Registration Dossier

Administrative data

Description of key information

Toxicity of calcium via the oral route is addressed by upper intake levels (UL) for adults determined by the Scientific Committee on Food (SCF), being UL = 2500 mg/d, corresponding to 36 mg/kg bw/d (70 kg person) for calcium.

Toxicity of calcium oxide via the dermal route is not considered as relevant in view of the anticipated negligible absorption through skin.

Toxicity of calcium oxide via inhalation (local effect, irritation of mucous membranes) is addressed by an IOELV of 1 mg/m³ respirable fraction (8h-TWA) (Commission Directive (EU) 2017/164 of 31 January 2017).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
3 August 2015-11 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
7 September, 2009
Deviations:
yes
Remarks:
Exposure period reduced to 14 days
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15-25ºC) dry, avoid CO2 uptake (the test material was kept under N2)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The study was conducted with albino rats. The rat was used because this species is normally used in toxicity studies of this type and is accepted by the relevant authorities. Wistar outbred (Crl:WI(Han)) were used. The Wistar rat strain was used because it is routinely used at the test facility for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: Approximately 7-8 weeks old
- Weight at study initiation: Mean body weight at the start of treatment on day 0 was 222 grams for males of the range finding study, and 239 and 165 grams for males and females of the main study, respectively.
- Fasting period before study:
- Housing: The rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the study. The animals were housed in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. After allocation, the animals were housed three (range finding study) or five (main study) animals to a cage. During exposure, the animals were kept individually in the exposure unit. Immediately after each exposure, the animals were returned to their home cages.
- Diet: ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: 12days for range-finding study and >= 13 days for main study.

DETAILS OF FOOD AND WATER QUALITY:
The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of VRF1 (FG)) diet is analysed by the supplier for nutrients and contaminants.
Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): Approx. 10/hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.34 - <= 2.74 µm
Remarks on MMAD:
The average (+/- standard deviation) mass median aerodynamic diameter (MMAD) of the low-, mid-, and high concentration test atmospheres were 2.34 (+/- 0.25), 2.67 (+/- 0.14) and 2.74 (+/- 0.22) µm, with corresponding average geometric standard deviations (+/- standard deviation) of 2.19 (+/- 0.27), 1.99 (+/- 0.11) and 2.16 (+/- 0.15), respectively. Thus, average particle size was within the range of 1 – 3 µm MMAD with a gsd in the range of 1.5 – 3.0, as recommended by OECD guideline 412.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Groenendijk Kunststoffen BV cylindrical polypropylene or steel column
- Method of holding animals in test chamber: Rodent tube
- Source and rate of air: compressed air
- Method of conditioning air: Humidified and filtered
- System of generating particulates/aerosols: Turntable dust feeder and eductor
- Temperature, humidity, pressure in air chamber: 22+/-3C, 30-70%, slight positive pressure
- Air flow rate: 1 litre/min per animal
- Method of particle size determination: Particle size distribution measurements were carried out using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure condition. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated.

TEST ATMOSPHERE
- Brief description of analytical method used: Filters weighed before and after loading with test atmosphere. Samples were taken at least three times per day for each exposure condition.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of the test material in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere at 4.6 Ln/min2 through fiber glass filters (Sartorius, 13400-47). During the 14-day study, samples of 207 (group 2), 92 (group 3) or 46 (group 4) Ln test atmosphere were obtained; during the 5-day range finding study, samples of 207 (group 2), 46 (group 3) or 23 (group 4) Ln test atmosphere were obtained. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter, by the volume of the sample taken. Samples were taken at least three times per day for each exposure condition. During preliminary experiments, it was determined whether correction of filter weights for possible hygroscopy or CO2 uptake was necessary. Although test material applied directly onto fiber glass filters increased about 35% in weight when kept at ambient conditions until a stable weight was reached (after two days), the weight gain occurred very slowly (<5% increase in weight within an hour after loading). This was confirmed by drying of a loaded gravimetric filter in a stream of dry nitrogen after sampling at a target concentration of 0.220 mg/L; the weight change of the test material on the filter was about 1.3% after drying for a day. Thus, it was concluded that correction of filter weights was not necessary as long as the gravimetric filters were weighed shortly after loading.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
Main study: 5 days/week over a 14-day period with a total number of 10 exposure days.
Dose / conc.:
0 mg/L air
Dose / conc.:
0.025 mg/L air (nominal)
Dose / conc.:
0.05 mg/L air (nominal)
Dose / conc.:
0.1 mg/L air (nominal)
No. of animals per sex per dose:
Main groups: 5 animals/sex/group.
Recovery groups (control and high dose groups): 5 animals/sex/group
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of a 5-day reange-finding study in which groups of 3 male rats were exposed to target concentrations of 0.025, 0.100 and 0.220 mg/L. Under the conditions of this range finding study, exposure to calcium dihydroxide at 0.220 mg/L resulted in substantial body weight loss, decreased food consumption, increased lung weights, and clinical abnormalities indicating respiratory distress. Changes at 0.100 mg/L were limited to slightly decreased growth and food intake; no exposure-related changes were observed at 0.025 mg/L. On the basis of these results the target concentrations for the main study were chosen at 0.025, 0.050 and 0.100 mg/L.
Two recovery groups, also consisting of 5 male and 5 female animals each, were simultaneously exposed with the main study animals of the control and top concentration groups,
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: On exposure days, each animal was observed daily in the morning, prior to exposure. All animals checked again after exposure. During exposure, a group-wise observation was made about half-way through the 6-h exposure period. On weekends, 1 check per day was made.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: See above

BODY WEIGHT: Yes
- Time schedule for examinations: In the range-finding study, the body weight of each animal was recorded 3 days before the start of the exposure. Subsequently the animals were weighed on the day of exposure (day 0) prior to the exposure and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.
In the main study, the body weight of each animals was recorded 4 days before the start of exposure. Subsequently, the animals were weighed prior to exposure on the first day (day 0) and every 3 or 4 days, including the day of scheduled sacrifice. The body weights measured on the day of scheduled sacrifice were used to calculate the organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption was measured per cage by weighing the feeders. The results were expressed in g per animal per day. For animals of the range finding study, food consumption was measured for a single period of 5 days, starting on day 0. For animals of the main study, the food consumption was measured over 7-day periods, starting on day 0 and finishing on the day of sacrifice.

