Lime (chemical), hydraulic

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria:
Calcium dihydroxide (read across to lime (chemical) hydraulic) - OECD TG 471 - negative with and without metabolic activation
Calcium carbonate - OECD TG 471 - negative with and without metabolic activation
 In vitro chromosome aberration study in mammalian cells:
Calcium dihydroxide (read across to lime (chemical) hydraulic) - OECD TG 473 - negative with and without metabolic activation
Calcium dihydroxide - non-mutagenic is a study performed using D824 cells
Calcium carbonate - OECD 473 - negative with and without metabolic activation
 In vitro gene mutation study in mammalian cells:
Calcium dihydroxide (read across to lime (chemical) hydraulic) - OECD TG 476 - negative
Calcium carbonate - OECD TG 476 - negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 2015 - 27 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

The thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared in-house from the livers of male Sprague- Dawley rats weighing ~ 250g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day.

Test concentrations with justification for top dose:

Preliminary toxicitiy test: 2.17 to 555.74 μg/mL
Mutagenicity test:
Experiment 1: 0, 8.68, 17.37, 34.73, 69.47, 138.94, 185.25, 231.57, 277.88 μg/mL (4-h;-S9); 0, 4.34, 8.68, 17.37, 34.73, 69.47, 138.94, 277.88, 555.75 μg/mL (4-h;+S9).
Experiment 2: 0, 17.25, 34.5, 69, 138, 184, 230, 276, 368, 460, 552 μg/mL (24-h; -S9); 0, 4.34, 8.68, 17.37, 34.73, 69.47, 138.94, 277.88, 555.75 μg/mL (4-h; +S9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: R0 medium
- Justification for choice of solvent/vehicle: Based on solubility checks performed in house for the OECD TG 473 study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) at 400 μg/mL and 150 μg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ~g/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ~g/ml) and 10% donor horse serum (giving R10 media). Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The cells were exposed to doses of the test material, vehicle and positive control, both with and without metabolic activation. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.

DURATION
- Preincubation period: Not applicable
- Exposure duration: Experiment 1: 4-h (+/- S9); Experiment 2: 24-h (-S9), 4-h (+S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): ~ 2 h

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): thiazolyl blue tetrazolium bromide (MTT)

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000 cells/well for mutant frequency; 2 cells/well for viability

DETERMINATION OF CYTOTOXICITY
- Method: The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control

OTHER EXAMINATIONS:
- Determination of polyploidy: No
- Determination of endoreplication: No
- Other: Calculation of Day 2 Viability (%V), Calculation of Relative Total Growth (RTG), Calculation of Mutation Frequency (MF) were performed, and the experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

OTHER:

Evaluation criteria:

For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance.
Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director’s discretion.

Statistics:

The experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS statistical package. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Please refer to discussion below.

RANGE-FINDING/SCREENING STUDIES:
The dose range of the test item used in the preliminary toxicity test was 2.17 to 555.75 μg/mL.
There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups, with the most marked reductions observed in the 4 and 24-hour dose groups in the absence of metabolic activation. Precipitate of the test item was observed at and above 17.37 μg/mL (see table below). Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were the approximate maximum practical dose level of 555.75 μg/mL, and limited by test item-induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical control data are attached.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Please refer to discussion below

Preliminary cytotoxicity test results

Dose(μg/mL)	% RSG (-S9)4-Hour Exposure	% RSG (+S9)4-Hour Exposure	% RSG (-S9)24-Hour Exposure
0	100	100	100
2.17	108	93	109
4.34	96	91	103
8.68	97	94	108
17.37	114	96	116
34.73	115	100	124
69.47	106	92	110
138.94	117	85	65
277.88	1	56	23
555.75	6	34	0

Mutagenicity Test

A summary of the results from the test is presented in Table 1 (attached)

Experiment 1

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7 (attached).

