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Toxicological information

Repeated dose toxicity: oral

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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

Reference
Name:
Unnamed
Type:
Constituent
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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Ca(OH)2 to achieve pH 11.2
Basis:
nominal in water
Remarks:
Doses / Concentrations:
Ca(OH)2 to achieve pH 12
Basis:
nominal in water
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.