3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

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:

Study Initiation (4 January 2019) to Experimental Completion (21 January 2019)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2018

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

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

EC Number:

230-512-1

EC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Cas Number:

7158-25-0

Molecular formula:

C15H18

IUPAC Name:

3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Materia inc.
- Lot/batch No.of test material: RP229-0714
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 28 March 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in orginial container as supplied by the Sponsor. Stored after a brief sweep with Nitrogen gas in a sealed bottle.
- Stability under test conditions: Assumed stable for the duration of the test.
- Solubility and stability of the test substance in the solvent/vehicle: In DMSO, the test item was soluble after 40 minutes sonication with heating (between 35 to 45°C), however this formulation crystalized when at room temperature. The test item was therefore maintained at a temperature between 35 to 45°C so the test item would remain soluble in the vehicle throughout its use in this test system.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The test item in the vehicle (DMSO) crystallised when at room temperature, however as the test item stock was maintained at a temperature between 35 to 45°C within in this test system, the use of the vehicle was considered to have had no adverse affect on the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared as a solution in DMSO (after sonication at heating between 35 to 45°C). The formulation was thereafter maintained at a temperature between 35 to 45°C throughout the study.

Method

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:

Type and composition of metabolic activation system: S9 fraction procured from Dr. G.P Meshram, Nagpur, and (Lot N ° MWR/ARI/S9F/01/18) was used in this study.

Composition of Co-factor Mix and S9 Mix:

Co-factor Mix
D- Glucose –6- phosphate : 0.80 g
 Nicotinamide adenine dinucleotide
Phosphate (NADP) : 1.75 g
Magnesium chloride : 0.90 g
Potassium chloride : 1.35 g
Sodium phosphate, dibasic: 6.40 g
Sodium phosphate, monobasic: 1.40 g
Distilled water : 450 mL
The prepared co-factor mix was dispensed into suitable volumes and stored below 0 °C.

S9 Mix (10 mL)
Constituent 5% v/v S9 mix (10 mL) 10% v/v S9 mix (10 mL)

Co-factor mix 9.5 mL 9.0 mL
S9 fraction 0.5 mL 1.0 mL
The S9 mix was prepared fresh by adding the required quantity of S9 fraction to thawed co-factors and maintained in an ice bath. Any remaining portions of S9 mix were discarded.

- source of S9 : S9 fraction (Aroclor 1254-induced (Analab, USA) 500 mg/kg bw) was procured from Dr. G.P Meshram, Nagpur, and (Lot N ° MWR/ARI/S9F/01/18) was used in this study.
- method of preparation of S9 mix: The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and Glucose-6-phosphate to form “S9 mix”. This mix was then added to the top agar in this activated assay.
- concentration or volume of S9 mix and S9 in the final culture medium: A volume of 0.1 mL of S9 mix (5% v/v S9 mix for the initial toxicity-mutation test and 10% v/v S9 mix for the confirmatory mutation test) was prepared for treatment and added aseptically to 2 mL of top agar, mixed well and poured onto MGA plates.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability). yes, an efficiency check report (as a CoA) was issued from the vendor for the batch used in this study. This confirmed acceptable metabolic activation of this batch to tester strians prior to use on this study.

Test concentrations with justification for top dose:

Initial toxicity mutation test concentrations:
All strains, TA1537, TA1535, TA98, TA100 and TA102 (+/- S9 mix, 5% v/v S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/ plate.

As the test item crystalized at room temperature in DMSO, the test item stock (50000 µg/mL) was maintained at a temperature between 35 to 45°C so the test item would remain soluble. Once treated, cultures were incubated at 37 ± 1°C.
In the initial toxicity mutation test the OECD 471 limit concentration of 5000 µg/plate was acheived based on solubility in DMSO. Normal growth was observed up to the guideline limit concentration of 5000 µg/plate in tester strain TA100 in the absence of metabolic activation, and in tester strain TA102 in the absence and presence of metabolic activation (5% v/v S9 mix); with partial and complete inhibition and up to 50 µg/plate in tester strains TA1537, TA1535 and TA98 in the absence of metabolic activation and up to 150 µg/plate in tester strains TA1537, TA1535, TA98, and TA100 in the presence of metabolic activation (5% v/v S9 mix).
No increase in the number of revertant colonies was observed either in the absence or presence of metabolic activation (5% v/v S9 mix) at any tested concentration, in any tester strain. Results revealed that there was no mutagenic effect in tester strains TA1537, TA1535, TA98, TA100 and TA102.

Based on these observations the below concentrations were selected for the confirmatory mutation test:

TA100 (-S9, 10% v/v S9 mix): 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ plate.
TA102 (+/-S9, 10% v/v S9 mix): 156.25, 312.5, 625, 1250, 2500 and 500 µg/ plate.
TA1537, TA1535 and TA98 (-S9, 10% v/v S9 mix): 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ plate.
TA1537, TA1535, TA98 and TA100 (+S9, 10% v/v S9 mix): 15.63, 31.25, 62.5, 125, 250 and 500 µg/ plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO for the test item.

- Justification for choice of solvent/vehicle: DMSO was selected as the vehicle for the study. The test item crystalized at room temperature in DMSO, therefore to maintain test item solubility the test item stock was maintained at a temperature between 35 to 45°C. DMSO was therefore selected as the vehicle for the study. Once treated, cultures were incubated at 37 ± 1°C under standard test conditions.

