Reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliable in vitro data from a gene mutation study in bacteria, a cytogenicity study in mammalian cells and a gene mutation study in mammalian cells are available for substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.). There is no evidence for an intrinsic genotoxic activity relevant to humans.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-01-24 to 1991-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 84/449 EEC, EEC Publication No. L251, September 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Metabolic activation system:

liver S-9 fraction derived from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

10.0, 33.3, 100.0, 333.3, 1000.0, 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (without metabolic activation, strains TA 1535, TA 100); 4-nitro-o-phenylene-diamine (without metabolic activation, strains TA 1537, TA 1538, TA 98); 2-aminoanthracene (with metabolic activation, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

NUMBER OF PLATES: For each strain and dose level, including the controls, a minimum of three plates were used.
NUMBER OF REPLICATIONS: 2

DETERMINATION OF BACTERIOTOXICITY
- To evaluate the toxicity of the test article a prestudy was performed with strains TA 98 and TA 100. 8 concentrations were tested (1.0 - 5000.0 µg/plate) for toxicity and mutation induction with each 3 plates. Two independents experiments were performed. The experimental conditions in the pre-experiment were the same as described for the main experiment. Toxicity of the test article may be evidenced by reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.

A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
- A test article is considered as mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
- Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method was available.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

A significant slight increase in the number of revertants was found in the strains TA 1537 and TA 1538 both in the presence of metabolic activation in experiment I and II at the highest dose judged as probable cytotoxic but not mutagenic response

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in strain TA 98 at 5000.0 µg/plate with and without S9 mix in experiment I

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

According to an independent expert judgement (Prof. Dr. C. Schlatter, Institute für Toxikologie der Eidgenössischen Technischen Hochschule und der Universität Zürich (Schwerzenbach, 10. September 1991)), the slight increased numbers of revertants in strains TA 1537 and TA 1538 at the highest tested concentration of 5000 µg/plate with metabolic activation, is hardly a real mutagenic effect because such activity is due to the structure of the test substance not plausible at all. The result is probably an unspecific result of the toxicity and/or disturbance of the osmolality in the test system and therefore of no relevance for humans and animals.

Conclusions:

Based on the results of this study it is concluded that substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according OECD Guideline 471, adopted May 26, 1983 and EU Method B.13/14, strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 of Salmonella typhimurium were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in DMSO at concentrations of 10 to 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9-mix, plate incorporation).  

Substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) was tested up to and including cytotoxic concentrations (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. 

There was no real evidence of induced mutant colonies over background.

A significant slight increase in the number of revertants was found in the strains TA 1537 and TA 1538 both in the presence of metabolic activation in experiment I and II at the highest investigated dose. However, according to an independent expert judgement (Prof. Dr. C. Schlatter, Institute für Toxikologie der Eidgenössischen Technischen Hochschule und der Universität Zürich (Schwerzenbach, 10.September 1991)), the slight increased numbers of revertants in strains TA 1537 and TA 1538 at the highest tested concentration of 5000 µg/plate with metabolic activation, is hardly a real mutagenic effect because such activity is due to the structure of the test substance not plausible at all. The result is probably an unspecific result of the toxicity and/or disturbance of the osmolality in the test system and therefore of no relevance for humans and animals.

The adopted OECD TG 471 (1997) respectively EU Method B.13/14 according to Regulation (EC) No 440/2008 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 and EU Method B.13/14 (Version Commission Directive 92/69/EEC) without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the reaction mass in this bacterial test system.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471and EU Method B.13/14 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-05-22 to 1991-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 4, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 88/302, L 133

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

7-1-86 Edition

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium, SEROMED, D-1000 Berlin, F.R.G) supplemented with 10 % FCS (fetal calf serum, SEROMED, D-1000 Berlin, F.R.G)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

liver S9 microsomal fraction derived from male rats induced with Aroclor 1254

Test concentrations with justification for top dose:

0.30, 1.00, 2.50 and 5.00 mg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0.6 mg/mL, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

7.7 µg/mL, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours, on day 2 after 24 hour subculturing of a log-phase culture
- Expression time (cells in growth medium): until day 9
- Selection time (if incubation with a selection agent): day 9
- Fixation time (start of exposure up to fixation or harvest of cells): day 17

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: 300000 to 471000

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY

The assay is considered acceptable if it meets the following criteria:
- The number of mutants colonies per 1.000.000 cells found in the negative and/or solvent controls fall within the laboratory historical control data range: 5-45 mutants/1.000.000 cells.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The plating efficiency (absolute value) of the negative and/or solvent control should exceed 50 %.