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Bronchoalveolar lavage and measurements:
At necropsy, the lungs of animals of the main groups were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations.

Biochemical determinations:
The volume of the supernatant was determined. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), and gammaglutamyltransferase (GGT) were determined.

Cellular determinations:
Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).

Since exposure-related changes were observed in animals of the main groups, investigation of bronchoalveolar lavage parameters (biochemical and cellular determinations) was extended to animals of the recovery groups.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals of the main study were sacrificed on the day after the last exposure in such a sequence that the average time of killing was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 14-day recovery period (males and females on 10 and 11 November 2015, respectively). The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.
Organ weights:
The following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weight and the terminal body weight: adrenals; spleen; brain; testes; heart; left lung lobe; kidneys; thymus; liver.


HISTOPATHOLOGY: Yes
Tissue preservation:
The tissues and organs of all animals of the main study mentioned above, the complete respiratory tract of all animals and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin). The left lung (after weighing) was infused with the fixative under ca. 15 cm water pressure to ensure fixation. The nose was decalcified and embedded in paraffin for all rats of all groups. The carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded.
Histopathological examination:
The nose, larynx, trachea and left lung lobe of all animals of the control and the high concentration group (groups 1 and 4) were processed for histopathological examination. These tissues were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. All preserved tissues of the animals of the control group and high concentration group were examined histopathologically (by light microscopy). The nasopharyngeal tissues were examined at six levels, with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level included the base of the epiglottis), the trachea at three levels (including a longitudinal section through the carina of the bifurcation), and the unlavaged left lung lobe was examined at three levels.
Statistics:
Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ data transformation method. Day 0 body weight data were used as covariate in the analysis of the posttreatment data unless removed during data preprocessing.

Pre-treatment body weight, organ weight and bronchoalveolar lavage data: ‘Generalised Anova/Ancova Test’ with ‘Automatic’ data transformation method.
Food consumption: no statistics were applied on food intake (only one cage/sex).

Incidences of histopathological changes: Fisher’s exact probability test.

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) was greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male rat in the low concentration group had skin encrustations and a skin wound, which were not related to the exposure.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of the high concentration group showed a slightly, but statistically significantly reduced growth during the exposure period; average body weight was about 5% below controls by day 14. Complete recovery of growth changes was observed during the 2-week recovery period. Body weight data in males of the low and mid concentration group also showed occasional differences when compared to controls, but a clear concentration-response relationship or a consistent trend over time was absent (Tables attached).
There were no statistically significant differences in body weights of the female rats between the test groups and controls. A statistically significantly reduced body weight gain in females of the high concentration group during a single period (day 21-24) of the recovery phase was considered to be a chance finding (Tables attached).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the first two weeks, food consumption in male rats of the high concentration group was about 10% lower than in unexposed controls. This finding proved to be fully reversible within the recovery period. Food consumption in female rats was comparable among the groups throughout the study period (Table attached).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly increased relative weight of the heart was observed in males of the low (9.0%) and high (6.7% above controls) concentration group sacrificed at the end of the exposure period. However, a dose-response relationship was not found and absolute heart weights were not affected. No changes were observed in absolute or relative organ weights of female animals of the main study groups.
At the end of the recovery period, an increased absolute and relative weight of the adrenals was found in animals of the high concentration group. Since similar changes were not observed at the end of treatment period and organ weights were still well within the historical control range, this finding was not considered to be related to the exposure to the test material. In addition, an increased absolute weight of the spleen in males of the high concentration recovery group was considered to be an incidental finding (unrelated to the treatment), since changes in relative organ weight were absent and similar effects at the end of exposure or in females were not observed. (Tables attached).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy no treatment-related macroscopic changes were observed. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups (Table attached).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic evaluation of the organs and tissues did not reveal any treatment-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure to the test material (Table attached).
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences in bronchoalveolar lavage (BAL) parameters were observed between animals of the main groups exposed to the test material and unexposed controls (Tables attached):
- Increased levels of single biochemical parameters in animals of the high concentration group: the concentrations of lactate dehydrogenase (LDH) and gamma-glutamyltransferase (GGT) were increased in males, while the concentration of alkaline phosphatase (ALP) was increased in females of the high concentration group at the end of the exposure period. An increased protein content in females of the mid concentration group was considered to be a chance finding, since a dose-response relationship was absent.
- Increased absolute and relative numbers of eosinophils in males of the high concentration group. The percentage distribution of other white blood cells was, however, unaffected in these animals, with a relative contribution of macrophages of 97.6% (i.e. very close to 100% which is normally observed in healthy animals). Female animals did not show any exposurerelated changes in total or differential white blood cell numbers.

Since statistically significant changes were observed in animals of the main groups, bronchoalveolar lavage parameters were also examined in animals of the recovery groups (control and high concentration). No exposure-related changes were observed in any of the parameters investigated at the end of the recovery period. A slightly increased protein level in BAL fluid of females of the high concentration group was considered to be a chance finding, since similar changes were not observed at the end of the treatment period and the finding was not substantiated by any changes in other parameters.
Key result
Dose descriptor:
NOAEC
Effect level:
0.107 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: BAL parameters

Actual concentration of the test material in the test atmospheres as determined by gravimetric analysis

 

Group 2

(0.025 mg/L)

Group 3

(0.050 mg/L)

Group 4

(0.100 mg/L)

Date

(dd-mm-yyyy)

