5H-Cyclopenta[h]quinazoline, 6,6a,7,8,9,9a-hexahydro-7,7,8,9,9-pentamethyl-

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:

The experimental phase of this study was performed between 23 May 2012 and 23 June 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

EC Number:

801-694-5

Cas Number:

1392325-86-8

Molecular formula:

C16H24N2

IUPAC Name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

Test material form:

other: Solid

Details on test material:

Identification: IFF TM 12-206 (FRET 10-0199)
Description: White solid block
Storage conditions: Approximately 4 °C, in the dark

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

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 applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

main test:
Experiment one: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: Salmonella strains TA100, TA1537 and TA98 (absence and presence of S9-mix) and TA1535 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strain TA1535 (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/ml and acetone at 100 mg/ml in solubility checks performed in house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Dimethyl
sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

Microsomal Enzyme Fraction
The S9 Microsomal fraction was prepared in-house (15 April 2012) from rats induced with Phenobarbitone/b‑Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15M KCl solution (1g liver to 3 ml KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/ml. Aliquots of the supernatant were frozen and stored at approximately -196oC. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

S9-Mix and Agar
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
S9: 5.0 ml; 1.65 M KCl/0.4 M MgCl2: 1.0 ml; 0.1 M Glucose-6-phosphate: 2.5 ml; 0.1 M NADP: 2.0 ml; 0.2 M Sodium phosphate buffer (pH 7.4): 25.0 ml; Sterile distilled water: 14.5 ml

A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS Ltd.

Tester Strains
Salmonella typhimurium TA1535, TA1537, TA98 and TA100 Escherichia coli WP2uvrA.

The strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987. All of the strains were stored at approximately
-196°C in a Statebourne liquid nitrogen freezer, model SXR 34. 
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37ºC for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Preparation of Test and Control Materials
The test item was fully soluble in dimethyl sulphoxide at 50 mg/ml and acetone at 100 mg/ml in solubility checks performed in‑house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Dimethyl sulphoxide was therefore selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 20 minutes at room temperature on the day of each experint. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirent of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino‑silicate pellets with a nominal pore diameter of 4 x 10-4microns.
Vehicle and positive controls were used in parallel with the test material. 

Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test item. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The assay was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 0.1 ml of test item formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test item and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37ºC the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test – Experiment 1 (Range-finding Test)
Seven concentrations of the test item (5, 15, 50, 150, 500, 1500, 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Additional dose levels and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the vehicle, test item formulation or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.
All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using an automatic colony counter.

Mutation Test – Experiment 2 (Main Test)
The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was amended slightly, based on results from the range finding test and the change in test methodology, and was as follows:
Salmonella strains TA100, TA1537 and TA98 (absence and presence of S9-mix) and TA1535 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate. Salmonella strain TA1535 (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate. Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate.
Additional dose levels and an expanded dose range were again selected in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method.
All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using an automatic colony counter.

METHOD OF APPLICATION: Experiment 1:in agar (plate incorporation). Experiment 2 pre-incubation

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnett's linear regression Analysis

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate in Experiment 1 and toxic limit in Experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in dimethyl sulphoxide at 50 mg/ml and acetone at 100 mg/ml in solubility checks performed in house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Dimethyl sulphoxide was therefore selected as the vehicle.
- Precipitation: A test item precipitate (oily in appearance) was noted under an inverted microscope (and on some occasions by eye) at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item was toxic to the tester strain TA100 from 1500 µg/plate and was non-toxic to WP2uvrA. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range finding test (plate incorporation), the test item caused a visible reduction in the growth of the Salmonella bacterial background lawns and/or substantial decreases in revertant colony frequency, initially from 1500 µg/plate. In the main
test (pre-incubation method) the test item induced a slightly stronger toxic response with weakened bacterial background lawns initially noted from 50 µg/plate in the absence of S9 mix and 500 µg/plate in the presence S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA in the
range-finding test (plate incorporation method). However, in the main test (pre-incubation method) weakened lawns were noted at 5000 µg/plate in the absence of S9-mix only. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9 mix and experimental methodology. The test item was tested up to the toxic limit or the maximum recommended dose level of
5000 µg/plate depending on bacterial strain type, presence or absence of S9-mix or experimental methodology.

Any other information on results incl. tables

Preliminary Toxicity Test

The test item was toxic to the tester strain TA100 from 1500 µg/plate and was non-toxic to WP2uvrA. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	121	103	110	110	93	102	110	80	65	26*	18*
+	TA100	100	96	90	98	93	100	103	87	52	39*	27*
-	WP2uvrA	45	44	34	37	40	44	43	29	34	35	28
+	WP2uvrA	50	45	44	45	43	44	38	30	44	30	35

*        Partial absence of bacterial background lawn

MutationTest

Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
104		19		44		36		9
100	(98)	24	(21)	49	(49)	41	(38)	11	(12)
90		21		53		36		15

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
99		25		32		40		19
110	(104)†	16	(23)	32	(33)	47	(35)	15	(15)
104		29		35		19		12
		15
		15	(14)†
		12

†         Experimental procedure repeated at a later date (with and/or without S9-mix) due to contamination (TA100) or insufficient number of non-toxic dose levels (TA1535) in the original test.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997).

Executive summary:

The substance is tested in the Ames test (OECD TG 471), using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and E. coli strain WP2uvrA.

In the range finding test (plate incorporation), the substance was tested up to a concentration of 5000 µg/plate and caused a visible reduction in the growth of the Salmonella bacterial background lawns and/or substantial decreases in revertant colony frequency from 1500 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA.

In the main test (pre-incubation method) the substance was tested up to a concentration of 5000 µg/plate in the first experiment and up to 1500 µg/plate (strain TA1535) or 5000 µg/plate (all other strains) in the second experiment. The substance induced a slight toxic response with weakened bacterial background lawns initially noted from 50 µg/plate in the absence of S9‑mix and 500 µg/plate in the presence S9-mix.

In the Escherichia coli strain WP2uvrA weakened lawns were noted at 5000 µg/plate in the absence of S9-mix only. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9‑mix and experimental methodology. 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation or exposure method.

Based on this, the substance is not mutagenic in the Ames test.