Inositol phosphates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 24, 2009 to April 22, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The procedures performed in this study were based on OECD Guideline 471, EU Method B.13/14, USA, EPA (TSCA) OPPTS harmonised guidelines, Japanese regulatory guidelines including METI, MHLW and MAFF. The GLP compliance is claimed through compliance with UK Good Laboratory Practice standards [Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)].

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Inositol phosphates

EC Number:

700-206-7

Molecular formula:

As this substance is a UVCB substance and consists varying degree of components, a single molecular formula is not applicable

IUPAC Name:

Inositol phosphates

Constituent 2

Reference substance name:

Synthetic inositol phosphates

IUPAC Name:

Synthetic inositol phosphates

Details on test material:

- Name of test material (as cited in study report): Synthetic Inositol phosphates
- Physical state: Amber coloured liquid
- Analytical purity: 24-31% Inositol phosphates in water
- Lot/batch No.: 95816206JH-012
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Not applicable

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% liver S9 fraction in standard co-factors

Test concentrations with justification for top dose:

Preliminary test (Range-finding test): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1,500 and 5,000 μg/plate

Mutation test:
Experiment 1 & 2: 50, 150, 500, 1,500 and 5,000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Test material was fully miscible (50 mg/mL) in sterile distilled water

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Concurrent negative controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation (S9)

Migrated to IUCLID6: 3 μg/plate for TA100, 5 μg/plate for TA1535 and 2 μg/plate for WP2uvrA-

Controls 2

Untreated negative controls:

yes

Remarks:

Concurrent negative controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation (S9)

Migrated to IUCLID6: 80 μg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Remarks:

Concurrent negative controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation (S9)

Migrated to IUCLID6: 0.2 μg/plate for TA98

Controls 4

Untreated negative controls:

yes

Remarks:

Concurrent negative controls

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation (S9)

Migrated to IUCLID6: 5 μg/plate for TA98

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, and 10 μg/plate for WP2uvrA-

Remarks:

with metabolic activation (S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (direct plate incorporation)


DURATION
- Preincubation period: Not applicable
- Exposure duration:
Preliminary test: 48 h (with and without S9) at 37 °C
Mutation test:
Experiment 1 & 2: 48 h (with and without S9) at 37 °C
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable


NUMBER OF REPLICATIONS: Triplicate

Evaluation criteria:

Criteria for determining positive result: Dose-related increase in the frequency of revertant colony 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.

A test material was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Statistics:

The experimental data was analysed using UKEMS recommended statistical guidelines.

Results and discussion

Test results

Test results 1

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not applicable
- Water solubility: 50 mg/mL
- Precipitation: No
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5,000 μg/plate.
- The test material caused no visible reduction in the growth of the bacteria at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5,000 μg/plate.
- No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- 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.


COMPARISON WITH HISTORICAL CONTROL DATA: No comparison performed.


ADDITIONAL INFORMATION ON CYTOTOXICITY: Not applicable

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test:

The test material was non-toxic to the strains (TA100 and WP2uvrA-) of bacteria.

Sterility check of S9-mix:

The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Table 1: Numbers of revertant colonies for the toxicity assay

With (+) or without (-) S9-mix	Strain	Dose (μg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	99	101	104	90	109	103	109	111	131	93	118
+	TA100	104	94	108	119	111	106	91	98	99	88	87
-	WP2uvrA-	21	20	22	24	32	27	19	34	31	21	18
+	WP2uvrA-	30	44	19	35	25	37	39	29	26	35	24

Mutation test:

No visible reduction in the growth of the bacterial background lawn was observed at any dose level. Therefore, the test material was tested upto the maximum recommended dose level of 5,000 µg/plate.

Neither the test material precipitate nor any significant increases in the frequency of revertant colonies were observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

Significant increases in the frequency of revertant colonies were observed with all the positive control chemicals which confirmed the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 2: Test Results: Experiment 1 – Without Metabolic Activation (see attached PDF titled 'Genetic toxicity-in vitro' page no. 16)

Table 3: Test Results: Experiment 1 – With Metabolic Activation (see attached PDF titled 'Genetic toxicity-in vitro' page no. 17)

Table 4: Test Results: Experiment 2 – Without Metabolic Activation (see attached PDF titled 'Genetic toxicity-in vitro' page no. 18)

Table 5: Test Results: Experiment 2 – With Metabolic Activation (see attached PDF titled 'Genetic toxicity-in vitro' page no. 19)

 

Applicant's summary and conclusion

Conclusions:

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

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

Executive summary:

A study was conducted to determine the potential mutagenicity of synthetic inositol phosphates using a bacterial/microsome test system. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFFand the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

 

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

 

The vehicle (sterile distilled water) 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 test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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.

 

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