Lime (chemical), hydraulic

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Calcium dihydroxide

EC Number:

215-137-3

EC Name:

Calcium dihydroxide

Cas Number:

1305-62-0

Molecular formula:

CaH2O2

IUPAC Name:

calcium dihydroxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Calcium dihydroxide
- Physical state: White solid
- Analytical purity: 98%
- Lot/batch No.: 5133
- Expiration date of the lot/batch: Not supplied
- Storage condition of test material: Room temperature, in the dark, under nitrogen, and over silica

Method

Target gene:

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

Species / strain

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.

Controls

Controls 1

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.

Controls 2

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.