There was evidence of marked toxicity following exposure to the test item in the absence of metabolic activation, as indicated by the %RSG and RTG values (Table 3). On this occasion, the levels of toxicity observed in the presence of metabolic activation were not as great as those observed in the preliminary toxicity test with only modest levels of toxicity observed (Table 6). However, this was not considered to have affected the purpose or integrity of the study as the maximum practical dose level due to pH had been tested. There was no evidence of any significant reductions in viability (%V), in either the absence or presence of metabolic activation, indicating that residual toxicity had occurred (Tables 3 and 6). Based on the RTG and %RSG values observed, it was considered that optimum levels of toxicity were achieved in the absence of metabolic activation. The toxicity observed at 277.88 μg/mL in the absence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, including the dose level that achieved optimum levels of toxicity in the absence of metabolic activation, and the maximum practical dose level due to pH in the presence of metabolic activation (Tables 3 and 6). Overall, precipitate of the test item was observed at and above 34.73 μg/mL.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13 (attached).

As was seen previously, there was evidence of marked toxicity in the absence of metabolic activation, and modest toxicity in the presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 9 and 12). On this occasion, the levels of toxicity observed in the presence of metabolic activation were more similar to those observed in the preliminary toxicity test. There was once again no evidence of any significant reductions in viability (%V), in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred (Tables 9 and 12). Based on the RTG and / or %RSG values observed,

optimum levels of toxicity were considered to have been achieved in the absence of metabolic activation (Table 9). The excessive toxicity observed at and above 276 μg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a very modest effect on the toxicity of the test item.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce any statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, including the dose level that achieved optimum levels of toxicity in the absence of metabolic activation, and the maximum practical dose level due to pH in the presence of metabolic activation (Tables 9 and 12). Precipitate of the test item was observed at and 34.5 μg/ml.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with OECD TG 476, Method B17 of Commission Regulation (EC) No. 440/2008 and US EPA OPPTS 870.5300, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose level ranges plated out for viability and expression of mutant colonies were as follows: Experiment 1 - 34.73 - 277.88 μg/mL (4 -h, -S9); 17.37 - 555.75 μg/mL (4 -h, +S9); Experiment 2 - 17.25 - 230 μg/mL (24-h, -S9); 17.37 - 555.75 (4 -h, +S9).

Results

The maximum dose levels used in the mutagenicity test were limited by a combination of pH increase and test item-induced toxicity. Precipitate of the test item was observed at and above 34.5 μg/mL in the Mutagenicity Test. The vehicle controls had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2015 - 03 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from male rats induced with phenobarbitone/beta-naphthoflavone

Test concentrations with justification for top dose:

Preliminary tox test: 2.17 to 556 μg/mL
Main Experiment:
4-h exposure (-S9-mix); 20-h culture in treatment-free media: 0*, 4.38, 8.75, 17.5*, 35*, 70*, 140*, MMC 0.2* μg/mL
4-h exposure (+S9-mix); 20-h culture in treatment-free media: 0*, 4.38, 8.75, 17.5*, 35*, 70*, 140, CP 1.25* μg/mL
24-h exposure (-S9-mix): 0*, 4.38, 8.75, 17.5*, 35*, 70*, 140*, MMC 0.1* μg/mL
* = Dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Medium (MEM)
- Justification for choice of solvent/vehicle: The test item was partly soluble/suspendable in MEM

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9, mitomycin C dissolved in MEM was used at 0.2 and 0.1 μg/mL for 4(20)-hour cultures and 24-hour continuous exposure cultures, respectively.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9, cyclophosphamide dissolved in DMSO was used at 1.25 μg/mL.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time.

SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Test performed in duplicate

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value. Where possible the first 150 consecutive well-spread metaphases from each culture were counted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell is scored as endoreduplicated

Evaluation criteria:

Evaluation criteria
The following criteria were used to determine a valid assay:
• The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.
Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1. The number of induced chromosome aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
3. There is no concentration-related increase at any dose level A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is outside the range of the laboratory historical control data.
2. At least one concentration exhibits a statistically significant increase in the frequency of cells with aberrations compared to the concurrent negative control.
3. The increase observed is considered dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploid and endoreduplications.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was a significant change in pH when the test item was dosed into media at the 10 mM dose level; however, there was no substantial change in osmolality. Based on the above information an extra dose level 555.75 μg/mL was assessed and gave a pH of 8.20 and osmolality value of 273 mOsm. It was considered that this would be a more appropriate maximum dose level.
- Precipitation: The qualitative assessment of the slides determined that precipitate observations were similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 140 μg/mL in all three exposure groups.
Precipitate observations were made at the end of exposure in blood-free cultures and was noted at 140 μg/mL, in the 4(20)-hour exposure group in the absence of S9, at and above 70 μg/mL, in the 4(20)-hour exposure group in the presence of S9 and at 140 μg/mL in the 24-hour continuous exposure group.


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 2.17, 4.34, 8.69, 17.38, 34.75, 69.5, 139, 278 and 556 μg/mL. The maximum dose was limited by pH issues at the maximum recommended dose level of 749 μg/mL.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 139 μg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9) and at and above 69.5 μg/mL in the 4(20)-hour exposure group with metabolic activation (S9) and 24-hour continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 556 μg/mL in all three of the exposure groups. The maximum dose level assessed for mitotic index values was 139 μg/mL in all three exposure groups due the presence of precipitate. The mitotic index data are presented in Table 1 (attached). The test item induced marked evidence of toxicity only in the 24-hour continuous exposure group.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and to some extent toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range (Appendix 2).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The mitotic index data for the Main Experiment are given in Table 2 and Table 3 (attached). They confirm the qualitative observations in that no marked dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 16% and 13% mitotic inhibition was achieved at 70 μg/mL and 140 μg/mL respectively. In the presence of S9, no clear dose-related inhibition of mitotic index was observed with a maximum of 20% being demonstrated at 35 μg/mL. Again, with no clear dose-relationship in the 24-hour continuous exposure group the maximum inhibition of mitotic index of 40% was noted at 35 μg/mL.

The chromosome aberration data are given in Table 4, Table 5 and Table 6 (attached).

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 7 (attached). The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

Interpretation of results (migrated information):
negative

The test item calcium dihydroxide, did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

An in vitro study for the detection of structural chromosomal aberrations was performed under GLP in accordance with OECD TG 473 using calcium dihydroxide.

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited predominantly on precipitate. The dose levels used for the main experiment were 4.38 - 140 μg/mL

Results

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was marginally toxic and did not induce a statistically significant increase in the frequency of cells with aberrations, using a dose range that was limited by the lowest precipitating dose level.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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.

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.

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 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 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.

Conclusions:

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.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented publication. The test substance calcium hydroxide is the dominant main constituent of lime (chemical) hydraulic, governing its toxicological propoerties. The study results can therefore be used by read-across.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Examination of the ability to induce chromosome aberrations in human dental pulp cells.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: human dental pulp cells, designated as D824 cells

Details on mammalian cell type (if applicable):

The enzymazically released cells were suspended in alpha-minimum essential medium (alpha-MEM) supplemented with 20 % fetal bovine serum, 100 µM L-ascorbic acid phosphate magnesium salt n-hydrate, 2mM L-glutamine, 0.22 % NaHCO3, 100 units/mL penicilline and 100 µg/mL streptomycin and passed through a 70 µm nylon cell strainer to remove cell aggregates. After counting the number of cells, 1.5x10^5 cells were plated into 75cm² flasks. When confluent, cells were harvested with a solution containing 0.25 % trypsin and 0.1 % EDTA and replated into new flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

30; 100 and 300 µM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test substance was dissolved at 30 mM in glycerol at 65 °C.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cells were treated for 2 hours in the presence of 5% PMS Migrated to IUCLID6: (50 µM)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: formalin (0.001 and 0.0003 µM)

Remarks:

CA in D824 cells induced by treatment for 30 hours.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): After treatment, cells were washed twice with fresh medium and subsequently incubated for a further 27-hour period, or cells were treated continuously for 30 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): 3 hours before the end of the incubation, colcemid was administered at 0.02 µg/mL, and metaphase chromosomes were prepared for analysis.