Average (mg/L)

sd

n

Average (mg/L)

sd

n

Average (mg/L)

sd

n

13-10-2015

0.025

0.001

3

0.051

0.007

3

0.104

0.006

3

14-10-2015

0.025

0.004

3

0.051

0.008

4

0.104

0.006

4

15-10-2015

0.025

0.001

3

0.051

0.023

4

0.101

0.007

3

16-10-2015

0.023

0.002

3

0.051

0.024

3

0.121

0.008

3

19-10-2015

0.025

0.001

3

0.053

0.003

3

0.127

0.017

3

20-10-2015

0.024

0.002

3

0.049

0.004

3

0.102

0.001

3

21-10-2015

0.023

0.003

3

0.050

0.006

3

0.104

0.007

3

22-10-2015

0.025

0.001

3

0.050

0.005

3

0.101

0.011

3

23-10-2015

0.026

0.003

3

0.047

0.004

3

0.101

0.004

3

26-10-2015

0.024

0.001

3

0.049

0.003

3

0.098

0.016

3

27-10-2015

0.027

0.003

3

0.053

0.016

3

0.110

0.021

3

Average

sd

0.025

0.001

 

 

0.050

0.002

 

 

0.107

0.009

 

 

 

Conclusions:
Under the conditions of the current study, inhalation exposure to calcium dihydroxide resulted in a few modest changes at the highest concentration tested (slightly decreased growth and food consumption in males and a minor increase in bronchoalveolar lavage parameters). No exposure-related changes were observed at lower concentrations. Since the changes at the high concentration - which were fully reversible within a 14-day recovery period - were considered not to constitute adverse effects, the No-Observed-Adverse-Effect-Concentration (NOAEC) for sub-acute exposure of rats to calcium dihydroxide was placed at 0.107 mg/L.
Executive summary:

The aim of the present study was to provide data on the toxicity of calcium dihydroxide upon repeated inhalation exposure as investigated in a sub-acute (14day) toxicity study in rats. Four main groups of 5 male and 5 female rats each were exposed nose-only to concentrations of 0 (control), 0.025 (± 0.001), 0.050 (± 0.002), or 0.107 (± 0.009) mg/L calcium dihydroxide for 6 hours/day, 5 days/week over a 14-day period, with a total number of 10 exposure days. The average particle size (MMAD) of the aerosol was 2.34 µm (gsd of 2.19), 2.67 µm (gsd of 1.99) and 2.74 µm (gsd of 2.16) for the low, mid and high concentration test atmospheres, respectively. Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 5 male and 5 female animals each, were exposed together with the animals of the control and high concentration groups, and were sacrificed after a 2-week recovery period following the exposure period. The target concentration levels for this sub-acute study were selected on the basis of the results of a preceding 5-day range finding study, in which groups of animals (males only) were exposed to calcium dihydroxide at target concentrations of 0 (control), 0.025, 0.100 or 0.220 mg/L. Exposure at 0.220 mg/L resulted in substantial body weight loss, decreased food consumption, increased lung weights, and clinical abnormalities indicating respiratory distress. Changes at 0.100 mg/L were limited to slightly decreased growth and food intake; no exposure-related changes were observed at 0.025 mg/L. The exposure to the test material was well tolerated by the animals. No treatmentrelated clinical abnormalities were observed. In males of the high concentration group, a slightly decreased growth and food consumption was observed during the exposure period. These changes were not observed in females and were fully reversible within the 14-recovery period. Exposure to the test substance resulted in minimal and reversible changes in single bronchoalveolar lavage (BAL) parameters in animals exposed to the high concentration. The changes consisted of minor elevations in the levels of ALP (females only), GGT and LDH (males only), and a very slight increase in the number of eosinophils (males only). Although these changes in BAL parameters may well be related to the exposure, they were not considered to be adverse changes, because: 1) the difference with controls was small, 2) they occurred in single parameters only without consistent elevations in other parameters or in the other sex, 3) the changes were transient and were no longer observed at the end of the recovery period, and 4) they were not associated with any concomitant changes in lung weight or any histopathological changes in the respiratory tract. Under the conditions of the current study, inhalation exposure to calcium dihydroxide resulted in a few modest changes at the highest concentration tested (slightly decreased growth and food consumption in males and a minor increase in bronchoalveolar lavage parameters). No exposure-related changes were observed at lower concentrations. Since the changes at the high concentration - which were fully reversible within a 14-day recovery period - were considered not to constitute adverse effects, the No-Observed-Adverse-Effect-Concentration (NOAEC) for sub-acute exposure of rats to calcium dihydroxide was placed at 0.107 mg/L.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Calcium oxide (Target substance) hydrolyses to calcium dihydroxide (Source) in aqueous systems, Both the Source and the Target will have a common mode of action upon inhalation, i.e. local effects due to irritation caused by alkalinity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Calcium dihydroxide (EC 215-137-3).
Target: Calcium oxide (EC 215-138-9). Purity/impurity profile as described in Section 1.2

