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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2007-03-29 to 2007-07-19
Reliability:
1 (reliable without restriction)

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)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- 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 / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
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
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: trp-; uvrA
Metabolic activation:
with and without
Metabolic activation system:
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 μg/plate.
The following concentrations were tested in experiment II: 117.2; 234.4; 468.75; 937.5; 1875 and 3750 μg/plate
Vehicle / solvent:
- 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
Untreated negative controls:
yes
Remarks:
(untreated controls, no test item or solvent added)
Negative solvent / vehicle controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation; strains TA1535 and TA100

Migrated to IUCLID6: (10 μg/plate), dissolved in deionised water
Untreated negative controls:
yes
Remarks:
(untreated controls, no test item or solvent added)
Negative solvent / vehicle controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD (10 or 50 μg/plate), dissolved in DMSO
Remarks:
without metabolic activation; strains TA1537, TA98
Untreated negative controls:
yes
Remarks:
(untreated controls, no test item or solvent added)
Negative solvent / vehicle controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation; strain WP2 uvrA

Migrated to IUCLID6: (3.0 μL/plate), dissolved in deionised water
Untreated negative controls:
yes
Remarks:
(untreated controls, no test item or solvent added)
Negative solvent / vehicle controls:
yes
Remarks:
1 M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA (2.5 μg/plate or 10 μg/plate), dissolved in DMSO
Remarks:
with metabolic activation; all strains
Details on test system and experimental 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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with calcium dihydroxide 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 μg/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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, during the described mutagenicity test and under the experimental conditions reported, the test item calcium dihydroxide 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, calcium dihydroxide is considered to be non-mutagenic in this in this Salmonella typhimurium and Escherichia coli reverse mutation assay.