NUMBER OF METAPHASES EVALUATED: 100 to 200

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was measured to select concentrations for analysis of chromosome aberrations. Cytotoxicity was determined as the number of cells treated with calcium hydroxide, relative to the number of cells in the control cultures x 100.
D824 cells after 6 to 8 passages were plated in triplicate onto 60-mm dishes at 8x10³ cells/cm², equivalent to 1.6x10^5 cells/dish, and incubated overnight. The cells were treated with calcium hydroxide (in absence or presence of metabolic activation) at varying concentrations for 3 hours. After two washings with 2 mL of fresh medium, cells were incubated for a further 24 hours and the number of cells was counted after harvesting cells with 0.25% trypsin.

EXAMINATIONS:
The aberrations scored were gaps, breaks, exchanges, dicentric chromosomes, ring chromosomes and fragmentations.

Evaluation criteria:

No data given

Statistics:

Statistical analysis was performed by qui-square test to assess the significance of the difference in the incidences of chromosome aberrations between control cultures and cultures treated with calcium hydroxide. The level of significance in the statistical analysis was determined at p<0.05.

Species / strain:

mammalian cell line, other: D824 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increases in the levels of chromosome aberrations were observed in cells treated with calcium hydroxide.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Calcium hydroxide did not induce chromosome aberrations after 3 h. Since it is known that some substances induce chromosome aberrations only after longer exposure, a protocol with a continuous treatment for 30 hours was employed additionally. Results show that calcium hydroxide did not enhance the level of chromosome aberrations in D824 cells even after prolonged exposure.

Conclusions:

Interpretation of results (migrated information):
negative

Calcium hydroxide failed to induce chromosome aberrations in the absence or presence of exogenous metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 December 2009 - 19 January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver homogenate metabolising system (10% liver S9 in standard co-factors))

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Mutation test - Experiments 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle: Dimethyl sulphoxide (DMSO)

- Justification for choice of vehicle: The test material was insoluble in sterile distilled water and acetone at 50 mg/mL and tetrahydrofuran at 200 mg/mL but was fully soluble in dimethyl sulphoxide and dimethyl formamide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls (spontaneous mutation rates)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 2 μg/plate for WP2uvrA-, 3 μg/plate for TA100, 5 μg/plate for TA1535 - without S9-mix

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 80 μg/plate for TA1537 - without S9-mix

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.2 μg/plate for TA98 - without S9-mix

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA) with S9-mix: 1 μg/plate for TA100; 2 μg/plate for TA1535 and TA1537; 10 μg/plate for WP2uvrA-

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 5 μg/plate for TA98 - with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and preincubation for Experiment 2:

Preliminary Toxicity Test: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1: Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counte
r.

Mutation Test - Experiment 2: The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 μg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. Manual counts were required after employing the pre-incubation method at 5000 μg/plate (absence of S9-mix only) because of a particulate test material precipitation.


DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 h (Experiment 1)


NUMBER OF REPLICATIONS: Plates were prepared in triplicate


NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 1 to 9.9 x 10^09 bacteria per mL.

Evaluation criteria:

There were several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results were considered first, statistical methods, as recommended by the UKEMS were also used as an aid to evaluation, however, statistical significance was not the only determining factor for a positive response.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix after employing the plate incorporation method of dosing (preliminary toxicity test and Experiment 1). However, after employing the pre-incubation method (Experiment 2) a particulate test material precipitate was noted from 1500 and at 5000 μg/plate, in the absence and presence of S9-mix, respectively.


RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.


COMPARISON WITH HISTORICAL CONTROL DATA: A history profile of vehicle and positive control values for 2007 and 2008 is presented in Appendix I.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 December 2009 to 15 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1 - 4(20) hour:
Without S9-mix: 0*, 31.25, 62.5, 125*, 250*,500*, 1000 (* Dose levels selected for metaphase analysis)
With S9-mix: 0*, 31.25, 62.5, 125*, 250*,500*, 1000 (* Dose levels selected for metaphase analysis)
Experiment 2 - 24 hour:
Without S9-mix: 0*, 31.25, 62.5, 125*, 250*,500*, 1000 (* Dose levels selected for metaphase analysis)

This study was run in parallel with a Mouse Lymphoma Assay (MLA) using L5178Y cells (Harlan Laboratories Ltd project No. 2974/0008) which has the capability of detecting clastogenic activity. The study was performed to meet the requirements of the OECD 476 Guideline. It was therefore considered that the results of the MLA study gave adequate scientific justification for the omission of the repeat of the with metabolic activation exposure group.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of vehicle: A solubility check process was performed to determine a suitable solvent; initially an aqueous vehicle (MEM culture medium) was investigated. However, this was unsuitable and therefore the next coice was DMSO, resulting in a suitable dosable suspension.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9
- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT (mutation assays): No selection agent
SPINDLE INHIBITOR (cytogenetic assays): Demecolcine
STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In the 24 hours continuous exposure group, dose level 250 µg/ml, A culture, only 80 metaphases were scored due to partial loss of pellet during the experiment. However due to no aberrations being seen in the 80 metaphases scored it was considered to have no impact on the study.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable
- Water solubility: Not applicable, the test substance was suspended in DMSO
- Precipitation:

Preliminary test:
A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 µg/ml, in the 4(20)-hour pulse exposure group in the absence of metabolic activation and at and above 62.5 µg/ml in the 4(20)-hour exposure group in the presence of metabolic activation and in the 24-hour continuous exposure group.

Main test:
The maximum dose level selected for metaphase analysis was limited by the presence of heavy precipitate on the slides at 1000 µg/ml in all three exposure groups. The precipitate was such that it was considered impractical to accurately assess the metaphases for the presence of aberrations and, therefore, the maximum dose level selected was 500 µg/ml.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/ml. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 µg/ml, in the 4(20)-hour pulse exposure group in the absence of metabolic activation and at and above 62.5 µg/ml in the 4(20)-hour exposure group in the presence of metabolic activation and in the 24-hour continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1000 µg/ml in all three of the exposure groups. The mitotic index data are presented in Table 1. The test material induced modest evidence of toxicity in all three of the exposure groups.
The selection for the main experiment was the maximum recommended 10 mM concentration and was 1000 µg/ml for all three exposure groups.


COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The qualitative assessment of the slides determined that the toxicity was less than that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test material, 1000 µg/ml in all three exposure groups. The mitotic index data are given in Tables 1, 2 and 3 (attached background information). They confirm the qualitative observations in that no clear dose-related inhibition of mitotic index was observed in any of the exposure groups.

The chromosome aberration data are given in Table 4, Table 5 and Table 6 (attached background information). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 7 (attached background information). The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

There was no evidence of a response in the presence of metabolic activation in this study or in the MLA performed on the test material (Harlan Laboratories Ltd Project No. 2974/0008). This was taken as scientific justification to confirm that the repeat of the exposure group with metabolic action was not required.

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 December 2009 to 17 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

A repeat of the 4-hour exposure with metabolic activation was not performed because there was no evidence of a response in the presence of metabolic activation in this study or in the Chromosome Aberration Test performed on the same test material.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Properly maintained: yes - The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Periodically "cleansed" against high spontaneous background: yes - Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of male Sprague-Dawley rats weighing approximately 250g. 20% S9-mix was prepared by mixing S9, NADP (5 mM), G6P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0. The final concentration of S9 was 2% throughout the study.

