Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
A number of supporting studies are reported in the literature where calcium has been administered to pregnant women. No teratogenic or reproductive effects were noted. However, the excessive consumption of antacid tablets during pregnancy can lead to neonatal hypercalcaemia. These effects were fully reversed following birth and hospital treatment.
These studies can be read across to SFL.
Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
26 February 2010 to 02 June 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study As this study is being used for read-across, the reliability has been amended to reflect this. Read-across from calcium carbonate to sugar factory lime (SFL) is justified on the following basis. SFL is primarily composed of inorganic substances. The major constituent is calcium carbonate, along with sand and a small amount of other inorganic salts (including calcium salts) and the remainder is composed of organic plant material. SFL is not classified for human health. Of its components, only calcium oxalate is classified as acutely toxic via the oral and dermal routes (category 4); however, this does not affect the overall classification of SFL. As a result, it is considered that the properties of SFL are governed by those of calcium carbonate. It is therefore considered appropriate for this data to be used for read-across purposes and any further testing would be scientifically unjustified.
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.
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
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.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
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.
Reproductive effects observed:
not specified
Conclusions:
No treatment-related effects were observed for reproduction, therefore, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Studies available for the main constituent and one other constituent.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Rationale for read across:

SFL is primarily composed of inorganic substances. The major constituent is calcium carbonate, along with silicon dioxide and a small amount of other inorganic salts (including calcium salts) and the remainder is composed of organic plant material. SFL is not classified for human health. Of its components, only calcium oxalate is classified as acutely toxic via the oral and dermal routes (category 4); however, it is present below the concentration limit that would result in classification of SFL. As a result, it is considered that the properties of SFL are governed by those of calcium carbonate.

It is therefore considered appropriate for this data to be used for read-across purposes and any further testing would be scientifically unjustified.

A 28 day repeat dose oral toxicity study combined with a reproduction/ developmental toxicity screening test was performed in the rat in accordance with OECD TG 422 (Dunster, 2010) and GLP. Calcium carbonate (nano) was administered by gavage to three groups, each of ten male and ten female Wistar rats, 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. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

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.

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.

Since this study can be read across to SFL, it is expected that SFL would not cause any reproductive toxicity.


Short description of key information:
Calcium carbonate showed no signs of reproduction/developmental toxicity in an OECD 422 reproduction/developmental screening toxicity test at doses up to 1000 mg/kg bw/day. The results of this study can be read across to SFL.

Justification for selection of Effect on fertility via oral route:
Study performed on main constituent of SFL

Effects on developmental toxicity

Description of key information
Calcium carbonate showed no signs of developmental toxicity in a prenatal developmental screening toxicity test. The results of this study can be read across to SFL.
Additional information

Rationale for read across:

SFL is primarily composed of inorganic substances. The major constituent is calcium carbonate, along with silicon dioxide and a small amount of other inorganic salts (including calcium salts) and the remainder is composed of organic plant material. SFL is not classified for human health. Of its components, only calcium oxalate is classified as acutely toxic via the oral and dermal routes (category 4); however, it is present below the concentration limit that would result in classification of SFL. As a result, it is considered that the properties of SFL are governed by those of calcium carbonate.

It is therefore considered appropriate for this data to be used for read-across purposes and any further testing would be scientifically unjustified.

 

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 TG 414. 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.

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.

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.

Since this study can be read across to SFL, it is expected that SFL would not cause any developmental toxicity.

 

Toxicity to reproduction: other studies

Additional information

All calcium carbonate toxicity studies are considered to be relevant to sugar factory lime (SFL) based on the fact that SFL is primarily composed of calcium carbonate and other calcium salts. SFL is not classified for human health. Of its components, only calcium oxalate is classified as acutely toxic via the oral and dermal routes (category 4); however, it is present below the concentration limit that would result in classification of SFL. As a result, it is considered that the properties of SFL are governed by those of calcium carbonate..

 

A study is available in the literature which investigates how dietary calcium and lead interact to modify maternal blood pressure, erythropoiesis and foetal neonatal growth in rats during pregnancy and lactation (Bogden et al, 1995). Female rats were fed dietary calcium carbonate at concentrations of 0.1, 0.5 and 2.5 g/100g. After one week the animals were mated and after pregnancy was confirmed the animals were fed drinking water containing either 0 or 250 mg/L of lead. This treatment was continued for the duration of pregnancy and for one week of lactation. Three control groups were fed the same diets without lead exposure.

The dietary calcium concentrations that were used did not interfere with the ability of the rats studied to maintain pregnancy and deliver normal pups.

 

A human study investigating the long term effect of calcium supplementation during pregnancy on the blood pressure of the offspring is reported in the literature (Belizan et al, 1997). The trial examined the effectiveness of 2 g of elemental calcium supplementation per day (four tablets of calcium carbonate 500 mg) for the prevention of hypertensive disorders of pregnancy. Supplementation was started at 20 weeks' gestation and continued until delivery.

The reports of women with anaemia, premature rupture of membranes, diabetes mellitus, third trimester haemorrhage, and receiving medical treatment, and the numbers of hospital admissions, inductions of labour, and caesarean sections were closely similar in the calcium and placebo groups.

At birth the weight, length, gestational age, and the rate of preterm delivery were closely similar between the two groups. A higher proportion of the newborn infants in the calcium group had an Apgar score <7 at the first minute (20/248 vs 10/251), and more newborns were admitted to the neonatal intensive care unit (38/248 vs 29/251) (not significant). However, these results were not statistically significant.

Therefore calcium supplementation at a dose of 2 g/day during pregnancy did not cause any adverse effects in either the mother or child.

 

Clinical cases are available in the literature which report the excessive ingestion of antacid tablets during pregnancy, potentially leading to the development of hypercalcaemia (milk-alkali syndrome). The amount of calcium carbonate ingested was significantly greater than that which would be consumed in the diet and from following the recommended guidelines for antacid consumption (one patient consumed 7,500 – 10,500 mg calcium carbonate/day from midway through the first trimester to the onset of labour). Following hospital treatment both the mother and child were reported to be fully recovered with no lasting side effects or teratogenic effects.

 

Justification for classification or non-classification

Under the conditions of the OECD TG 422 study, calcium carbonate administered to male and female rats up to a dose level of 1000 mg/kg bw/day for a period of up to 48 days is not toxic to reproduction and has no effect on fertility or development. No treatment-related effects were observed for reproduction; hence, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day. The prenatal developmental toxicity study also demonstrated that calcium carbonate was neither foetotoxic 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.

Based on these results, calcium carbonate is not a reproductive or developmental toxicant. Since this study can be read across to SFL, SFL would not warrant classification under either the DSD or CLP.

Additional information

Categories Display