Reaction mass of 9-icosyl-9-phosphabicyclo[3.3.1]nonane and 9-icosyl-9-phosphabicyclo[4.2.1]nonane

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Materials and methods

Test guideline

Qualifier:

no guideline followed

Deviations:

not applicable

Principles of method if other than guideline:

This work was carried out to obtain preliminary information on the mutagenicity of RM-17 catalyst using short term in vitro tests. These include a test for the induction of mutation in a series of micor-organisms with and without microsomal activation and a chromosome study in cultures of rat liver celss.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 9-icosyl-9-phosphabicyclo[3.3.1]nonane and 9-icosyl-9-phosphabicyclo[4.2.1]nonane

EC Number:

915-206-4

Molecular formula:

C28H55P

IUPAC Name:

Reaction mass of 9-icosyl-9-phosphabicyclo[3.3.1]nonane and 9-icosyl-9-phosphabicyclo[4.2.1]nonane

Details on test material:

- Name of test material (as cited in study report): RM-17
- Substance type: no data
- Physical state: no data
- Analytical purity: not stated.
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: 9-eicosyl-9-phosphabicyclo[421]nonane, mixture with [331] isomer
- Isomers composition: no data
- Purity test date: not stated.
- Lot/batch No.: not stated.
- Expiration date of the lot/batch: not stated.
- Stability under test conditions: not stated.
- Storage condition of test material: not stated.

Method

Target gene:

Bacterial mutation study
E.coli: tryptophan
S.typhymurium: histidine
Yeast study: histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1538, TA 92, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: ready poured petri plates containing 25 mL of a minimal agar medium based on Vogel-Bonner.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not stated
- Periodically checked for karyotype stability: not stated
- Periodically "cleansed" against high spontaneous background: not stated

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: ready poured petri plates containing 25 mL of a minimal agar medium based on Vogel-Bonner.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not stated
- Periodically checked for karyotype stability: not stated
- Periodically "cleansed" against high spontaneous background: not stated

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):

- Type and identity of media: ready poured petri plates containing 25 mL of a minimal agar medium based on Vogel-Bonner.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not stated
- Periodically checked for karyotype stability: not stated
- Periodically "cleansed" against high spontaneous background: not stated

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

Saccharomyces cerevisiae

Details on mammalian cell type (if applicable):

- Type and identity of media: a complete YEPD medium prepared containing 1% yeast extract, 2% Difco Bactopeptone and 2% glucose.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not stated
- Periodically checked for karyotype stability: not stated
- Periodically "cleansed" against high spontaneous background: not stated

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9: Aroclor 500 mg/kg

Test concentrations with justification for top dose:

Bacterial mutation study: 0.2, 2.0, 20 and 200 µg RM-17 per plate.
Saccharomyces gene conversion assay: 0.001, 0.01, 0.1 and 1,0 mg/mL of liquid suspension

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: not stated

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

E.coli WP2 and WP2 uvr A

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

no

Remarks:

with and without S9

Controls 2

Untreated negative controls:

yes

Remarks:

S.typhimurium TA 1538 and TA 92

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

no

Remarks:

with and without S9

Controls 3

Untreated negative controls:

yes

Remarks:

S.typhimurium TA 98 and TA 100

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with and without S9

Controls 4

Untreated negative controls:

yes

Remarks:

Saccharomyces cerevisiae JD1

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

with and without S9

Controls 5

Untreated negative controls:

yes

Remarks:

Saccharomyces cerevisiae JD1

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

BACTERIAL MUTATION STUDY
Culture media:
Ready poured petri plates containing 25 mL of a minimal agar medium based on Vogel-Bonner.
Preparation of bacterial cultures:
Cultures of each bacterial strain are prepared in 10 mL nutrient broth and incubated overnight at 37°C in a shaking water bath. The cultures are then centrifuged, washed twice in phosphate buffer at pH 7.0 and the organisms resuspended in 5 mL buffer.
Microbial mutation assays in soft agar overlay:
For plate incorporation assays, the following are added, in order, to 2 mL of molton top agar in a test tube at 45°C: 0.1 mL of an overnight bacterial culture in phosphate buffer, 20 µL of the compound or solvent to be tested, and 0.5 mL of the S9 mix, where appropriate. The contents are mixed and poured onto minimal agar plates distributing the top agar uniformly across the plate by gentle tilting. The top agar is allowed to solidify and the plates are incubated at 37°C. The mutant colonies are counted after 2 days incubation and the presence of a background lawn confirmed.
A wide range of concentrations of each compound are tested both in the presence and absence of the S9 mix. Control plates are set up with the solvent both with and without the S9 mix.
All tests are carried out in quadruplicate and the mean number of colonies on the test plates are compared with the control means.