Test concentrations with justification for top dose:

4 hour and 24 hour exposures: 0, 7.81, 15.63, 31.25, 62.5, 125 and 250 µg/mL

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: - the positive control in the absence of metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: - the positive control in the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4-hour exposure with and without metabolic activation: The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
24-hour exposure without metabolic activation: The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker for 24 hours.
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 5-trifluorothymidine (TFT) in 96-well microtitre plates. The microtitre plates were then incubated for 10-14 days.

STAIN: To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

SELECTION AGENT (mutation assays): 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Each treatment was performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at six dose levels of the test material (7.81 to 250 µg/mL), vehicle and positive controls.

NUMBER OF CELLS EVALUATED: At the end of the treatment period the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

Evaluation criteria:

The daily cell counts were used to obtain a Percentage Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgement. Large colonies were defined as those that cover approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

The normal range for mutant frequency per survivor is 50-200 x 10^-6 for the TK+/- locus in L5178Y cells. For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The extended exposure time had no effect on the toxicity of the test material.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

4-HOUR EXPOSURE WITH AND WITHOUT METABOLIC ACTIVATION:
There was no evidence of any marked toxicity following exposure to the test material in either the absence or presence of metabolic activation, as indicated by the %RSG and RTG values. There was also no evidence of any reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that no residual toxicity had occurred. The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in either the absence or presence of metabolic activation.

24-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION:
There was no evidence of any marked toxicity following exposure to the test material, as indicated by the %RSG and RTG values. There was also once again no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no effect on the toxicity of the test material. The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the 4 hour exposure experiment a precipitate of the test material was observed at and above 62.5 µg/mL in both the absence and presence of metabolic activation. However, the purpose and integrity of the study was considered unaffected.
In the 24 hour exposure experiment a precipitate of test material was observed at and above 15.63 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 3.91 to 1000 µg/mL for all three of the exposure groups.
In the 4-hour exposure groups, both in the absence and presence of metabolic activation (S9), there was no evidence of any marked dose related reductions in the relative suspension growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of metabolic activation, there was evidence of very modest reductions in %RSG values of cells treated with test material at and above 500 µg/mL. However, it was considered that this was due to some of the cell pellet being lost with the precipitate during the washing phase and not true test material induced toxicity. A precipitate of the test material was observed at and above 31.25 µg/mL in the 4-hour exposure groups, and at and above 15.63 µg/mL in the 24-hour exposure group, and increased with increase in dose concentration in all three of the exposure groups. In the subsequent mutagenicity test, with no evidence of any marked test material-induced toxicity, the maximum dose was limited to 250 µg/mL due to the nature of the precipitate observed at and above 500 µg/mL that resulted in handling problems during the washing stage of the test.

Table 1: Preliminary toxicity test – results for the Relative Suspension Growth (%RSG)

Dose (µg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
3.91	93	94	102
7.81	94	96	101
15.63	101	92	108
31.25	95	94	112
62.5	93	107	100
125	97	91	113
250	99	94	117
500	97	91	85
1000	94	92	76

Table 2: Summary of results for 4 hour exposure (main experiment)

Treatment (µg/mL)	4-Hours-S-9			Treatment (µg/mL)	4-Hours+S-9
-	%	RTG	MF§	-	%	RTG	MF§
0	100	1.00	67.20	0	100	1.00	60.28
7.81	101	1.12	70.00	7.81	106	1.25	58.93
15.63	107	1.23	71.96	15.63	110	1.12	72.14
31.25	114	1.28	56.60	31.25	111	1.21	60.34
62.5	104	1.17	61.11	62.5	100	1.15	60.31
125	99	1.30	55.11	125	109	1.13	66.41
250	113	1.38	57.94	250	105	1.16	65.04
Linear trend		-	NS	Linear trend		-	NS
EMS	-	-	-	CP	-	-	-
400	72	0.52	566.79	2	69	0.36	746.57