EVALUATION OF RESULTS

- A test article is classified as positive if it induces either a significant concentration-related increase in the mutant frequency or a reproducible and significant positive response for at least one of the test points.
- A test article producing neither a significant concentration related increase in the mutant frequency nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:

- The test article is classified as mutagenic if it induces reproducibly with at least one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
- The test article is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.
- However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range normally found (5-45 mutants per 1.000.000 cells) a seemingly concentration-related increase of the mutations within this range will be regarded as to be irrelevant.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test article substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) was tested in two independent experiments, using identical procedures, both with and without liver microsomal activation. The following concentrations were used:

without S9 mix: 0.30; 1.00; 2.50 and 5.00 mg/mL

with S9 mix: 0.30; 1.00; 2.50 and 5.00 mg/mL.

Toxic effects of the test article were observed as a reduction of plating efficiency with 5.0 mg/mL. In the first experiment without metabolic activation the plating efficiency was also reduced with 1.0 mg/mL and 2.5 mg/mL and the highest concentration of 5.0 mg/mL could not be evaluated due to toxic effects of the test article.

Taking into account the mutation rates found in the groups treated with the test article compared to the negative and solvent controls it can be concluded that no biologically relevant increase of mutations was observed. The test article did not induce reproducible concentration-related increase in mutant colony numbers. However, high mutation rates beyond the range of the historical control values (5 - 45 mutants/10⁶ cells) were observed in experiment II at 0.30 mg/mL with and without metabolic activation (62.5 and 47.5 mutants/10⁶ cells) and at 2.5 mg/mL without metabolic activation (48.0 mutants/10⁶ cells). As these increases were neither concentration dependent nor reproduced in experiment I and were only slightly above the historical controls they are regarded as irrelevant.

In this study in both experiments (with and without S9 mix) the range of the negative controls was from 15.0 up to 29.5 mutants per 10⁶ cells; the range of the groups treated with the test article was from 14.0 up to 62.5 mutants per 10⁶ cells.

EMS (0.6 mg/mL) and DMBA (7.7 mg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion, it can be stated that in this mutagenicity assay the test article substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) did not induce gene mutations at the HGPRT locus in V79 cells.

Conclusions:

Based on the results of this study it is concluded that substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not mutagenic in the mammalian cell HPRT gene mutation assay using Chinese hamster V79 cells.

Executive summary:

In a mammalian cell gene mutation assay (HPRT-test) according to OECD Guideline 476, adopted April 4, 1984, EU method B.17 and EPA OTS 798.5300, Chinese hamster V79 cells cultured in vitro were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in DMSO at concentrations of  0.30, 1.00, 2.50, and 5.00 µg/mL in the presence and absence of mammalian metabolic activation (S9 -mix).  

Substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) was tested up to cytotoxic concentrations. The positive controls did induce the appropriate response. 

There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476, EU Method B.17 and EPA OTS 798.5300 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-04-21 to 1993-08-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69, L 383 A, Annex V, December 29, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Code of Federal Regulations, Titel 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, "In vitro mammalian cytogenetics"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No specific target gene. The chromosome aberration assay is an assay for the detection of structural chromosomal aberrations. These aberrations are frequently lethal to the damaged cells. However, cytogenetic damage in somatic cells is an indicator of a potential to induce more subtle chromosomal damage that is compatible with cell division. Similar damage induced in germinal cells may lead to heritable cytogenetic abnormalities. Heritable cytogenetic abnormalities are known to have deleterious effects in man, e.g. induction of neoplastic events or birth defects.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium, SEROMED, D-1000 Berlin, F.R.G) supplemented with 10 % FCS (fetal calf serum, Boehringer Mannheim, D-68261 Mannheim)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

liver S9 microsomal fraction derived from male rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I:
- not reported due to technical problems during the performance

Experiment II and III with and without S9 mix:
- 18 h preparation interval: 100, 250, 500, 1000, 2500, 5000 µg/mL
- 28 h preparation interval: 500, 1000, 2500, 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: As the test article is not soluble in appropriate vehicles an aqueous suspension (culture medium) had to be tested. Therefore, all concentrations given have to be regarded as nominal values. In all test article groups evaluated particles of the test article were observed.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

purity > 98 %, 0.6 mg/mL, without metabolic activation

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

purity >/= 99.5 %, 0.93 µg/mL, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- with S9 mix:
- Exposure duration: 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours and 28 hours after start of treatment
- without S9 mix:
- Exposure duration: 18 hours and 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours and 28 hours after start of treatment

SPINDLE INHIBITOR: colcemid
STAIN: Giemsa

NUMBER OF REPLICATIONS: three, however first experiment not reported due to technical problems during the performance.

