Bis(6-methylalkyl-5-en-2-yl)dihydro-heteropolycycle

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

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:

Dec. 2006 - Jan. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by a combination of phenobarbital 80 mg/kg + ß-naphthoflavone 100 mg/kg

Test concentrations with justification for top dose:

Dose finding test: 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate in absence and presence of S9-mix in TA100 and WP2uvrA. Sa03 precipitated on the plates at dose levels of 1000 µg/plate and upwards.

First mutation assay: 10, 33, 100, 333, 1000 ug/plate in absence and presence of 10 % (v/v) S 9-mix in TA1535, TA1537 and TA98.

First mutation assay: 10, 33, 100, 333, 1000 ug/plate in absence and presence of 10 % (v/v) S 9-mix in TA1535, TA1537, TA98, TA100 and WP2uvrA.

Vehicle / solvent:

Dimethyl formamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Remarks:

-S9: sodium azide, 9-aminoacridine, 2-nitrofluorene, methylmethanesulfonate, 4-nitroquinoline; +S9: 2-aminoanthracene

Details on test system and experimental conditions:

TEST SYSTEM

The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation
assay have been shown to be rapid and adequate indicators for the mutagenic activity of a wide
range of chemical compounds.

The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100.
The Escherichia coli strain used was WP2uvrA. The strains TA1537 and TA98 are capable of
detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting
base-pair substitution mutagens.

The characteristics of the different Salmonella typhimurium strains were as follows:

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

*: R-factor = plasmid pKM101 (increases error-prone DNA repair)


EXPERIMENTAL CONDITIONS

Preparation of bacterial cultures
Sampies of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid
LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the
cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/ml). Freshly grown cultures
of each strain were used for a test.

Agar plates
Agar plates (diameter 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (B. Braun, Melsungen, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Acros Organics).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid L TD) and 0.5% (w/v) sodium
chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred
into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in the dark at 34.5 - 38.4°C (protocolled range 37.0 ± 1.0°C).
Temporary deviations of maximally 1 hour (in the range of 34.0 - 38.5°C) occurred due to
addition of plates (which were at room temperature) to the incubator or due to opening and
closing the incubator door. Based on laboratory historical data these deviations are considered
not to affect the study integrity.

Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9 -mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were
successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of
one of the tester strains, 0.1 ml of a dilution of the test substance in dimethyl formamide and
either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of
non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar
tube was poured onto a selective agar plate. After solidification of the top agar, the plates were
inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 h. After this period revertant colonies
(histidine independent (His +) for Salmonella typhimurium bacteria and tryptophan independent
(Trp+) for Escherichia coli) were counted.


Colony counting
The revertant colonies (histidine independent or tryptophan independent) were counted
manually if less than 40 colonies per plate were present. If more than 40 colonies were present,
these could be counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with
sufficient test article precipitate to interfere with automated colony counting were counted
manually and evidence of test substance precipitate on the plates was recorded. The condition
of the bacterial background lawn was evaluated, both macroscopically and microscopically by
using a dissecting microscope.

Evaluation criteria:

Acceptability of the assay:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three time the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the premilary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test, if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test, if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98 and TA100, E.coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: other: all strains/cell types tested reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Dose range finding test:

The dose range finding test is reported as a part of the first experiment of the mutation test: Table 1 of report R 0700061. The individual data are presented in Appendix II of report R 0700061.

Precipitation of Sa03 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.

To determine the toxicity of Sa03, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The definitions are stated in Appendix I of R 0700061.

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with Sa03 under all conditions tested.

 

Mutation assay:

Based on the results of the dose finding test, Sa03 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9 -mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537, and TA97 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The results are shown in Table 1 and 2, the individual data are presented in Appendix II of the report R 070061.

Precipitation of Sa03 on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate.

Toxicity: In both mutation assays, there was no reduction of the bacterial background lawn and no biological relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9 -mix.

Mutagenicity: In both mutation assays, no increase in the number of revertants was observed upon treatment with Sa03 under all conditions tested.

Conclusions:

Interpretation of results (migrated information): negative with and without S9-mix.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within NOTOX laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Sa03 is not mutagenic in the Salmonella thyphimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of Sa03 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).
Sa03 was tested in the Salmonella typhimurium reverse mutation assay with four
histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).
The study procedures described in this report were based on the most recent OECD and EEC guidelines.

Sa03 was dissolved in dimethyl formamide.
In the dose range finding test, Sa03 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Sa03 precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, Sa03 was tested in the first mutation assay at a concentration range of 10 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, Sa03 was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Sa03 precipitated on the plates at the top dose of 1000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Sa03 did not induce a significant dose-related increase in the number of revertant (His+)
colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
In this study, the negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolie activation system functioned properly.

Based on the results of this study it is concluded that Sa03 is not mutagenic in the
Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The test substance showed no adverse effects in OECD 471 study with and without metabolic activation. The substance does not need to be classified for genetic toxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.