Table 3: Summary of results for 24 hour exposure

Treatment (µg/mL)	24-Hours-S-9
-	%	RTG	MF§
0	100	1.00	88.92
7.81	109	1.10	83.34
15.63	111	1.05	68.82
31.25	116	1.29	55.07
62.5	112	1.08	111.52
125	121	1.26	57.35
250	109	1.00	76.21
Linear trend		-	NS
EMS	-	-	-
150	62	0.44	758.03

 

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Calcium carbonate (nano) did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional groups/mechanism of action.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target: Lime (chemical), hydraulic [CAS 85117-09-5; See section 1.2 for information on purity.
Source: calcium dihydroxide [CAS 1305-62-0; EC 215-137-3] 98%

3. ANALOGUE APPROACH JUSTIFICATION
Lime (chemical) hydraulic dissociates in aqueous solutions to calcium- and hydroxyl ions, whereas calcium carbonate will be decomposed into calcium and CO2, and calcium silicate remains largely undissolved.Calcium dihydroxide represents the main constituent of lime (chemical) hydraulic that governs any potential toxicological effects (pH shift). Calcium dihydroxide is therefore considered as functionally equivalent to the target substance (lime (chemical), hydraulic).

4. DATA MATRIX
Source: No studies available
Target: OECD TG 476 - negative with and without metabolic activation

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional groups/mechanism of action.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target: Lime (chemical), hydraulic [CAS 85117-09-5; See section 1.2 for information on purity.
Source: calcium dihydroxide [CAS 1305-62-0; EC 215-137-3] 98%

3. ANALOGUE APPROACH JUSTIFICATION
Lime (chemical) hydraulic dissociates in aqueous solutions to calcium- and hydroxyl ions, whereas calcium carbonate will be decomposed into calcium and CO2, and calcium silicate remains largely undissolved.Calcium dihydroxide represents the main constituent of lime (chemical) hydraulic that governs any potential toxicological effects (pH shift). Calcium dihydroxide is therefore considered as functionally equivalent to the target substance (lime (chemical), hydraulic).

4. DATA MATRIX
Source: No studies available
Target: OECD TG 473 - negative with and without metabolic activation

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Common functional groups/mechanism of action.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Target: Lime (chemical), hydraulic [CAS 85117-09-5; See section 1.2 for information on purity.
Source: calcium dihydroxide [CAS 1305-62-0; EC 215-137-3] 98.2%

3. ANALOGUE APPROACH JUSTIFICATION
Lime (chemical) hydraulic dissociates in aqueous solutions to calcium- and hydroxyl ions, whereas calcium carbonate will be decomposed into calcium and CO2, and calcium silicate remains largely undissolved.Calcium dihydroxide represents the main constituent of lime (chemical) hydraulic that governs any potential toxicological effects (pH shift). Calcium dihydroxide is therefore considered as functionally equivalent to the target substance (lime (chemical), hydraulic).

4. DATA MATRIX
Source: No studies available
Target: OECD TG 471 - negative with and without metabolic activation

Reason / purpose for cross-reference:

read-across source

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

Conclusions:

I

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria:

No studies are available for lime (chemical) hydraulic, however a study is available for the read-across susbtance, calcium dihydroxide. In a reliable GLP OECD TG 471 study [Sokolowski, 2007] calcium dihydroxide (purity = 98.2 %) was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The concentrations tested were 117.2, 234.4, 468.75, 937.5, 1875 and 3750 µg/plate. No mutations occurred.

Similarly, in a reliable GLP OECD TG 471 study [Thompson, 2010], calcium carbonate (nano) was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The concentrations tested were 50, 150, 500, 1,500 and 5,000 μg/plate. No mutations occurred.