NUMBER OF CELLS EVALUATED: In each experimental group two parallel cultures were set up. Per culture 100 well spread metaphases per culture were scored for cytogenetic damage. Only metaphases with characteristic chromosome numbers of 22 +/- 1 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)

OTHER EXAMINATIONS:
- Determination of polyploidy: polyploid cells (% polyploid metaphases; in the case of this aneupoid cell line polyploid means a near tetrapoid karyotype).

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY

The assay is considered acceptable if it meets the following criteria:
- The number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.00 % - 4.00 %
- The positive control substances should produce significant increases of the number of cells with structural chromosomal aberrations.

EVALUATION of RESULTS

- A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
- A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

The evaluation of results can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The test article substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) was tested in three independent experiments, however experiment I was not reported due to technical problems during the performance.

The tested concentrations in experiment II and III with and without metabolic activation were:

- 18 h preparation interval: 100, 250, 500, 1000, 2500, 5000 µg/mL

- 28 h preparation interval: 500, 1000, 2500, 5000 µg/mL

In experiment III in the absence of S9 mix at both preparation intervals the concentrations of 5000, 2500, and 1000 µg/mL could not be evaluated due to cytotoxic effects (cell lysis, reduced mitotic index).

In the absence of S9 mix, in both experiments the mitotic indices were reduced after treatment with the test article at each fixation interval except in experiment III at preparation interval 28 h were 500 µg/mL had to be evaluated due to cytotoxicity in the test groups of higher concentrations.

In the presence of S9 mix in experiment II the mitotic indices were not reduced (500 and 2500 µg/mL; 18 h preparation interval) or not substantially reduced (5000 µg/mL, 18 h and 28 h preparation intervals) after treatment. In experiment III the mitotic indices were reduced at both preparation intervals and with all concentrations evaluated.

The following concentrations were evaluated (18 h preparation interval: 3 concentrations; 28 h preparation interval: highest evaluable concentration):

Experiment II without metabolic activation:

- 18 h preparation interval: 500, 2500, 5000 µg/mL

- 28 h preparation interval: 2500 µg/mL

Experiment II with metabolic activation:

- 18 h preparation interval: 500, 2500, 5000 µg/mL

- 28 h preparation interval: 5000 µg/mL

Experiment III without metabolic activation:

- 18 h preparation interval: 100, 250, 500 µg/mL

- 28 h preparation interval: 500 µg/mL

Experiment III with metabolic activation:

-- 18 h preparation interval: 250, 2500, 5000 µg/mL

-- 28 h preparation interval: 5000 µg/mL

In none of the experiments in the absence and presence of S9 mix the test article significantly increased the frequency of cells with aberrations. The aberration rates of the cells after treatment with the test article (exp. II: 0.0 % - 2.0 %; exp. III: 0.0 % - 1.5 %) were in the range of the vehicle control values (exp. II: 0.5 % - 2.0 %; exp. III: 1.5 % - 3.0 %) and in the range of the laboratory historical control data (0.0 % - 4.0 %).

In experiment II the statistical evaluation revealed no significant differences between the treatment group (2.0 % aberrant cells; 500 µg/mL with S9 mix) versus the corresponding control (0.5 % aberrant cells).

In both experiments, no biologically relevant increase in the rate of polyploid metaphases (exp. II: 1.0 % - 2.5 %; exp. III: 0.5 % - 2.5 %) as compared to the rates of the controls (exp. II: 1.0 % - 3.0 %; exp. III: 0.5 % - 4.5 %) were found after treatment with the test article.

In both experiments, ethylmethanesulphonate (0.6 mg/mL) and CPA (0.93 µg/mL) were used as positive controls and showed a distinct increase in cells with structural chromosomal aberrations.

In conclusion, it can be stated that in this chromosomal aberration assay, the test article substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.

