3	17.8 ± 0.3*	18.6 ± 0.3^a	18.5 ± 0.3^d
0-42	17.3 ± 0.2	17.5 ± 0.2*	18.1 ± 0.2^a	18.2 ± 0.2^b
	Pregnant rats from days 0-20
	(45)	(47)	(44)	(48)
0-6	19.6 ± 0.4	20.4 ± 0.4	20.8 ± 0.4^a	21.0 ± 0.3^b
6-12	22.8 ±0.4	23.3 ± 0.3	23.9 ± 0.4^d	24.2 ± 0.4^a
12-18	23.5 ± 0.4	24.3 ± 0.3	24.1 ± 0.5	24.2 ± 0.3
18-20	23.7 ± 0.5	24.0 ± 0.4	23.7 ± 0.6	23.0 ± 0.5
0-20	22.2 ± 0.4	22.8 ± 0.3	23.0 ± 0.4	23.1 ± 0.3

* Because one animal was autopsied in extremis on day 40 in the 0.75% group the number of rats was reduced to 68 for the data for days 36 -42 and 0 -42.

Number of animals are indicated in parantheses. Values are means ± SEM. superscripts indicate differences between control and test values ( ^aP ≤0.05; ^bP≤0.01; ^cP ≤0.001; ^d 0.05 P 0.01 )

Table 2: Mean body weight gains and initial body weights of rats given excess dietary calcium

Concentration of Calcium in the Diet (%)
Days	0.5	0.75	1.00	1.25
	Body weight  gain (g) of non-pregnant rats 6 weeks before mating
	(69)	(69)	(69)	(69)
0-6	8.1 ± 1.4	9.9 ± 1.2	9.1 ± 1.1	7.7 ± 1.4
6-12	22.1 ± 1.1	21.1 ± 0.9	21.7 ± 1.1	20.1 ± 1.1
12-18	12.1 ± 0.8	14.7 ± 0.9^a	16.1 ±1.0^a	16.3 ± 0.7^a
18-24	16.3 ± 0.8	14.0 ± 0.7^a	15.7 ± 0.9	14.1 ± 0.7^a
24-30	9.0 ± 0.6	9.8 ± 0.8	11.9 ± 0.9^a	10.6 ± 0.7
30-36	11.0 ±0.9	9.2 ± 0.8	10.1 ± 1.0	9.6 ± 0.8
36-42	5.3 ± 0.7	5.8 ± 0.9*	4.9 ± 0.8	6.4 ± 0.6
0-42	83.9 ± 2.9	85.1 ± 3.1*	89.5 ± 3.0	84.7 ± 2.6
	Initial body weight (g) of non pregnant rats
0	217.7 ± 1.0	217.6 ± 1.0	217.4 ± 1.0	217.7 ± 1.0
	Body weight gain (g) of pregnant rats
	(45)	(47)	(44)	(48)
0-6	29.8 ±1.2	29.7 ± 1.3	29.8 ± 1.1	29.4 ± 1.0
6-12	30.4 ± 1.0	32.0 ± 0.8	30.2 ± 0.7	31.9 ± 0.9
12-18	45.2 ± 1.8	51.1 ± 1.4^a	48.0 ± 2.6	48.9 ± 1.5
18-20	25.8 ± 1.1	27.9 ± 0.9	26.2 ± 1.1	25.1 ± 1.0
0-20	131.2 ± 3.1	140.7 ± 2.6	134.2 ± 3.9	135.3 ± 3.0
	Initial body weight (g) of pregnant rats
0	297.6 ± 3.6	300.8 ± 3.7	306.5 ± 4.8	300.0 ± 3.8

* Because one animal was autopsied in extremis on day 40 in the 0.75% group the number of rats was reduced to 68 for days 36-42 and 0 -42

Values marked with superscripts indicate significant differences between control and test values ^aP ≤0.05; ^bP≤0.01; ^cP ≤0.001; ^d 0.05 P 0.10

Table 3: Maternal and reproductive outcome at autopsy and foetal data from rats given excess dietary calcium