METHOD OF APPLICATION: A top agar (0.6% agar, 0.6% NaCl) is autoclaved and stored in bottles in volumes of 50 mL at room temperature. Before use the agar is melted by heating in a water bath and supplemented with either 1 mL 0.5 mM tryptophan and 2.5 mL nutrient broth for the E.coli strains or 5 mL o.5 mM histidine-0.5 mM biotin for the S.typhimurium strains.

DURATION
The cultures were incubated at 37°C for 24 hours before the revertant colonies were counted.

YEAST STUDY
Selection of suitable cultures:
A YEPD broth culture is inoculated from a dried stock culture and incubated overnight at 28°C. From this initial culture, a series of flasks containing 5 mL YEPD broth are each seeded with 200 yeast cells. These cultures are grown to a stationary phase by incubation, with continuous mechanical agitation, at 28°C for 4 days. Cells which are able to grow and form colonies on the respective synthetic media are judged to originate from conversion at the his4 or trp5 loci (or rarely, at both loci) and colony counts are carried out to assess the spontaneous conversion rate in each broth culture.
In vitro studies with S.cerevisiae JD1:
Broth cultures of yeast cells having a low spontaneous conversion rate are selected and the cells washed twice in phosphate buffer at pH 7.0. The cells are resuspended in buffer at a concentration of 2.5 x 10 (7) cells per mL.
20 µL of the compound or solvent to be tested are added to 2 mL aliquots of the cell suspension to give a wide range of final concentrations of the compound.
After 1 h exposure at room temperature without the S9 mix or after 1 h incubation in a shaking water bath at 37°C with 0.5 mL of the S9 mix, 0.1 mL samples of each suspension are seeded on appropriate medium to determine the number of mutants.

In these assays at least two replicate studies are carried out on different days to confirm reproducibility of the results.

METHOD OF APPLICATION: A complete YEPD medium is preprepared containing 1% yeast extract, 2% Difco Bactopeptone and 2% glucose. For solid medium YEPD agar is used.
For detecting revertants of S.cerevisiae, the synthetic "complete" medium of Wickerham is used, as modified by Roman. Histidine or tryptophan is omitted from the synthetic medium, where relevant, in order to isolate histidine or tryptophan prototrophs.

DURATION
After 1 hour incubation the cultures were seeded in appropriate culture medium for the selection of revertant colonies. After 3 days at 30°C the number of revertant colonies were counted. Four experiments were carried out; experiments A and B in the presence and C and D in the absence of S9 fraction.

Evaluation criteria:

BACTERIAL MUTATION STUDY
The added trace of tryptophan or histidine in the top agar allows the bacteria on the plate to undergo several divisions. This initial growth is necessary in many cases for mutations to be expressed. The resultant slight background lawn allows any inhibition of growth by the compound to be detected.

YEAST STUDY
The number of viable yeast cells surviving the treatment is obtained by plating dilutions of the suspension on complete medium. The colonies are counted after 4 days incubation at 28°C and the number of prototrophs per 10(6) survivors are determind.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1538, TA 92, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 200 µg per plate

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 200 µg per plate

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not applicable

Test results 3

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:

not applicable

Test results 4

Species / strain:

Saccharomyces cerevisiae

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
Saccharomyces gene conversion assay: The addition of the test material to liquid suspension cultures of S.cerevisiae JD1, with or without the incorporation of rat liver microsomal fraction (S9) did not induce a consistent increase in mitotic gene conversion.
A marginal increase in gene conversion occurred at the histidine locus in cultures exposed to 0.001 and 0.01 mg/mL the test material (Experiment A). This was not detected at the two higher concentrations or at the tryptophan locus; it was not apparent in the replicate study (Experiment B) and is considered to be a chance effect.
The S.cerevisiae culture used in Experiments B and D showed more than a 10-fold increase in the number of spontaneous revertants at the tryptophan locus. The results with the positive control compounds in these two experiments demonstrate the loss of sensitivity of the test system when this occurs. The spontaneous increase in revertants at the tryptophan locus is a common occurance with the JD1 strain, but as the histidine locus is not affected results at this locus are still valid and are therefore reported.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Bacterial mutation study:

The addition of the test material to agar layer cultures of S.typhimurium strain TA 1538, TA 92, TA 98 and TA 100 and E.coli strains WP2 and WP2 uvr A with or without the incorporation of rat liver microsomal fraction (S9) did not lead to an increase in the reversion frequency in any of the strains.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The results indicate that the test material is not a microbial mutagen under the conditions of the tests described.

Executive summary:

The mutagenic activity of the test material was investigated in agar layer cultures of Salmonella typhimurium TA 1538, TA 92, TA 98 and TA 100, Escherichia coli Saccharomyces cerevisiae JD1. Assays were carried out both with and without the incorporation of rat liver microsomal enzymes (S9).

The incorporation of test material in agar cultures of the bacterial tester strains or in liquid cultures of Saccharomyces cerevisiae did

not influence the mutation frequency either in the presence or absence of rat liver microsomal enzymes.

It is concluded that the test material did not elicit a mutagenic response in the test-systems used.