Reaction mass of Limestone and dicalcium silicate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The test substance of the study is calcium hydroxide, however the results are supporting also information on "Reaction mass of limestone and dicalcium silicate", in which calcium hydroxide is the main constituent governing the toxicological properties

Data source

Reference

Reference Type:

publication

Title:

Systemic and local effects of long-term exposure to alkaline drinking water in rats

Author:

Merne, M.E.T.; et al.

Year:

2001

Bibliographic source:

Int. J. Exp. Path 82, 213-219

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of Long-Evans rats (6 animals of each sex) received drinking water supplemented with Ca(OH)2 and adjusted to pH 11.2 or 12.0 for 52 weeks. The same number of animals served as control.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Calcium dihydroxide

EC Number:

215-137-3

EC Name:

Calcium dihydroxide

Cas Number:

1305-62-0

Molecular formula:

CaH2O2

IUPAC Name:

calcium dihydroxide

Details on test material:

- Name of test material (as cited in study report): Calcium hydroxide
No further details are given.

Test animals

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: In groups 1, 2 and 3 the experiment was started when the rats were 12 weeks old, and in groups 4, and 5 at the age of 6 weeks.
- Housing: rats were housed in solid-polycarbonate bottom metal cages with alderwood bedding in groups of three
- Diet: ad libitum; standard laboratory animal feed (SDS, RM3; essex, UK)
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 45-55
- Photoperiod: 12 hours dark/light cycle
No further details are given.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Exposure group 2 and 4: Calcium dihydroxide was used to raise water pH (adjusted to pH 11.2 or 12.0, measured using a pH meter)
- Exposure group 3 and 5: NaOH was used to raise water pH (adjusted to pH 11.2 or 12.0, measured using a pH meter)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Ca(OH)2 to achieve pH 11.2
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Ca(OH)2 to achieve pH 12
Basis:
nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:
pH 7 (untreated/ control)
Basis:
nominal in water

No. of animals per sex per dose:

48 rats were divided into 4 study groups (group 2-5) and 1 control group (group 1).
Group 1 to 3: 6 male and 6 female rats, respectively.
Group 4 and 5: 3 male and 3 female rats, respectively.

Control animals:

yes

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
All animals were weighed at the completion of the experiment.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
FOOD EFFICIENCY: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was monitored intermittently in all groups.
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

- After completion of the study, the rats were killed using carbon dioxide, and necropsy samples were taken from the oral buccal mucosa, palate, tongue, oesophargus, stomach, intestines, liver and kidneys.
- In group 4 and 5, additional samples were obtained from the salivary glands, masseter muscle, thyroid, hypothalamus and ovary or testes.
GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes
- Tissues samples (as stated above) were subjected to histopathological examination. Samples were fixed in 10 % neutral formalin, embedded in paraffin, cut into 5-μm sections and stained with
haematoxylin and eosin for routine light microscopic evaluation.
- For immunohistochemistry (IHC), formalin-fixed, paraffin-embedded samples were sectioned to 5-μm thickness and mounted on organosilan-coated slides.
- Sections were stained using the avidin-biotin complex technique and an automatic appliance, according to the manufacturer's instructions.
- Antibodies used were pankeratin, cytokeratin (CK) 19, CK5, CK4, proliferating cell nuclear antigen (PCNA), intercellular adhesion molecule-1 (ICAM-1), CD44, CD68 and S-100, as well as heat shock
protein (HSP) 60, HSP 70 and HSP 90.
- Oral mucosa biopsy samples were also subjected to immunohistochemical analysis of the expression of a number of proteins regulating keratinocyte differentiation, cell proliferation, cell adhesion, and cell
stress.

Other examinations:

no data

Statistics:

no data

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
- All animals survived the experiment and remained in good condition until the end of the experiment.
BODY WEIGHT AND WEIGHT GAIN
- At the study end, animals in the exposed groups had lower body weights (up to 29 % less) than controls despite equal food and water intake, suggesting a systemic response to the alkaline treatment.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Food intake was equal in the study and control rats.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
- No differences between the groups were observed.
HISTOPATHOLOGY: NON-NEOPLASTIC
- The epithelium of the oral buccal mucosa, palate, and tongue of the rats from the study groups was similar in morphology and structure to that of controls.
- No defined mucosal atrophy or ulcerations were observed. In samples obtained from extra-oral tissues, no marked changes were seen after alkaline exposure.
- Alkaline exposure did not affect cell proliferation in the oral epithelium. The up-regulation of HSP70 protein expression in the oral mucosa of Ca(OH)2 exposed rats may indicate a protective response
against alkaline water. Intracellular adhesion molecule-1 (ICAM-1) positivity was lost in 6/12 and 0/6 rats treated with Ca(OH)2 at pH 11.2 and 12, respectively. A loss in CD44 expression was seen in the
study groups exposed to alkaline water with pH 12.

Effect levels

Dose descriptor:

NOAEL

Basis for effect level:

other: The study is not appropriate for derivation of a NOAEL.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The results suggest that the oral mucosa of rats is resistant to the effects of highly alkaline drinking water. However, high alkalinity may have some unknown systemic effects leading to growth retardation. The study is not appropriate for derivation of a NOAEL.