Concentration of Calcium in the Diet (%)
Parameter	0.5	0.75	1.00	1.25
No. of pregnant females	45	47	44	48
No. of corpora lutea/female	16.27 ± 0.4	16.83 ± 0.40	16.52 ± 0.27	15.93 ± 0.32
Implantation frequency  (% female)*$	82.01 ± 3.73	90.29 ± 1.89	86.51 ± 3.46	93.41 ± 2.28^b
No of implants/female*	13.58 ± 0.68	15.26 ± 0.43	14.25 ±  0.60	15.15 ± 0.50
No of viable foetuses/female*	11.82 ± 0.66	13.53 ± 0.46	12.82 ± 0.65	13.54 ± 0.58
No of viable foetuses/litter*	6.29 ± 0.43	6.49 ± 0.33	6.95 ± 0.49	6.83 ± 0.37
Viable female foetuses/litter*	5.53 ± 0.39	7.04 ± 0.38^b	5.86 ± 0.37	6.71 ± 0.43^a
Resorptions/female (mean %)*‡	12.50 ± 1.98	11.15 ± 1.73	12.92 ± 3.19	13.81 ± 3.09
No of early deaths/litter$	1.71	1.70	1.41	1.56
No. of late deaths/litter$	0.04	0.02	0.02	0.04
No of early and late deaths/litter$‡	1.76	1.72	1.43	1.60
No of litters totally resorbed	0	0	1	2
Body weight (g)
Males	3.84	3.90	3.83	3.79
Females	3.61	3.69	3.66	3.58
Crown rump length (cm)
Males	4.0	4.0	4.0	4.0
Females	3.9	3.9	3.9	3.9
No of runts
Males	5	5	3	3
Females	4 (4)	4 (1)	1 (1)	3 (2)

* Means ± SEM

$ Statistical analysis was performed on these data after application of the Freman-Tukey arcsine transformation

‡ The number of resorptions includes early deaths and late deaths per litter

§ The number of litters is indicated in parentheses

Values marked with superscripts indicate significant differences between control and test values ^aP ≤0.05; ^bP≤0.01; ^cP ≤0.001; ^d 0.05 P 0.10

Table 4: Incidence of specific external variations in foetuses from rats given excess dietary calcium

Concentration of Calcium in the Diet (%)
Parameter	0.5	0.75	1.00	1.25
No. foetuses (litters examined)	532 (45)	636 (47)	564 (43)	650 (46)
No of foetuses (litters with variations)	2 (2)	2 (2)	1 (1)	8 (6)
Heamorrhages, external	1 (1)	-	1 (1)	2 (2)
Cleft palate	-	1 91)	-	2 (2)
Agnethia	1 (1)	-	-	1 (1)
Astomia	1 (1)	-	-	-
Thread like tail	-	1 (1)	-	1 (1)
Kinked tail	-	-	-	2 (2)
Oedema	-	-	-	3 (2)
Malformed head	-	-	-	1 (1)
Beaked snout	-	-	-	1 (1)
Protruding tongue	-	-	-	1 (1)
Open eye	-	-	-	1 (1)
Diaphragmatic cleft*	-	-	-	1 (1)
Omphalocele	-	-	-	1 (1)
Club foot	-	-	-	1 (1)
Seollosis	-	-	-	1 (1)
Imperforate anus	-	-	-	2 (2)

The number of litters is indicated parentheses

* This parameter includes exposed heart and lungs

Table 5: Incidence of specific external variations in foetuses from rats given excess dietary calcium

Concentration of Calcium in the Diet (%)
Parameter	0.5	0.75	1.00	1.25
No. foetuses (litters examined)	251 (44)	305 (47)	273 (43)	313 (46)
No of foetuses with specific stenebral variations
Incomplete ossification	74 (31)	84 (35)	88 (36)	95 (35)
Bipartite	4 (4)	3 (3)	4 (3)	10 (10)^d
Unossified	50 (27)	49 (27)	40 (18)	54 (31)
Malagnified	8 (7)	2 (2)	10 (9)	11 (11)
Fused	1 (1)	0 (0)	0 (0)	1 (1)

