Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

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:

2004-07-19 to 2004-08-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Pre-incubation method amended due to reduced background growth at lowest concentration (10 ug/plate)

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 2001-06-13; Date of signature 2002-01-14

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

EC Number:

939-227-3

Cas Number:

84238-39-1

Molecular formula:

Not applicable (a generic Molecular formula cannot be provided for this substance)

IUPAC Name:

Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

Method

Target gene:

- Histidine for Salmonella typhimurium

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Dose selection: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate.
- Experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate.
- Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate.

Vehicle / solvent:

- On the day of the experiment, the test material was dissolved in ethanol (> 99 %; Merck, D-64293 Darmstadt).
- The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
- The test material precipitated weakly at 5000 μg/plate in experiment I and at 2500 μg/plate and above in experiment II. Undissolved particles of test item were not considered to influence data recording.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA 100 without metabolic activation Migrated to IUCLID6: at least 99 % purity dissolved in deionised water (10 μg/plate)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (> 99 % purity dissolved in DMSO) 10μg/plate

Remarks:

TA 1537 and TA 98 without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 102 without metabolic activation Migrated to IUCLID6: > 99.0 % purity dissolved in deionised water (4.0 μL/plate)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (> 99 % purity dissolved in DMSO) 2.5 μg/plate (10.0 μg/plate for TA 102)

Remarks:

TA 1535, TA 1537, TA 98, TA 100 and TA 102 with metabolic activation

Details on test system and experimental conditions:

CHARACTERISATION
- Bacterial strain cultures were obtained from Trinova Biochem GmbH (35394 Giessen, Germany)
- Regular checking of the properties of the strains was performed with respect to membrane permeability, ampicillin and tetracycline resistance, and spontaneous mutation rates.

STORAGE
- Strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (Merck, D-64293 Darmstadt) in liquid nitrogen.

PRE-CULTURES
- From thawed ampoules of the strains, 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium.
- A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100 and TA 102.
- 20 μL tetracycline (2 μg/mL) was also added to strain TA 102.
- The nutrient medium contained 8g/L Merck Nutrient Broth and 5 g/L sodium chloride.
- Bacterial cultures were incubated in a shaking water bath for 4 hours at 37 ºC.

AGAR
- Plates with minimal agar were obtained from E Merck, D-64293 Darmstadt.
- Overlay agar (Merck, D-64293 Darmstadt) contained 6.0 g/L Merck Agar, 6.0 g/L sodium chloride, 10.5 mg/L L-histidine x HCl x water and 12.2 mg/L Biotin.
- Sterilisations were performed at 121 ºC in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9 was prepared from 8-12 week old male Wistar Hanlbm rats (220-320 g) induced by applications of 80 mg/kg bw phenobarbital i.p. (Destin, D-22335 Hamburg) and beta-naphthoflavone p.o. (Aldrich, D-89555 Steinheim) on three consecutive days.
- Livers were prepared 24 hours after the last treatment.
- S9 fractions were produced by dilution of the liver homogenate with potassium chloride solution (1+3) followed by centrifugation at 9000 g.
- Aliquots of the supernatant were frozen and stored in ampoules at minus 80 ºC.
- Small numbers of ampoules were kept at minus 20 ºC for up to one week.
- Protein concentration in the S9 preparation was 29.0 mg/mL (lot number R 230404).

S9 MIX
- An appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution.
- The amount of S9 supernatant was 15 % v/v in the S9 mix.
- Co-factor concentrations in the S9 mix were 8mM (magnesium chloride), 33 mM (potassium chloride), 5 mM (glucose-6-phosphate), 5 mM (NADP) in 100 mM sodium orthophosphate buffer (pH 7.4).
- During the experiment, the S9 mix was stored in an ice bath.

PRE-EXPERIMENT FOR TOXICITY
- Eight concentrations of TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested in triplicate for toxicity and mutation induction.
- Experimental conditions were identical to those of experiment I.

EXPERIMENTAL PERFORMANCE
- The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control).
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation).
- 100 μL bacterial suspension
- 2000 μL overlay agar.
- In the pre-incubation assay, 100 μL test solution, solvent (50 μL in strains TA 100 and TA 102) and positive control, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 ºC for 60 minutes.
- After pre-incubation, 2.0 mL overlay agar (45 ºC) was added to each tube and the mixture was poured onto minimal agar plates.
- After solidification, the plates were incubated upside down for at least 48 hours at 37 ºC in the dark.

DATA RECORDING
- Colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben).
- The counter was connected to an IBM AT compatible PC with printer, which provided the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.
- Due to the reduced background growth of the test item, colonies were counted manually at higher concentrations.

Evaluation criteria:

CRITERIA FOR ACCEPTABILITY OF THE ASSAY
- Regular background growth in the negative and solvent control.
- Spontaneous reversion rates in the negative and solvent control are in the range of historical data.
- Positive control substances produce a significant increase in mutant colony frequencies.

EVALUATION OF RESULTS
- A test item is considered a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in a second independent experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Statistics:

- Statistical analysis of the data was not required.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table below

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Results tables and historical control data are presented in the report annex (attached).
- Plates incubated with the test item showed reduced background growth at higher concentrations with and without S9 mix in all strains used.
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration level (with or without the presence of S9 mix).
- There was no tendency to higher mutation rates with increasing concentration in the range below the generally acknowledged border of biological relevance.
- In experiment II, the data in the negative control of strain TA 102 was slightly above the historical control range. However, the deviation was small and the effect was considered to be based on biologically irrelevant fluctuations in the number of colonies.
- Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.
- The historical range of positive controls was exceeded in strains TA 1535 (experiment I) and TA 100 (experiment II) without metabolic activation and in strain TA 1537 (experiment I) with metabolic activation but this effect was considered to indicate the sensitivity of the strains rather than assay compromise.

Remarks on result:

other:

Any other information on results incl. tables

Toxic effects (evident as a reduction in the number of revertants) were observed at the concentrations shown in the table below:

 

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9
TA 1535	No toxic effects	2500-5000 μg/plate	5000 μg/plate	1000, 5000 μg/plate
TA 1537	1000-2500 μg/plate	No toxic effects	2500-5000 μg/plate	100-5000 μg/plate
TA 98	No toxic effects	No toxic effects	1000-5000 μg/plate	1000-5000 μg/plate
TA 100	5000 μg/plate	5000 μg/plate	No toxic effects	No toxic effects
TA 102	5000 μg/plate	5000 μg/plate	1000-5000 μg/plate	1000-5000 μg/plate

 

Applicant's summary and conclusion

Conclusions:

The test material did not induce gene mutation by base pair changes or frameshifts in the genome of the strains used.