 In vitro chromosome aberration study in mammalian cells:

No study is available for lime (chemical) hydraulic, however studies are available for the read-across substance, calcium dihydroxide. An in vitro study for the detection of structural chromosomal aberrations was performed under GLP in accordance with OECD TG 473 using calcium dihydroxide (purity = 98 %) [Morris, 2015].

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9 at a 2% final concentration) and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited predominantly on precipitate. The dose level range used for the main experiment were 4.38 - 140 μg/mL.

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was marginally toxic and did not induce a statistically significant increase in the frequency of cells with aberrations, using a dose range that was limited by the lowest precipitating dose level. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

The ability of calcium dihydroxide to induce chromosome aberrations in human dental pulp cells (D824) has been investigated [Nishimura, 2008]. The D824 cells were exposed to calcium dihydroxide at test concentrations of 30, 100 and 300 µM in glycerol for 3 or 30 hours. Calcium dihydroxide did not induce chromosome aberrations after treatment for either time period and was considered to be non-mutagenic under the conditions of the test.

In a reliable GLP OECD TG 473 study [Lacey & Durwood, 2010], calcium carbonate (nano) was tested for its ability to induce structural chromosomal aberrations in cultured mammalian cells (human lymphocytes) in the presence and absence of metabolic activation. Calcium carbonate (nano) did not induce any statistically significant increases in the frequency of cells with aberrations or in the numbers of polyploid cells, in either the absence or presence of metabolic activation and was therefore considered to be non-mutagenic under the conditions of the test.

 In vitro gene mutation study in mammalian cells:

No studies are available for lime (chemical) hydraulic, howver a study is available for the read-across substance, calcium dihydroxide. A GLP study was conducted according to OECD TG 476, Method B17 of Commission Regulation (EC) No. 440/2008 and US EPA OPPTS 870.5300, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances, in order to assess the potential mutagenicity of calcium dihydroxide (purity = 98 %) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line [Flanders, 2015].

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose level ranges plated out for viability and expression of mutant colonies were as follows: Experiment 1 - 34.73 - 277.88 μg/mL (4 -h, -S9); 17.37 - 555.75 μg/mL (4 -h, +S9); Experiment 2 - 17.25 - 230 μg/mL (24-h, -S9); 17.37 - 555.75 (4 -h, +S9).

The maximum dose levels used in the mutagenicity test were limited by a combination of pH increase and test item-induced toxicity. Precipitate of the test item was observed at and above 34.5 μg/mL in the Mutagenicity Test. The vehicle controls had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment and was therefore considered to be non-mutagenic under the conditions of the test.

In a reliable GLP OECD TG 476 study [Flanders, 2010], calcium carbonate (nano) was tested for its ability to induce mutations in mouse lymphoma L5178Y cells in the presence and absence of metabolic activation. Calcium carbonate (nano) did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.

Other data:

Several comet assays have been performed using calcium dihydroxide-containing endodontic materials [Ribeiro, 2004; Ribeiro, 2005; da Silva, 2007]. The results of these assays were all negative with respect to DNA breakage, demonstrating lack of genotoxic potential and may be read across to lime (chemical) hydraulic.

Finally, calcium is an essential mineral nutrient omnipresent in the human body, with daily dietary requirements for adults of approximately 1000 mg, varying by developmental status and age.

On the basis of the available data, it may be concluded that all grades of lime (chemical) hydraulic including those containing up to 40% calcium carbonate are not genotoxic.

Justification for classification or non-classification

The results of in vitro gene mutation studies in bacteria, in vitro chromosome aberration studies in mammalian cells and in vitro gene mutation studies in mammalian cells performed using calcium dihydroxide or calcium carbonate were all negative. It is concluded that both calcium dihydroxide and calcium carbonate are not genotoxic and neither chemical nor mixtures of these chemicals warrants classification for mutagenicity under CLP. This conclusion is read across to lime (chemical) hydraulic.