3. ANALOGUE APPROACH JUSTIFICATION
Calcium oxide will hydrolyse rapidly in the respiratory tract to calcium dihydroxide. The pH for a solution of calcium dihydroxide (12.4) is almost identical to that of 12.3 for calcium oxide (See RSS for water solubility; Fox 2010). Both substances are classified as Skin Irritant 2 (H315: Causes skin irritation) and STOT SE3 (H335: May cause respiratory irritation).
The Scientific Committee on Occupational Exposure Limits in their recommendation for calcium oxide and calcium hydroxide (SCOEL/SUM/137, February 2008) recognised that the effects of inhalation of calcium oxide or calcium dihydroxide are local only and are due to the high alkalinity of the solutions formed. Systemic effects of calcium oxide and calcium dihydroxide are negligible at normal occupational exposure levels, since exposure by inhalation (7.1 and 5.4 mg Ca/day, respectively) adds a negligible body burden of calcium compared to the tolerable upper intake level of 2500 mg/day.
Based the almost identical pH, the rapid hydrolysis of calcium oxide to calcium dihydroxide and similar skin and respiratory irritation potentials, it is concluded that it is acceptable to read-across from calcium dihydroxide (Source) to calcium oxide (Target) for repeated dose inhalation toxicity.
Reason / purpose for cross-reference:
read-across source
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEC
Effect level:
0.107 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: BAL parameters
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
107 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
3 August 2015-11 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
7 September, 2009
Deviations:
yes
Remarks:
Exposure period reduced to 14 days
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15-25ºC) dry, avoid CO2 uptake (the test material was kept under N2)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The study was conducted with albino rats. The rat was used because this species is normally used in toxicity studies of this type and is accepted by the relevant authorities. Wistar outbred (Crl:WI(Han)) were used. The Wistar rat strain was used because it is routinely used at the test facility for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: Approximately 7-8 weeks old
- Weight at study initiation: Mean body weight at the start of treatment on day 0 was 222 grams for males of the range finding study, and 239 and 165 grams for males and females of the main study, respectively.
- Fasting period before study:
- Housing: The rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the study. The animals were housed in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. After allocation, the animals were housed three (range finding study) or five (main study) animals to a cage. During exposure, the animals were kept individually in the exposure unit. Immediately after each exposure, the animals were returned to their home cages.
- Diet: ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: 12days for range-finding study and >= 13 days for main study.

DETAILS OF FOOD AND WATER QUALITY:
The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of VRF1 (FG)) diet is analysed by the supplier for nutrients and contaminants.
Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): Approx. 10/hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.34 - <= 2.74 µm
Remarks on MMAD:
The average (+/- standard deviation) mass median aerodynamic diameter (MMAD) of the low-, mid-, and high concentration test atmospheres were 2.34 (+/- 0.25), 2.67 (+/- 0.14) and 2.74 (+/- 0.22) µm, with corresponding average geometric standard deviations (+/- standard deviation) of 2.19 (+/- 0.27), 1.99 (+/- 0.11) and 2.16 (+/- 0.15), respectively. Thus, average particle size was within the range of 1 – 3 µm MMAD with a gsd in the range of 1.5 – 3.0, as recommended by OECD guideline 412.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Groenendijk Kunststoffen BV cylindrical polypropylene or steel column
- Method of holding animals in test chamber: Rodent tube
- Source and rate of air: compressed air
- Method of conditioning air: Humidified and filtered
- System of generating particulates/aerosols: Turntable dust feeder and eductor
- Temperature, humidity, pressure in air chamber: 22+/-3C, 30-70%, slight positive pressure
- Air flow rate: 1 litre/min per animal
- Method of particle size determination: Particle size distribution measurements were carried out using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure condition. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated.

TEST ATMOSPHERE
- Brief description of analytical method used: Filters weighed before and after loading with test atmosphere. Samples were taken at least three times per day for each exposure condition.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of the test material in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere at 4.6 Ln/min2 through fiber glass filters (Sartorius, 13400-47). During the 14-day study, samples of 207 (group 2), 92 (group 3) or 46 (group 4) Ln test atmosphere were obtained; during the 5-day range finding study, samples of 207 (group 2), 46 (group 3) or 23 (group 4) Ln test atmosphere were obtained. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter, by the volume of the sample taken. Samples were taken at least three times per day for each exposure condition. During preliminary experiments, it was determined whether correction of filter weights for possible hygroscopy or CO2 uptake was necessary. Although test material applied directly onto fiber glass filters increased about 35% in weight when kept at ambient conditions until a stable weight was reached (after two days), the weight gain occurred very slowly (<5% increase in weight within an hour after loading). This was confirmed by drying of a loaded gravimetric filter in a stream of dry nitrogen after sampling at a target concentration of 0.220 mg/L; the weight change of the test material on the filter was about 1.3% after drying for a day. Thus, it was concluded that correction of filter weights was not necessary as long as the gravimetric filters were weighed shortly after loading.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
Main study: 5 days/week over a 14-day period with a total number of 10 exposure days.
Dose / conc.:
0 mg/L air
Dose / conc.:
0.025 mg/L air (nominal)
Dose / conc.:
0.05 mg/L air (nominal)
Dose / conc.:
0.1 mg/L air (nominal)
No. of animals per sex per dose:
Main groups: 5 animals/sex/group.
Recovery groups (control and high dose groups): 5 animals/sex/group
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on the results of a 5-day reange-finding study in which groups of 3 male rats were exposed to target concentrations of 0.025, 0.100 and 0.220 mg/L. Under the conditions of this range finding study, exposure to calcium dihydroxide at 0.220 mg/L resulted in substantial body weight loss, decreased food consumption, increased lung weights, and clinical abnormalities indicating respiratory distress. Changes at 0.100 mg/L were limited to slightly decreased growth and food intake; no exposure-related changes were observed at 0.025 mg/L. On the basis of these results the target concentrations for the main study were chosen at 0.025, 0.050 and 0.100 mg/L.
Two recovery groups, also consisting of 5 male and 5 female animals each, were simultaneously exposed with the main study animals of the control and top concentration groups,
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: On exposure days, each animal was observed daily in the morning, prior to exposure. All animals checked again after exposure. During exposure, a group-wise observation was made about half-way through the 6-h exposure period. On weekends, 1 check per day was made.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: See above

BODY WEIGHT: Yes
- Time schedule for examinations: In the range-finding study, the body weight of each animal was recorded 3 days before the start of the exposure. Subsequently the animals were weighed on the day of exposure (day 0) prior to the exposure and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.
In the main study, the body weight of each animals was recorded 4 days before the start of exposure. Subsequently, the animals were weighed prior to exposure on the first day (day 0) and every 3 or 4 days, including the day of scheduled sacrifice. The body weights measured on the day of scheduled sacrifice were used to calculate the organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption was measured per cage by weighing the feeders. The results were expressed in g per animal per day. For animals of the range finding study, food consumption was measured for a single period of 5 days, starting on day 0. For animals of the main study, the food consumption was measured over 7-day periods, starting on day 0 and finishing on the day of sacrifice.

