H112323

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 May. 13, 1991 to Sep. 12, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD Guideline 471 and 472 and UK Department of Health Guidelines (DoH, 1989) in compliance with GLP

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:

1991

Report date:

1991

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UK Department of Health Guidelines (DoH, 1989)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Substance H112323
- Physical state: Dark blue powder
- Storage condition of test material: Ambient 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 specified

Species / strain 2

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Plate incorporation test (Phase I):
a: without metabolic activation:
200, 500, 1000, 2500, 5000, 8710.8 µg/plate
b: with metabolic activation:
200, 500, 1000, 2500, 5000, 8710.8 µg/plate

Preincubation test (Phase II):
a: with metabolic activation:
200, 500, 1000, 2500, 5000, 8710.8 µg/plate

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9

Positive control substance:

other: 2-Aminoanthracene (2AA) ( for strain TA 1535, TA 1537, TA 98, TA 100, WP2P, WP2P uvrA)

Remarks:

-

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9

Positive control substance:

other: N-Methyl-N'-nitro-N-nitrosoguanidine(MNNG) ( for strain TA 1535, TA100, WP2P and WP2P uvrA )

Remarks:

-

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9

Positive control substance:

other: Acridine Mutagen ICR191 (for strain TA 1537)

Remarks:

-

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9

Positive control substance:

other: Daunomycin HCl (DR) (for strain TA 98)

Remarks:

-

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period:
- Incubation period: 3 d at 37 °C in the dark

NUMBER OF REPLICATIONS: Three


DETERMINATION OF CYTOTOXICITY
Method: A reduction in the number of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators.
Thinning of the bacterial lawn was evaluated microscopically.

Evaluation criteria:

a) a statistically significant dose-related increase in the mean number of revertant colonies is obtained;
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) which is statistically significant, is observed at least one dose level.

Statistics:

- One-tailed Student's t-test
- Dunnett's method

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

bacteria, other: E. coli WP2P and WP2P uvrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

bacteria, other: E. coli WP2P and WP2P uvrA

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium, other: TA1537, TA98, TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Not observed

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation (TA1537, TA98, TA100, TA1535)
negative without metabolic activation (TA1537, TA98, TA100, WP2P and WP2P uvrA)
positive with metabolic activation (WP2P and WP2P uvrA)
positive without metabolic activation (TA1535)

Under the test conditions, test substance gave negative (non-mutagenic), response with S. typhimurium strains TA1537, TA98, TA100 in the presence and absence of an auxiliary metabolising system (S9), with strain TA1535(+S9), and with E. coli strains WP2P and WP2P uvrA (-S9). With strains TA1535 (-S9) and WP2P and WP2P uvrA (+S9), the test substance gave a weak positive i.e. mutagenic response.

Executive summary:

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, 472 and UK Department of Health Guidelines (DoH, 1989) in compliance with GLP.

Four strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and two strains of E. coli (WP2P and WP2P uvrA were used in the bacterial mutagenicity assay.

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in strains TA1537, TA98, TA100, WP2P and WP2P uvrA in the absence of an auxiliary metabolising system (S9), or in the four Salmonella strains in the presence of S9. With both E. coli strains in the presence of S9 and with TA1535 in the absence of S9, small but reproducible statistically significant increases in colony numbers were observed.

In each experiment, the positive controls responded as expected indicating that the assay was performing satisfactorily.

Under the test conditions, test substance gave negative (non-mutagenic), response with S. typhimurium strains TA1537, TA98, TA100 in the presence and absence of an auxiliary metabolising system (S9), with strain TA1535(+S9), and with E. coli strains WP2P and WP2P uvrA (-S9). With strains TA1535 (-S9) and WP2P and WP2P uvrA (+S9), the test substance gave a weak positive i.e. mutagenic response.