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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
Reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide
EC Number:
915-069-0
Molecular formula:
not applicable
IUPAC Name:
Reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide

Method

Species / strain
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.
Controls
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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.