Terpene Dimers

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:

10. Jul. 2018 to 19. Jul. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Terpene Dimers

EC Number:

948-032-2

IUPAC Name:

Terpene Dimers

Test material form:

liquid

Details on test material:

Batch no. DF1710011

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium LT2, Strains: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the first experiment: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate
The following nominal concentrations were prepared for the second experiment: 5 µL/plate, 2.5 µL/plate, 1.25 µL/plate, 0.63 µL/plate, 0.31 µL/plate, 0.16 µL/plate and 0.08 µL/plate

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, demineralised water, acetone

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-Amino-anthracene (CAS-No.: 613-13-8), 4-Nitro-1,2-phenylene diamine (CAS-No.: 99-56-9)

Details on test system and experimental conditions:

Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre. On the day of the test, the bacteria cultures were checked for growth visually. The incuba-tion chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0 °C.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium LT2, Strains: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item Terpene Dimers showed no increase in the number of revertants in all bac-teria strains in both experiments. Nearly all negative and all strain-specific positive control values were within the 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 Terpene Dimers is not muta-genic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Any other information on results incl. tables

Mean Revertants First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	73	107	43	48	107	106	225	253	10	12
	sd	9.0	13.5	3.8	5.8	30.6	8.7	16.2	15.1	2.5	1.2
DMSO	Mean	88	123	40	44	87	91	215	221	15	11
	sd	17.4	16.7	2.6	5.8	11.6	14.7	24.4	12.2	2.6	1.2
Acetone	Mean	82	102	45	45	105	103	260	272	11	14
	sd	10.6	16.6	6.7	4.0	15.5	8.3	12.0	13.9	1.0	2.6
Positive Controls*	Mean	613	475	199	239	1195	1001	1312	1240	416	363
	sd	169.8	57.9	24.1	30.0	44.1	0.0	73.3	361.7	107.6	66.0
	f(I)	6.97	3.86	4.98	5.43	11.17	11.00	6.10	5.61	41.60	33.00
5 µL/plate	Mean	86	119	38	43	96	112	275	239	15	10
	sd	4.9	21.0	6.7	3.0	21.6	11.9	15.5	14.2	4.2	0.6
	f(I)	1.05	1.17	0.84	0.96	0.91	1.09	1.06	0.88	1.36	0.71
1.5 µL/plate	Mean	80	94	36	39	101	98	238	237	10	12
	sd	14.0	8.0	7.6	3.5	5.0	3.5	17.8	33.6	0.0	3.8
	f(I)	0.98	0.92	0.80	0.87	0.96	0.95	0.92	0.87	0.91	0.86
0.5 µL/plate	Mean	83	97	44	41	112	93	275	206	13	14
	sd	6.4	2.3	6.0	3.5	4.0	7.6	38.0	6.9	4.6	1.5
	f(I)	1.01	0.95	0.98	0.91	1.07	0.90	1.06	0.76	1.18	1.00
0.15 µL/plate	Mean	90	96	45	44	110	105	239	235	13	13
	sd	5.5	14.4	5.7	4.4	15.1	20.2	26.6	19.4	1.2	2.9
	f(I)	1.10	0.94	1.00	0.98	1.05	1.02	0.92	0.86	1.18	0.93
0.05 µL/plate	Mean	80	96	42	43	91	115	242	203	14	10
	sd	7.8	11.6	2.5	6.1	2.6	23.9	36.7	23.2	3.5	1.5
	f(I)	0.98	0.94	0.93	0.96	0.87	1.12	0.93	0.75	1.27	0.71

Mean Revertants Second Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	81	106	43	42	93	100	341	397	10	12
	sd	11.1	9.5	6.9	7.2	9.5	7.2	32.3	16.7	2.0	1.7
DMSO	Mean	102	113	42	39	92	95	408	389	11	10
	sd	4.4	6.9	8.2	4.0	5.3	8.1	13.9	25.7	1.2	2.0
Acetone	Mean	80	120	40	40	87	86	411	397	10	9
	sd	5.9	9.3	2.1	7.9	6.0	8.7	24.4	116.6	1.5	1.5
Positive Controls*	Mean	570	735	549	445	1091	1001	1176	1235	199	122
	sd	137.4	135.4	32.3	28.1	37.8	0.0	28.8	20.1	64.0	19.1
	f(I)	5.59	6.50	13.07	11.41	11.73	10.54	2.88	3.17	19.90	12.20
5 µL/plate	Mean	69	77	41	37	90	96	445	345	11	10
	sd	13.0	2.1	6.1	3.5	6.0	3.8	60.0	20.1	1.5	0.6
	f(I)	0.86	0.64	1.03	0.93	1.03	1.12	1.08	0.87	1.10	1.11
2.5 µL/plate	Mean	74	85	43	38	100	105	428	401	11	11
	sd	5.8	3.8	13.9	4.0	15.0	18.5	26.2	52.2	2.3	2.5
	f(I)	0.93	0.71	1.08	0.95	1.15	1.22	1.04	1.01	1.10	1.22
1.25 µL/plate	Mean	72	87	47	42	93	98	445	419	13	10
	sd	10.0	4.6	15.5	4.6	1.2	19.6	40.1	11.5	2.9	3.5
	f(I)	0.90	0.73	1.18	1.05	1.07	1.14	1.08	1.06	1.30	1.11
0.63 µL/plate	Mean	64	73	43	43	97	83	371	411	13	11
	sd	7.2	12.2	6.0	7.0	11.3	4.2	50.3	16.2	2.1	2.1
	f(I)	0.80	0.61	1.08	1.08	1.11	0.97	0.90	1.04	1.30	1.22
0.31 µL/plate	Mean	92	79	35	38	87	95	440	421	11	10
	sd	9.9	19.7	3.8	7.5	5.6	13.3	58.9	30.0	3.5	2.1
	f(I)	1.15	0.66	0.88	0.95	1.00	1.10	1.07	1.06	1.10	1.11
0.16 µL/plate	Mean	87	99	38	42	91	95	413	429	10	11
	sd	10.4	13.8	1.0	9.0	12.9	23.2	8.3	47.7	1.5	5.6
	f(I)	1.09	0.83	0.95	1.05	1.05	1.10	1.00	1.08	1.00	1.22
0.08 µL/plate	Mean	78	90	44	41	93	98	423	241	10	12
	sd	6.4	3.6	3.1	4.2	14.5	3.5	14.0	30.3	1.0	4.5
	f(I)	0.98	0.75	1.10	1.03	1.07	1.14	1.03	0.61	1.00	1.33

Applicant's summary and conclusion

Conclusions:

In all experiments, no precipitation of the test item Terpene Dimers was observed at any of the tested concentrations up to 5 µL/plate. In the first and the second experiment, the test item caused no cytotoxicity towards all bacteria strains. The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.

Executive summary:

Two valid experiments were performed.  

The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item Terpene Dimers was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation. 

Experiment 1:

In the first experiment,the test item (dissolved in acetone) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test itemshowed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2:

Based on the results of the first experiment,the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test itemshowed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that Terpene Dimers is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.