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Bronchoalveolar lavage and measurements:
At necropsy, the lungs of animals of the main groups were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations.

Biochemical determinations:
The volume of the supernatant was determined. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), and gammaglutamyltransferase (GGT) were determined.

Cellular determinations:
Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).

Since exposure-related changes were observed in animals of the main groups, investigation of bronchoalveolar lavage parameters (biochemical and cellular determinations) was extended to animals of the recovery groups.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals of the main study were sacrificed on the day after the last exposure in such a sequence that the average time of killing was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 14-day recovery period (males and females on 10 and 11 November 2015, respectively). The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.
Organ weights:
The following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weight and the terminal body weight: adrenals; spleen; brain; testes; heart; left lung lobe; kidneys; thymus; liver.


HISTOPATHOLOGY: Yes
Tissue preservation:
The tissues and organs of all animals of the main study mentioned above, the complete respiratory tract of all animals and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin). The left lung (after weighing) was infused with the fixative under ca. 15 cm water pressure to ensure fixation. The nose was decalcified and embedded in paraffin for all rats of all groups. The carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded.
Histopathological examination:
The nose, larynx, trachea and left lung lobe of all animals of the control and the high concentration group (groups 1 and 4) were processed for histopathological examination. These tissues were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. All preserved tissues of the animals of the control group and high concentration group were examined histopathologically (by light microscopy). The nasopharyngeal tissues were examined at six levels, with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level included the base of the epiglottis), the trachea at three levels (including a longitudinal section through the carina of the bifurcation), and the unlavaged left lung lobe was examined at three levels.
Statistics:
Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ data transformation method. Day 0 body weight data were used as covariate in the analysis of the posttreatment data unless removed during data preprocessing.

Pre-treatment body weight, organ weight and bronchoalveolar lavage data: ‘Generalised Anova/Ancova Test’ with ‘Automatic’ data transformation method.
Food consumption: no statistics were applied on food intake (only one cage/sex).

Incidences of histopathological changes: Fisher’s exact probability test.

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) was greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male rat in the low concentration group had skin encrustations and a skin wound, which were not related to the exposure.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of the high concentration group showed a slightly, but statistically significantly reduced growth during the exposure period; average body weight was about 5% below controls by day 14. Complete recovery of growth changes was observed during the 2-week recovery period. Body weight data in males of the low and mid concentration group also showed occasional differences when compared to controls, but a clear concentration-response relationship or a consistent trend over time was absent (Tables attached).
There were no statistically significant differences in body weights of the female rats between the test groups and controls. A statistically significantly reduced body weight gain in females of the high concentration group during a single period (day 21-24) of the recovery phase was considered to be a chance finding (Tables attached).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the first two weeks, food consumption in male rats of the high concentration group was about 10% lower than in unexposed controls. This finding proved to be fully reversible within the recovery period. Food consumption in female rats was comparable among the groups throughout the study period (Table attached).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
A slightly increased relative weight of the heart was observed in males of the low (9.0%) and high (6.7% above controls) concentration group sacrificed at the end of the exposure period. However, a dose-response relationship was not found and absolute heart weights were not affected. No changes were observed in absolute or relative organ weights of female animals of the main study groups.
At the end of the recovery period, an increased absolute and relative weight of the adrenals was found in animals of the high concentration group. Since similar changes were not observed at the end of treatment period and organ weights were still well within the historical control range, this finding was not considered to be related to the exposure to the test material. In addition, an increased absolute weight of the spleen in males of the high concentration recovery group was considered to be an incidental finding (unrelated to the treatment), since changes in relative organ weight were absent and similar effects at the end of exposure or in females were not observed. (Tables attached).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy no treatment-related macroscopic changes were observed. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups (Table attached).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic evaluation of the organs and tissues did not reveal any treatment-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure to the test material (Table attached).
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences in bronchoalveolar lavage (BAL) parameters were observed between animals of the main groups exposed to the test material and unexposed controls (Tables attached):
- Increased levels of single biochemical parameters in animals of the high concentration group: the concentrations of lactate dehydrogenase (LDH) and gamma-glutamyltransferase (GGT) were increased in males, while the concentration of alkaline phosphatase (ALP) was increased in females of the high concentration group at the end of the exposure period. An increased protein content in females of the mid concentration group was considered to be a chance finding, since a dose-response relationship was absent.
- Increased absolute and relative numbers of eosinophils in males of the high concentration group. The percentage distribution of other white blood cells was, however, unaffected in these animals, with a relative contribution of macrophages of 97.6% (i.e. very close to 100% which is normally observed in healthy animals). Female animals did not show any exposurerelated changes in total or differential white blood cell numbers.

Since statistically significant changes were observed in animals of the main groups, bronchoalveolar lavage parameters were also examined in animals of the recovery groups (control and high concentration). No exposure-related changes were observed in any of the parameters investigated at the end of the recovery period. A slightly increased protein level in BAL fluid of females of the high concentration group was considered to be a chance finding, since similar changes were not observed at the end of the treatment period and the finding was not substantiated by any changes in other parameters.
Key result
Dose descriptor:
NOAEC
Effect level:
0.107 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: BAL parameters

Actual concentration of the test material in the test atmospheres as determined by gravimetric analysis

 

Group 2

(0.025 mg/L)

Group 3

(0.050 mg/L)

Group 4

(0.100 mg/L)

Date

(dd-mm-yyyy)

Average (mg/L)

sd

n

Average (mg/L)

sd

n

Average (mg/L)

sd

n

13-10-2015

0.025

0.001

3

0.051

0.007

3

0.104

0.006

3

14-10-2015

0.025

0.004

3

0.051

0.008

4

0.104

0.006

4

15-10-2015

0.025

0.001

3

0.051

0.023

4

0.101

0.007

3

16-10-2015

0.023

0.002

3

0.051

0.024

3

0.121

0.008

3

19-10-2015

0.025

0.001

3

0.053

0.003

3

0.127

0.017

3

20-10-2015

0.024

0.002

3

0.049

0.004

3

0.102

0.001

3

21-10-2015

0.023

0.003

3

0.050

0.006

3

0.104

0.007

3

22-10-2015

0.025

0.001

3

0.050

0.005

3

0.101

0.011

3

23-10-2015

0.026

0.003

3

0.047

0.004

3

0.101

0.004

3

26-10-2015

0.024

0.001

3

0.049

0.003

3

0.098

0.016

3

27-10-2015

0.027

0.003

3

0.053

0.016

3

0.110

0.021

3

Average

sd

0.025

0.001

 