Conclusions:

Based on the results of this study it is concluded that substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not clastogenic in the in vitro Chromosome aberration assay using Chinese hamster V79 cells.

Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration assay) according to OECD Guideline 473, adopted May 26, 1983, EU method B.10, December 29, 1992 and EPA, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, Chinese hamster V79 cells cultured in vitro were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in water at concentrations of 100, 250, 500, 1000, 2500 and 5000 µg/L (18 h preparation interval) and 500, 1000, 2500 and 5000 µg/mL (28 h preparation interval) in the presence and absence of mammalian metabolic activation (S9 -mix). 

Substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) was tested up to and including cytotoxic concentrations. The positive controls did induce the appropriate response. 

There was no evidence of structural chromosomal aberrations induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473, EU Method B.10 and the EPA Guideline for in vitro cytogenetic mutagenicity (chromosomal aberration) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Reliable in vivo data from a mouse bone marrow micronucleus assay is available for substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.). There is no evidence for an intrinsic genotoxic activity relevant to humans.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-01-28 to 1991-03-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Code of Federal Regulations, Titel 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay"

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf CH-4414 Füllinsdorf/Basel, Switzerland
- Age at study initiation: minimum 10 weeks at start of acclimatization
- Weight at study initiation: appr. 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: Appr. 18 hours before treatment the animals received no food but water ad libitum.
- Housing: single housing in Makrolon Type I cages with wire mesh top
- Diet: ad libitum, pelleted standard diet Altromin 1324, D-4937 Lage/Lippe
- Water: ad libitum, tap water
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative nontoxicity for the animals
- Concentration of test material in vehicle: 250 mg/mL
- Amount of vehicle (if gavage or dermal): dose volume was 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in carboxymethlycellulose (CMS, 1 %). At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted. All animals received a single standard dose volume of 20 mL/kg body weight orally.

Duration of treatment / exposure:

single application

Post exposure period:

24, 48 and 72 hours

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 (6 males, 6 females)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral: gavage
- Doses / concentrations: 30 mg/kg body weight dissolved in physiological saline, dose volume: 10 mL/kg body weight

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In three subsequent pre-experiments 4 animals per dose received orally 2000, 3000, 4000, or 5000 mg test substance/kg body weight. No animal died within 72 hours p.a.. The animals expressed symptoms of indisposition (reduced spontaneous activity, eyelid closure, apathy). Higher dosing was not attainable: appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/mL; application volumes higher than 20 mL/kg body weight were not justifiable for the rodents used.

The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24, 48, and 72 hours post-treatment with a single dose animals were sacrificed by cervical dislocation.

DETAILS OF SLIDE PREPARATION: After sacrifice, the femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.


METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100 x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.


OTHER: Five animals per sex and group were evaluated. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.

A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.

This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biologically and statistical significance should be considered together.

Statistics:

Mann-Whitney test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical symptoms

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 3000, 4000 and 5000 mg/kg body weight
- Solubility: Appropriate suspensions (homogeneity, viscosity) could be obtained only up to 250 mg/mL; application volumes higher than 20 mL/kg body weight were not justifiable for the rodents used.
- Clinical signs of toxicity in test animals: in all dose groups reduced spontaneous activity, eyelid closure, apathy in all dose groups
- Evidence of cytotoxicity in tissue analyzed: not analysed
- Rationale for dose selection: maximum attainable dose


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.). The mean values of micronuclei observed after treatment with substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: The ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.
- Appropriateness of dose levels and route: maximum attainable dose tested
- Statistical evaluation: no significant increase of micronucleus frequency
- Positive control: Cyclophosphamide (30 mg/kg body weight administered per os) was used as positive control which showed a distinct increase of micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) is considered to be non-mutagenic in this micronucleus assay.

Conclusions:

Based on the results of this study it is concluded that substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not clastogenic in the in vivo mouse bone marrow micronucleus assay.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD Guideline 474, adopted May 26, 1983, EU method B.12, EEC Directive 84/449 and EPA, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, groups of 6 mice per sex and bone marrow harvest time were given a single oral dose of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in carboxymethylcellulose at a dose of 5000 mg/kg body weight. Bone marrow cells were harvested at 24h, 48 h, and 72 h after application.

Substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) was tested at an adequate dose. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed symptoms of indisposition (reduced spontaneous activity, eyelid closure, apathy). The ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. The positive controls did induce the appropriate response. 