- Justification for percentage of solvent in the final culture medium: 5% v/v (0.1 mL of DMSO, used as vehicle was added aseptically to 2 mL of top agar, mixed well and poured onto MGA plates).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate in the initial toxicity mutation test, Triplicate in the main confirmatory mutation assay
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating that appropriate numbers of bacteria were plated.
- Test substance added in medium; Treatments were performed using the plate incorporation technique.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: None
- Exposure duration/duration of treatment: Plates were maintained in triplicate for each test item concentration, negative and positive controls. The numbers of revertant colonies were recorded after 48 h incubation at 37 ± 1 ºC.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, Cytotoxicity is detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn (Teo et al., 2003).
- Any supplementary information relevant to cytotoxicity: In the initial toxicity mutation test, partial and complete inhibition in bacterial background lawn pattern with a reduction in the number of revertant colonies was observed at the higher tested concentrations in some tester strains.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : A result was considered positive if a concentration-related increase over the range tested and/or a reproducible increase in the number of revertant colonies per plate at one or more test concentration in at least one strain in the absence or presence of metabolic activation was observed.

Evaluation criteria:

A result is considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.

Statistics:

A simple linear regression analysis was performed for strains: TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Not specifed
- Data on osmolality: Not specified
- Possibility of evaporation from medium: None, non-volatile substance
- Water solubility: Non water soluble
- Precipitation and time of the determination: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES (if applicable): In an initial toxicity-mutation test, bacterial cultures were exposed to the test item at 8 concentrations (two plates/concentration) from 1.5 to 5000 ug/plate. Based on the results, a confirmatory mutation test (three plates/concentration) was then performed a selected test concentrations.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : yes, all values for the negative control were within laboratory historical control ranges. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

Ames test:
- Signs of toxicity : Cytotoxicity observed in some tester strains
- Individual plate counts: yes (see below tables)
- Mean number of revertant colonies per plate and standard deviation : yes (see below tables)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Any other information on results incl. tables

Individual Plate Count (Confirmatory Mutation Test)
Absence of Metabolic Activation

Concentration of TCPD (Distilled Tricyclopentadiene) (µg/plate)		Number of Revertant Colonies
		TA1537			TA1535			TA98			TA100#			TA102#
		R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3
NC (DMSO)		11	13	5	12	11	15	26	19	29	116	125	113	268	220	243
3.91	156.25#	9	13	12	12	14	19	30	18	25	122	123	145	247	234	232
7.81	312.5#	8	8	10	12	10	13	33	25	23	147	131	105	243	236	221
15.63	625#	4	8	9	13	21	9	22	26	33	123	147	98	249	229	237
31.25	1250#	8	7	13	9	11	13	28	30	14	133	142	135	221	237	227
62.5	2500#	5	10	4	9	10	7	21	12	20	128	140	142	231	260	250
125	5000#	2	0	1	6	6	4	12	10	16	99	129	137	238	215	244
PC		242	285	424	217	224	221	321	375	378	662	627	664	905	1016	895
2Aa		-	-	-	-	-	-	-	-	-	120	139	133	-	-	-

Presence of Metabolic Activation (10% v/v S9 mix)

Concentration of TCPD (Distilled Tricyclopentadiene) (µg/plate)		Number of Revertant Colonies
		TA1537			TA1535			TA98			TA100			TA102#
		R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3
NC (DMSO)		9	6	9	15	13	10	17	23	31	153	108	116	232	206	231
15.63	156.25#	13	6	2	19	11	15	19	25	21	128	105	110	227	240	227
31.25	312.5#	10	6	4	12	14	13	25	24	17	108	146	123	221	255	227
62.5	625#	5	9	11	12	17	11	33	23	20	116	130	102	250	238	246
125	1250#	19	4	5	22	12	8	23	22	26	144	150	124	215	218	240
250	2500#	4	5	5	7	7	6	16	19	14	123	48	58	255	248	217
500	5000#	0	0	0	5	2	3	14	9	10	29	21	47	217	232	249
PC		291	284	284	262	262	489	688	688	684	868	732	818	810	896	1036

The mean number of histidine revertant colonies (Confirmatory test)

Concentration of TCPD (Distilled Tricyclopentadiene)  (µg/plate)	Reduction in Number of Revertant Colonies in the Absence of Metabolic Activation
	TA1537	TA1535	TA98	TA100*	TA102*
NC (DMSO)	NA	NA	NA	NA	NA
3.91	-17.17	-18.39	1.38	-10.17	2.46
7.81	10.34	7.89	-9.44	-8.19	4.24
15.63	27.61	-13.10	-9.44	-3.96	2.19
31.25	3.52	13.18	2.72	-15.82	6.30
62.5	34.54	31.57	28.37	-15.82	-1.37
125	84.49	57.93	48.64	-3.11	4.65
PC	NA	NA	NA	NA	NA
2AA	NA	NA	NA	-10.74	NA

Concentration of TCPD (Distilled Tricyclopentadiene)  (µg/plate)	Reduction in Number of Revertant Colonies in the Absence of Metabolic Activation (10% v/v S9 mix)
	TA1537	TA1535	TA98	TA100	TA102*
NC (DMSO)	NA	NA	NA	NA	NA
15.63	12.50	-18.39	8.45	9.02	-3.74
31.25	16.63	-2.60	7.06	0.00	-5.08
62.5	-4.13	-5.21	-7.01	7.69	-9.72
125	-16.63	-10.50	0.00	-10.87	-0.60
250	41.63	47.36	31.01	39.26	-7.62
500	100.00	73.72	53.53	74.27	-4.34
PC	NA	NA	NA	NA	NA

Applicant's summary and conclusion

Conclusions:

It is concluded that TCPD (Distilled Tricyclopentadiene) is non-mutagenic to five strains of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102, when tested under the specified experimental conditions.