The number of litters is indicated parentheses

Superscript indicates differences between control and test values (^d0.05  P 0.10)

Table 6: Incidence of sternebral variations in foetuses of rats given excess dietary calcium

Concentration of Calcium in the Diet (%)
Parameter	0.5	0.75	1.00	1.25
Sternebral variations*
Total no.	137	138	142	171
Mean no./litter	3.11	2.94	3.30	3.72
Foetuses with one or more sternbral variations
Total no.	105	118	112	138
Mean no./litter$	2.39	2.51	2.60	3.00
Foetuses affected (%)	41.83	38.69	41.03	44.09
Litters with foetuses with one or more variations
Total no.	37	42	38	39
Litters affected (%)	84.09	89.36	88.37	84.78
No of foetuses examined	251	305	273	313
No of litters examined	44	47	43	46

* Statistical analysis was not performed on this parameter

$ Statistical analysis was performed on these data after application of the Freeman-Tukey arcsine transformation

Conclusions:

Both the non-pregnant and pregnant rats in the 0.75, 1.00 and 1.25% groups ate slightly more than did the control group during most of the intervals measured, but not all the increases were statistically significant. There was no consistent pattern of increase or decrease in weight gain. No dose-related changes were found in maternal clinical findings, the average number of implantations, resorptions and viable foetuses, or foetal length or weight. Under the conditions of the study, there were no statistically significant increases as compared with the control group in the litter incidence regarding specific external, visceral or skeletal variations of the foetuses. Dietary calcium was neither foetotoxic nor teratogenic at the concentrations used.

Therefore, as no adverse effects were noted at this dose level (1.25% Ca in diet), the NOAEL for teratogenic and maternal toxic effects in rats is in excess of 1963 - 2188 mg/kg bw/day of calcium carbonate.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

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

A study in accordance with OECD Guideline 422 (Dunster, 2010) is available. The read-across substance and component of the test substance Calcium carbonate (nano) was administered by gavage to rats (Wistar, 10 animals/sex/group) for up to forty-eight consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. There were no treatment related effects observed on mating, fertility or gestation length at any dose level. The offspring litter size, viability, growth and development were all comparable to controls and no adverse effects were noted. Since no treatment-related effects were observed for reproduction, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.

A prenatal developmental toxicity study was performed to evaluate the developmental effects of moderate dietary calcium increases in rats fed nutritionally adequate diets (Shackelford et al, 1993). The test method used was not reported; however, it is considered to be similar to OECD Guideline 414. Female rats (CR CD/VAF) were given 0.50 (control), 0.75, 1.00 or 1.25% dietary calcium as calcium carbonate in AIN-76A diets for 6 weeks before mating, during mating and for 20 days of gestation. On gestation day 20, the animals were killed and caesarean sections were performed. No dose-related changes were found in maternal clinical findings, the average number of implantations, resorptions and viable foetuses, or foetal length or weight. There were no statistically significant increases as compared with the control group in the litter incidence regarding specific external, visceral or skeletal variations of the foetuses. Therefore, dietary calcium was neither fetotoxic nor teratogenic at the concentrations used. Since no adverse effects were noted at the highest dose level tested (1.25% Ca in diet), the NOAEL for teratogenic and maternal toxic effects in rats is in excess of 1.25% Ca, equivalent to approximately 1963 - 2188 mg/kg bw/day of calcium carbonate. A number of supporting studies are reported in the literature where calcium has been administered to pregnant women (Bogden et al, 1995 and Belizan et al, 1997). No teratogenic or reproductive effects were noted, indicating that calcium supplementation up to doses of 2 g/day are considered to be non-toxic. Based on the lack of effect upon reproduction or offspring development observed in these studies at dose levels well in excess of normal human exposure doses, calcium carbonate is not a significant risk to the reproductive process and further studies are unlikely to show any significant effects.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for reproductive and developmental toxicity under Regulation (EC) No 1272/2008, as amended for the eighteenth time in Regulation (EU) 2022/692.