 

0.050

0.002

 

 

0.107

0.009

 

 

 

Conclusions:
Under the conditions of the current study, inhalation exposure to calcium dihydroxide resulted in a few modest changes at the highest concentration tested (slightly decreased growth and food consumption in males and a minor increase in bronchoalveolar lavage parameters). No exposure-related changes were observed at lower concentrations. Since the changes at the high concentration - which were fully reversible within a 14-day recovery period - were considered not to constitute adverse effects, the No-Observed-Adverse-Effect-Concentration (NOAEC) for sub-acute exposure of rats to calcium dihydroxide was placed at 0.107 mg/L.
Executive summary:

The aim of the present study was to provide data on the toxicity of calcium dihydroxide upon repeated inhalation exposure as investigated in a sub-acute (14day) toxicity study in rats. Four main groups of 5 male and 5 female rats each were exposed nose-only to concentrations of 0 (control), 0.025 (± 0.001), 0.050 (± 0.002), or 0.107 (± 0.009) mg/L calcium dihydroxide for 6 hours/day, 5 days/week over a 14-day period, with a total number of 10 exposure days. The average particle size (MMAD) of the aerosol was 2.34 µm (gsd of 2.19), 2.67 µm (gsd of 1.99) and 2.74 µm (gsd of 2.16) for the low, mid and high concentration test atmospheres, respectively. Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 5 male and 5 female animals each, were exposed together with the animals of the control and high concentration groups, and were sacrificed after a 2-week recovery period following the exposure period. The target concentration levels for this sub-acute study were selected on the basis of the results of a preceding 5-day range finding study, in which groups of animals (males only) were exposed to calcium dihydroxide at target concentrations of 0 (control), 0.025, 0.100 or 0.220 mg/L. Exposure at 0.220 mg/L resulted in substantial body weight loss, decreased food consumption, increased lung weights, and clinical abnormalities indicating respiratory distress. Changes at 0.100 mg/L were limited to slightly decreased growth and food intake; no exposure-related changes were observed at 0.025 mg/L. The exposure to the test material was well tolerated by the animals. No treatmentrelated clinical abnormalities were observed. In males of the high concentration group, a slightly decreased growth and food consumption was observed during the exposure period. These changes were not observed in females and were fully reversible within the 14-recovery period. Exposure to the test substance resulted in minimal and reversible changes in single bronchoalveolar lavage (BAL) parameters in animals exposed to the high concentration. The changes consisted of minor elevations in the levels of ALP (females only), GGT and LDH (males only), and a very slight increase in the number of eosinophils (males only). Although these changes in BAL parameters may well be related to the exposure, they were not considered to be adverse changes, because: 1) the difference with controls was small, 2) they occurred in single parameters only without consistent elevations in other parameters or in the other sex, 3) the changes were transient and were no longer observed at the end of the recovery period, and 4) they were not associated with any concomitant changes in lung weight or any histopathological changes in the respiratory tract. Under the conditions of the current study, inhalation exposure to calcium dihydroxide resulted in a few modest changes at the highest concentration tested (slightly decreased growth and food consumption in males and a minor increase in bronchoalveolar lavage parameters). No exposure-related changes were observed at lower concentrations. Since the changes at the high concentration - which were fully reversible within a 14-day recovery period - were considered not to constitute adverse effects, the No-Observed-Adverse-Effect-Concentration (NOAEC) for sub-acute exposure of rats to calcium dihydroxide was placed at 0.107 mg/L.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Calcium oxide (Target substance) hydrolyses to calcium dihydroxide (Source) in aqueous systems, Both the Source and the Target will have a common mode of action upon inhalation, i.e. local effects due to irritation caused by alkalinity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Calcium dihydroxide (EC 215-137-3).
Target: Calcium oxide (EC 215-138-9). Purity/impurity profile as described in Section 1.2

3. ANALOGUE APPROACH JUSTIFICATION
Calcium oxide will hydrolyse rapidly in the respiratory tract to calcium dihydroxide. The pH for a solution of calcium dihydroxide (12.4) is almost identical to that of 12.3 for calcium oxide (See RSS for water solubility; Fox 2010). Both substances are classified as Skin Irritant 2 (H315: Causes skin irritation) and STOT SE3 (H335: May cause respiratory irritation).
The Scientific Committee on Occupational Exposure Limits in their recommendation for calcium oxide and calcium hydroxide (SCOEL/SUM/137, February 2008) recognised that the effects of inhalation of calcium oxide or calcium dihydroxide are local only and are due to the high alkalinity of the solutions formed. Systemic effects of calcium oxide and calcium dihydroxide are negligible at normal occupational exposure levels, since exposure by inhalation (7.1 and 5.4 mg Ca/day, respectively) adds a negligible body burden of calcium compared to the tolerable upper intake level of 2500 mg/day.
Based the almost identical pH, the rapid hydrolysis of calcium oxide to calcium dihydroxide and similar skin and respiratory irritation potentials, it is concluded that it is acceptable to read-across from calcium dihydroxide (Source) to calcium oxide (Target) for repeated dose inhalation toxicity.
Reason / purpose for cross-reference:
read-across source
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Key result
Dose descriptor:
NOAEC
Effect level:
0.107 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: BAL parameters
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
107 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose oral toxicity:

No studies have been performed using calcium oxide. There are five animal studies available in the literature for various calcium salts, but none of these allow the derivation of a DNEL. In addition, results are available from an OECD TG 422 study performed using calcium carbonate [Dunster, 2010]. No effects related to treatment were observed at any dose up to the limit tested of 1000 mg/kg bw/day.

