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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Remarks:
read across to hydrolysis product
Adequacy of study:
key study
Study period:
2007-03-29 to 2007-07-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Due to the fact that the substance hydrolyses rapidly within less than one second under formation of Hydrogen (volatilises) and calciumhydroxide, studies with the metabolite staying in the system are relevant for the substance itself. In fact if Calcium dihydride would be used as test substance, calcium hydroxide would be tested as well. Therefore the data from calciumdihydroxide are directly relevant for calcium diydride.
3. ANALOGUE APPROACH JUSTIFICATION
See above. As the substance hydrolyses instantly under formation of calcium di hydroxide in an aqueaous test system, such as the Ames assay, the data on genotxicity of the metabonate are ditrectly relevant for calcium dihydride.
(for the hydrolytical stability see section 5.1.2).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): PRECAL 50S (Calcium hydroxide (hydrated lime))
- Physical state: solid
- Storage condition of test material: at room temperature, moisture protected

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
other: TA1537: his C 3076; rfa-; uvrB-; TA98: his D 3052; rfa-; uvrB-;R-factor; TA:1535: his G 46; rfa-; uvrB- and
TA100: his G 46; rfa-; uvrB-;R-factor
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
other: trp-; uvrA
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix
Test concentrations with justification for top dose:
In the pre-experiment/experiment I the concentration range of the test item was 0.3; 1.0; 3.0; 10; 33; 100; 300; 600; 1875 and 3750
Mg/plate.
The following concentrations were tested in experiment II: 117.2; 234.4; 468.75; 937.5; 1875 and 3750 Mg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: On the day of the experiment, the test item was suspended in 1 M N-(2-Hydroxyethyl)piperazine-
N-2-ethane sulfonic acid (HEPES, SERVA, D-69115 Heidelberg, analytical grade).
- Justification for choice of solvent/vehicle: The solvent has been chosen according to its solubility properties.
Controlsopen allclose all
Negative controls:
yes
Remarks:
untreated control
Solvent controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
10 micro-g/plate dissolved in deionised water
Negative controls:
yes
Remarks:
untreated controls
Solvent controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD (10 or 50 Mg/plate), dissolved in DMSO
Remarks:
(10 or 50 Mg/plate), dissolved in DMSO
Negative controls:
yes
Remarks:
untreated control
Solvent controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
(3.0 ML/plate), dissolved in deionised water
Negative controls:
yes
Remarks:
untreated controls
Solvent controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
(2.5 Mg/plate or 10 Mg/plate), dissolved in DMSO
Details on test system and conditions:
METHOD OF APPLICATION: In agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 60 minutes
- Exposure duration: The plates were incubated upside down for at least 48 hours at 37 °C in the dark.
NUMBER OF REPLICATIONS: For each strain and dose level, including the controls three plates were used.
DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with all strains. 8 concentrations were tested for toxicity
and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described
below for the experiment I (plate incorporation test). Toxicity of the test item resulted in a reduction in the number of spontaneous
revertants or a clearing of the bacterial background lawn.
DATA RECORDING:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software
program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both the
individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as
compared to the spontaneous reversion rates. Due to precipitation of the test item and widespread bacteria colony growth some
plates were counted manually.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of
twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding
solvent/vehicle control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent
second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic
potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant and the substance hence not
regarded to be mutagenic in the bacterial reverse mutation assay.
Statistics:
According to the OECD guideline 471 a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with PRECAL
50S at any concentration level, neither in presence nor in absence of metabolic activation (S9 mix). There was also no tendency of
higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
RANGE-FINDING/SCREENING STUDIES:
The assay was performed with and without liver microsomal activation. Based on the results of the pre-tests (reported as
Experiment I), an additional plate incorporation assay was performed with the maximum dose level suitable for the reverse
mutation assay of 3750 Mg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA: yes; used data represent the laboratory's historical control data from May
2005 until June 2006 representing approx. 200 experiments (WP2 uvrA the historical data are based on approx. 100
experiments). The historical control data of the solvent control are pooled from all commonly used solvents like deionized water,
DMSO, ethanol, and THF not including control data for HEPES.

Applicant's summary and conclusion

Conclusions:
An Ames test was conducted with calcium hydroxide according to OECG TG 471. Species S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A (met. act.: with and without) were used with the following concentrations: 0.3; 1.0; 3.0; 10; 33; 100; 300; 600; 1875 and 3750 µg/plate in the experiment I and 117.2; 234.4; 468.75; 937.5; 1875 and 3750 µg/plate in the experiment II. The results regarding genotoxicity of Ca(OH)2 were negative under the testing conditions. In conclusion, during the described mutagenicity test and under the experimental conditions reported, the test item did not induce
gene mutations by base pair changes or frameshifts in the genome of the strains used up to and including the highest testable
concentration.
Therefore, PRECAL 50S is considered to be non-mutagenic in this in this Salmonella typhimurium and Escherichia coli reverse
mutation assay. Due to the rapid hydrolysis if calciumhydride in aqueous systems (t1/2 < 1 s) under formation of calcium dihydroxide this result is directly relevant for the target substance.
Executive summary:

An Ames test was conducted with calcium hydroxide according to OECG TG 471. Species S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A (met. act.: with and without) were used with the following concentrations: 0.3; 1.0; 3.0; 10; 33; 100; 300; 600; 1875 and 3750 µg/plate in the experiment I and 117.2; 234.4; 468.75; 937.5; 1875 and 3750 µg/plate in the experiment II. The results regarding genotoxicity of Ca(OH)2 were negative under the testing conditions. In conclusion, during the described mutagenicity test and under the experimental conditions reported, the test item did not induce

gene mutations by base pair changes or frameshifts in the genome of the strains used up to and including the highest testable

concentration.

Therefore, PRECAL 50S is considered to be non-mutagenic in this in this Salmonella typhimurium and Escherichia coli reverse

mutation assay. Due to the rapid hydrolysis if calciumhydride in aqueous systems (t1/2 < 1 s) under formation of calcium dihydroxide this result is directly relevant for the target substance.