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474, EU Method B.12 and the EPA Guideline for in vivo mammalian bone marrow cytogenetics tests (micronucleus assay).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a reverse gene mutation assay in bacteria according OECD Guideline 471, adopted May 26, 1983 and EU Method B.13/14, strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 of Salmonella typhimurium were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in DMSO at concentrations of 10 to 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9-mix, plate incorporation). The substance was tested up to and including cytotoxic concentrations (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. A significant slight increase in the number of revertants was found in the strains TA 1537 and TA 1538 both in the presence of metabolic activation in experiment I and II at the highest investigated dose. However, according to an independent expert judgement (Prof. Dr. C. Schlatter, Institute für Toxikologie der Eidgenössischen Technischen Hochschule und der Universität Zürich (Schwerzenbach, 10.September 1991)), the slight increased numbers of revertants in strains TA 1537 and TA 1538 at the highest tested concentration of 5000 µg/plate with metabolic activation, is hardly a real mutagenic effect because such activity is due to the structure of the test substance not plausible at all. The result is probably an unspecific result of the toxicity and/or disturbance of the osmolality in the test system and therefore of no relevance for humans and animals. The adopted OECD TG 471 (1997) respectively EU Method B.13/14 according to Regulation (EC) No 440/2008 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 and EU Method B.13/14 (Version Commission Directive 92/69/EEC) without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the reaction mass in this bacterial test system. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471and EU Method B.13/14 for in vitro mutagenicity (bacterial reverse gene mutation) data.

In a mammalian cell cytogenetics assay (Chromosome aberration assay) according to OECD Guideline 473, adopted May 26, 1983, EU method B.10, December 29, 1992 and EPA, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, Chinese hamster V79 cells cultured in vitro were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in water at concentrations of 100, 250, 500, 1000, 2500 and 5000 µg/mL (18 h preparation interval) and 500, 1000, 2500 and 5000 µg/mL (28 h preparation interval) in the presence and absence of mammalian metabolic activation (S9-mix). The substance was tested up to and including cytotoxic concentrations. There was no evidence of structural chromosomal aberrations induced over background. The positive controls did induce the appropriate response. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473, EU Method B.10 and the EPA Guideline for in vitro cytogenetic mutagenicity (chromosomal aberration) data.

 

In a mammalian cell gene mutation assay (HPRT-test) according to OECD Guideline 476, adopted April 4, 1984, EU method B.17 and EPA OTS 798.5300, Chinese hamster V79 cells cultured in vitro were exposed to substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in DMSO at concentrations of 0.30, 1.00, 2.50, and 5.00 µg/mL in the presence and absence of mammalian metabolic activation (S9-mix). The substance was tested up to and including cytotoxic concentrations. There was no evidence of induced mutant colonies over background. The positive controls did induce the appropriate response. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476, EU Method B.17 and EPA OTS 798.5300 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

In an in vivo NMRI mouse bone marrow micronucleus assay according to OECD Guideline 474, adopted May 26, 1983, EU method B.12, EEC Directive 84/449 and EPA, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicology, Revision July 1, 1986, groups of 6 mice per sex and bone marrow harvest time were given a single oral dose of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) in carboxymethylcellulose at a dose of 5000 mg/kg body weight. Bone marrow cells were harvested at 24h, 48 h, and 72 h after application. The substance was tested at an adequate dose. In pre-experiments this dose level was estimated to be the maximum attainable dose. The animals expressed symptoms of indisposition (reduced spontaneous activity, eyelid closure, apathy). The ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. The positive controls did induce the appropriate response. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474, EU Method B.12 and the EPA Guideline for in vivo mammalian bone marrow cytogenetics tests (micronucleus assay).

Justification for selection of genetic toxicity endpoint
In vivo study selected. In addition, full range of in vitro studies available. All studies according to OECD and EU guidelines, no deviations, GLP.

Justification for classification or non-classification

There is no evidence for a clastogenic or mutagenic potential of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 % a.i.) relevant to humans obtained from the results of a reverse gene mutation assay in bacteria, a mammalian cell cytogenetics assay (Chromosome aberration assay), a mammalian cell gene mutation assay (HPRT-test) and an in vivo mouse bone marrow micronucleus assay.  

In accordance with CLP Regulation (EC) No 1272/2008, substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" is not classified for genetic toxicity and labelling is not required.