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 different intervention studies of long duration in adults, some of which were placebo controlled and in which total daily calcium intakes of 2500 mg from both diet and supplements were generally tolerated without adverse effects.

The document prepared by the Scientific Committee on Food describes a number of potential adverse effects following calcium supplementation. These might include the following:

1). Kidney function: Some studies have indicated that perimenopausal women with total calcium intakes between 2 and 3 g/day may show a tendency for compromised glomerular function as indicated by increases in serum creatinine. However, no such effect was observed in another study with women receiving comparable calcium amounts. Therefore, it is not clear whether this finding can be attributed to the ingestion of calcium.

2). Milk-alkali syndrome: Manifestation of the milk-alkali syndrome through the combined intake of calcium both from food and especially from supplements and of absorbable alkalinising substances is facilitated by renal insufficiency, alkalosis and dehydration due to vomiting and anorexia and/or the use of thiazide diuretics, which increase renal tubular calcium reabsorption. All reported cases of milk-alkali syndrome in association with the prolonged or acute ingestion of calcium supplements used calcium carbonate as the nutrient source. In these reports the supplemental calcium intakes were reported as between 1.0 and 23 g/day. These patients also differ in their medical history, use and duration of use of drugs and alkali consumption, and their diets. Their dietary calcium intakes are often not known.

The use of calcium carbonate supplements in doses up to 2000 mg/day, and thereby achieving total daily calcium intakes up to more than 3000 mg/day, for preventive purposes in presumably healthy subjects, has not provoked the development of the milk-alkali syndrome, whereas the administration of large amounts (11.2 g calcium/day) of calcium carbonate in addition to large amounts of milk (1.8 g calcium/day) over 7 days to 20 gastric/duodenal ulcer patients resulted in reversible hypercalcaemia (2.8 mmol/L) in nine patients and renal insufficiency in all.

In conclusion, based on the available evidence, a calcium dose which by itself might cause milk-alkali syndrome cannot be identified. The primary cause of milk-alkali syndrome appears to be when significantly large doses of calcium are ingested and is more prevalent in those people suffering from renal insufficiency.

3). Kidney stones: Observational studies on the relationship between total calcium intake and kidney stone incidence and interventional studies with calcium supplements do not allow definition of a calcium intake on a population basis which promotes kidney stone formation. On dietary calcium intakes in the range of the recommended dietary intake the risk of nephrolithiasis is determined by other dietary components and by genetic factors.

4). Interaction with minerals: Single-dose experiments demonstrate interference of both dietary and supplemental calcium with the absorption of other minerals. However, this effect is not demonstrable in long-term observational and interventional studies at dietary calcium intakes in the range of recommended intakes and at supplemental calcium of up to 2000 mg/day in adults and up to 1200 mg/day in one study with infants.

 In conclusion, calcium ingestion in humans up to the tolerable upper intake level of 2500 mg calcium per day does not cause any adverse effects. Effects are only seen when this dose is significantly exceeded. For a 70 kg person, this upper intake level is equivalent to a dose of 36 mg Ca/kg bw/day.

Repeated dose inhalation toxicity:

Results from a 14-day repeated dose inhalation study in rats, which included a 5-day range-finding test are available for the read-across substance calcium dihydroxide (van Triel, 2016).

In the 5-day study, rats were exposed to calcium dihydroxide at measured levels of 0.027, 0.110 or 0.224 mg/L. Findings at the mid concentration level were limited to decreased food intake and slight body weight loss (the latter probably related to the former); these effects were considered not to be evidence of significant toxicity. Animals of the high concentration group showed more severe effects: several clinical signs of discomfort (altered breathing and encrustations on the nose/eyes), markedly reduced food consumption, substantial loss of body weight, and an increased weight of the lungs at necropsy. The histopathology results of the respiratory tract tissues showed no adverse effects. The No-Observed-Adverse-Effect-Concentration (NOAEC) for sub-acute (5-day) exposure of rats to calcium dihydroxide was placed at 0.110 mg/L.

In the 14-day study, rats were exposed to calcium dihydroxide at measured levels of 0.025, 0.05 or 0.107 mg/L. Inhalation exposure to calcium dihydroxide at 0.107 mg/L resulted in a few modest changes (slightly decreased food consumption and weight gain in males and a minor increase in bronchoalveolar lavage parameters). No exposure-related changes were observed at the lower concentrations. Macroscopic examination at scheduled termination revealed no treatment-related gross changes. In addition, microscopic examination of the tissues of the respiratory tract did not reveal any histopathological changes which were attributable to the exposure to the test material. Since the changes at the high concentration, which were fully reversible within a 14-day recovery period, were considered not to constitute adverse effects, the NOAEC for sub-acute (14-day) exposure of rats to calcium dihydroxide was placed at 0.107 mg/L (the highest concentration tested).

These results indicate that the severity of effects seen in an inhalation study using calcium dihydroxide are concentration-dependent rather than duration-dependent, as would be expected in the case where local effects are caused by the alkalinity of the substance in solution.

A sub-chronic (13week) repeated dose inhalation study is available for calcium carbonate (nano) in rats. Five main groups of 10 male and 10 female rats each were exposed by nose-only inhalation exposure to 0 (control), 0.026 (±0.002), 0.123 (±0.006), 0.212 (±0.013) or 0.399 (±0.019) mg/L calcium carbonate (nano)for 6 hours/day, 5 days/week over a 13-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 10 male and 10 female animals each were exposed together with the animals of the control and top concentration groups, and were sacrificed after a 4week recovery period following the exposure period.

