5(or 6)-methyl-7(or 8)-(1-methylethyl)bicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-05-17 to 2000-06-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

8-isopropyl-6-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

EC Number:

267-308-7

EC Name:

8-isopropyl-6-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Cas Number:

67845-30-1

Molecular formula:

C13H20O

IUPAC Name:

8-isopropyl-6-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Constituent 2

Reference substance name:

7-isopropyl-5-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

EC Number:

299-795-7

EC Name:

7-isopropyl-5-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Cas Number:

93904-56-4

Molecular formula:

C13H20O

IUPAC Name:

7-isopropyl-5-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

impurity 1

Reference substance name:

rel-(1S,2R,4R)-1-isopropyl-4-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Cas Number:

36208-34-1

Molecular formula:

C13H20O

IUPAC Name:

rel-(1S,2R,4R)-1-isopropyl-4-methylbicyclo[2.2.2]oct-5-ene-2-carbaldehyde

Test material form:

liquid

Specific details on test material used for the study:

Name: Lierral
Batch Number: 9000365675
Aggregate state at room temperature: liquid
Colour: colourless to pale yellow
Purity: 99.5% (sum of three peaks)
Storage: room temperature
Expiration date: December 20, 2000

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Bacterial suspensions were transferred into flasks containing nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA98, TA100, and TA102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA102. The nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl.
- Properly maintained: Yes. The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen. Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the testing laboratory.

Additional strain / cell type characteristics:

other: All strains possess the rfa- mutation. TA1537, TA98, TA1535 and TA100 possess the uvrB- mutation; TA102 possess the uvrB+ mutation. TA98, TA100 and TA 102 all have the R-factor plasmid pKM101.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Experiment 1: 3, 10, 33, 100, 333 and 1000 µg/plate
- Experiment 2: 1, 3, 13, 33, 100 and 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

- EXPERIMENT 1
METHOD OF APPLICATION: in agar (plate incorporation)
The following materials were mixed in a test tube and poured onto selective agar plates:
- 100 µL of test solution at each dose level, solvent or positive control;
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation);
- 100 µL bacteria suspension (prior to use, bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C); and
- 2000 µL overlay agar (containing per litre: 6.0 g Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCI x H₂O and 12.2 mg Biotin).

DURATION
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of background bacterial lawn


- EXPERIMENT 2
METHOD OF APPLICATION: pre-incubation
The following materials were mixed in a test tube and incubated:
- 100 µL of test solution at each dose level, solvent or positive control;
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation); and
- 100 µL bacteria suspension (prior to use, bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C).
- After pre-incubation, 2.0 mL overlay agar (as above, 45 °C) was added to each tube. The mixture was poured on minimal agar plates.

DURATION
- Pre-incubation period: 37 °C for 60 minutes.
- Exposure duration: after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of background bacterial lawn

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control;
- The spontaneous reversion rates in the negative and solvent control are in the range of historical data; and
- The positive control substances should produce a significant increase in mutant colony frequencies.

EVALUATION OF RESULTS
- A test material is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
- A test material producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.
- A biologically relevant response is described as follows: a test material is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA98, TA100, and TA102 or thrice in strains TA1535 and TA1537.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test material regardless whether the highest dose induced the criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Distinct toxic effects, evident as a reduction in the number of revertants, were observed as summarised in Table 1.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Reduced background growth was observed in strains TA1535, TA1537, TA98 and TA100 at 1000 µg/plate in the first experiment and in TA1537 and TA98 in the second experiment.

No substantial or reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no reproducible tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Positive controls showed a distinct increase in induced revertant colonies.

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

Any other information on results incl. tables

Table 1: Summary of Toxic Effects (µg/plate)

Strain	Experiment 1		Experiment 2
	-S9	+S9	-S9	+S9
TA 1535	333 to 1000	1000	33 to 333	333
TA 1537	333 to 1000	333 to 1000	333	333
TA 98	100 to 1000	1000	333	333
TA 100	100 to 1000	333 to 1000	100 to 333	333
TA 102	/	/	333	/

/ = no relevant toxic effects observed

Table 2: Experiment 1

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA102	TA98	TA1537
-	Solvent 0 3 10 33 100 333 1000	132 183 113 110 87 48 0 0	12 10 12 9 9 10 4 0	256 279 251 265 202 152 153 149	32 26 31 23 25 10 11 3	8 7 8 7 4 4 3 1
+	Solvent 0 3 10 33 100 333 1000	159 167 100 129 82 90 0 0	11 14 14 12 14 15 9 2	279 281 276 300 299 255 219 239	31 32 25 32 29 26 17 9	13 10 14 11 13 11 6 0
Positive Controls
-	Name	SA	SA	MMS	4-NOPD	4-NOPD
	Concentration (µg/plate)	10	10	5 (µl/plate)	10	50
	Mean no. colonies/plate	1002	1200	1156	253	49
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	10	2.5	2.5
	Mean no. colonies/plate	1132	238	1088	800	130

SA = Sodium azide

2AA = 2-Aminoanthracene

4-NOPD = 4-Nitro-o-phenylene-diamine

MMS = Methyl methane sulfonate

 

Table 3: Experiment 2

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA102	TA98	TA1537
-	Solvent 0 1 3 10 33 100 333	117 159 131 176 137 159 56 18	13 9 13 11 14 6 5 7	244 219 208 282 257 193 166 104	21 20 21 17 23 9 14 10	7 7 9 5 7 5 4 0
+	Solvent 0 1 3 10 33 100 333	152 169 162 212 203 200 166 53	11 10 5 7 7 11 10 1	220 277 176 203 243 194 144 131	36 40 28 35 37 36 25 0	10 8 8 14 13 11 8 0
Positive Controls
-	Name	SA	SA	MMS	4-NOPD	4-NOPD
	Concentration (µg/plate)	10	10	5 (µl/plate)	10	50
	Mean no. colonies/plate	527	926	1095	155	62
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2.5	2.5	10	2.5	2.5
	Mean no. colonies/plate	981	207	1101	677	128

SA = Sodium azide

2AA = 2-Aminoanthracene

4-NOPD = 4-Nitro-o-phenylene-diamine

MMS = Methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

The test material is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay in both the presence and absence of metabolic activation.

Executive summary:

The mutagenic potential of the test material was assessed in a bacterial reverse mutation assay conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.14.

The study was performed according to the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2) using the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Negative, solvent and positive controls were conducted concurrently. Each concentration, including the controls, was tested in triplicate. The test material was tested at the following concentrations in DMSO:

- Experiment 1:3, 10, 33, 100, 333 and 1000 µg/plate

- Experiment 2:1, 3, 13, 33, 100 and 333 µg/plate

Distinct toxic effects, evident as a reduction in the number of revertants, occurred in almost all strains at higher concentrations. Reduced background growth was observed in strains TA1535, TA1537, TA98 and TA100 at 1000 µg/plate in the first experiment and in TA1537 and TA98 in the second experiment.

No substantial or reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no reproducible tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The positive controls showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test material is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay in both the presence and absence of metabolic activation.