The exposure conditions were close to their respective targets. The aerodynamic particle size distribution of the test atmospheres was highly comparable across the groups with an average mass median aerodynamic diameter (MMAD) in the range of 1.29 – 1.35 µm and a geometric standard deviation (gsd) of 1.52 – 1.54. The relative contribution of nanoparticles (< 100 nm) in the various test atmospheres was determined to be very low. Scanning electron microscopy of aerosol samples confirmed that the particles were primarily present in agglomerates, which varied in size (ranging 60 nm – 30 µm, with trace amounts of primary particles) and shape, with little to no difference between the groups.

The exposure to the test material was well tolerated by the animals. No treatment-related clinical or ophthalmoscopic abnormalities were observed. A transient decrease in growth (females) or food consumption (males), observed in the top concentration group shortly after the initiation of exposure, was no longer observed after a few weeks. Haematology results, clinical chemistry analysis and necropsy findings did not show any treatment-related changes. No indications for systemic toxicity of inhaled calcium carbonate (nano) were observed in this study. 

Exposure to the test material resulted in local changes in the lower airways. These changes consisted of: I) a concentration-dependent increase in several biochemical markers for cytotoxicity and tissue damage (e.g. ALP, GGT, LDH, total protein) in bronchoalveolar lavage (BAL) fluid of animals of the mid, high and top concentration main groups; II) slight changes in differential white blood cell numbers in BAL fluid of animals of the high and top concentration main groups, characterized by an increase in the number of neutrophils and – for females of the top concentration group only – a slight increase in the number of lymphocytes; and III) an increased lung weight in males and females of the top concentration main group. These findings were not accompanied by any microscopic changes in the lungs; histopathology did not reveal any treatment-related changes in the respiratory tract (or in any other tissues) [1]. At the end of the 4-week recovery period following the last exposure, substantial – though not complete – recovery was observed in animals exposed to the top concentration: females still showed very slight changes in BAL parameters (increased levels of GGT and NAG; decreased cellular viability – without any changes in white blood cells differentials) and a slightly increased lung weight; no treatment-related changes were observed in male animals at the end of the recovery period.

Given the convergence of changes in pulmonary toxicological endpoints at the top concentration level – increased lung weights accompanied by increases in BALderived inflammation and cytotoxicity biomarkers, which (in females) were not fully reversible within a 4-week recovery period–exposure to 0.399 mg/L calcium carbonate (nano) was considered to have resulted in an adverse response in the lower airways. Exposure to 0.212mg/L calcium carbonate (nano) resulted in very limited alterations in BAL parameters only. These findings were not substantiated by any concomitant changes in lung weight or treatment-related histopathology. Therefore, the findings at the high concentration level were considered to be of no toxicological relevance and were judged as non-adverse.

Under the conditions of the current study, inhalation exposure to 0.399 mg/L Calcium carbonate (nano) resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BALderived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4week recovery period after the last exposure. Based on these observations, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects of sub-chronic inhalation exposure to calcium carbonate (nano) was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the No-Observed-Effect-Concentration (NOEC) for systemic effects was 0.399 mg/L.

[1] BAL measurements are usually a rather sensitive toxicological read-out parameter, and it is not uncommon to observe treatment-related changes at concentrations below a level at which histopathology is induced.

Based on the adopted Recommendation from the Scientific Committee on Occupational Exposure Limits (SCOEL) for Calcium oxide (CaO) and Calcium hydroxide (Ca(OH)2) the European Commission has established an IOELV of 1 mg/m³ respirable fraction (8h-TWA) for both calcium dihydroxide and calcium oxide. (Commission Directive (EU) 2017/164 of 31 January 2017). This is considered to be protective against adverse effects in case of long-term exposure to calcium dihydroxide and calcium oxide.

Effects upon inhalation of calcium oxide are purely local, i.e. irritation provoked by a pH shift. Hence, this value will also be protective for grades of calcium oxide containing up to 35% calcium carbonate, where the increased amount of carbonate would be expected to produce a lower pH shift compared to the oxide.

Repeated dose dermal toxicity:

No data are available. Based on its physico-chemical properties, absorption through the skin of calcium oxide is not expected to occur to any significant extent and performing a study is considered scientifically unjustified.

Justification for classification or non-classification

No systemic toxicological findings could be detected in rats after repeated administration of calcium carbonate by the oral route for a period of 28 days at up to 1000 mg/kg bw/day (equivalent to 400 mg Ca/kg bw/day). A number of potential adverse effects have been reported following calcium supplementation e.g. effects on kidney function, milk-alkali syndrome, kidney stones and interactions with minerals. However, these effects are more prevalent in those people suffering from renal insufficiency and following the ingestion of high doses of calcium (well above the recommended classification limits for STOT RE as defined in the Guidance on the Application of Regulation (EC) No 1272/2008). Therefore, a classification as STOT RE (oral) is not justified and no classification is proposed.

For inhalation, the NOAEC values after 5-day or 14-day repeat dose exposures to the read across substance calcium dihydroxide were 0.110 mg/L and 0.107 mg/L (the highest concentration tested), respectively. It is concluded that local effects due to exposure to calcium dihydroxide are related to pH shift and are concentration-dependent rather than dose dependent. The concentration at which significant toxicity was observed was 0.224 mg/L in the 5-day repeat dose study.

Per the guidance on the application of the CLP criteria (Version 4.1 – June 2015), STOT-RE is assigned based on findings of ‘significant’ or ‘severe’ toxicity, typically in a 90-day study. In such a study, the limiting concentration above which classification is not required is 0.2 mg/L. Usually, this limit can be adjusted to allow the use of shorter or longer duration studies to conclude on classification by application of factor, essentially based on Haber’s rule for inhalation, which states, essentially, that the effective dose is directly proportional to the exposure concentration and the duration of exposure. However, in this case, where the local effects are caused by a pH shift and, based on the findings in the 5- and 14-day studies, are independent of the exposure duration, then it is justified to maintain the 90-day guidance value, and hence calcium dihydroxide should not be classified for STOT-RE (inhalation) because the dose level at which significant toxic effects were observed was 0.224 mg/L, which is > 0.2 mg/L. This conclusion is read-across to calcium oxide.