3,4-dihydrocoumarin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydrocoumarin

EC Number:

204-354-9

EC Name:

3,4-dihydrocoumarin

Cas Number:

119-84-6

Molecular formula:

C9H8O2

IUPAC Name:

3,4-dihydro-2H-1-benzopyran-2-one

Method

Target gene:

Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537)

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):

All Salmonella typhimurium strains were obtained from Trinova BioChem GmbH and were stored as lyophilizates in the refrigerator at 2 – 8 °C.
On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate per strain at 3:30 pm (exp. 1) and 3:40 pm (exp. 2).
These overnight cultures were placed in the heating chamber at 37 ± 1 °C for 16 h 30 min (exp. 1) and 16h 20 min (exp. 2).
For the last two hours the overnight cultures were shaken on an orbital shaker (150 rpm) at 37 ± 1 °C.
Afterwards, the overnight cultures were ready for use in the experiment.
During the test, the overnight cultures were stored at room temperature (20 ± 5 °C) to pre-vent changes in the titre.

Metabolic activation:

with and without

Metabolic activation system:

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

Experiment 1:
In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
And no relevant signs of toxicity were observed in all bacteria strains.
The results of this experiment showed that none of the tested concentrations showed a relevant or dose-dependent 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 presence and absence 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 test item concentrations.
The bacterial background lawn was visible and not affected. Signs of mild toxicity were shown by a reduction in spontaneous revertants in several strains and concentrations could be observed.
The results of this experiments showed that the test item caused no increase in the num-ber of revertants in all bacteria strains compared to the solvent control, in both the pres-ence and absence of metabolic activation. The test item did not induce a dose-related in-crease in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.

Test concentrations with justification for top dose:

It is not specificed in the final report.

Vehicle / solvent:

DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this sol-vent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

On the day of the start of each experiment, a stock solution containing 50 mL/L of the test item in DMSO was prepared.

The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal test item concentrations were prepared for experiment 1:
5, 1.5, 0.5, 0.15 and 0.05 µL/plate.

The following nominal test item concentrations were prepared for the second experiment:
5, 2.5, 1.25, 0.63, 0.31, 0.16 and 0.08 µL/plate.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other: 4-Nitro-1,2-pheny-lene diamine, 2-Amino-anthracene

Details on test system and experimental conditions:

Per bacteria strain and concentration, three plates with (+S9) and three plates without met-abolic activation (-S9) were used.

For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

Experiment 1
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5, 1.5, 0.5, 0.15, 0.05 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Method: plate incorporation method

Experiment 2
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Method: pre-incubation method

Plate incorporation method:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
1. 100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 μL of the stock solu-tion were applied to achieve a final concentration of 0.5 μL/plate.
2. 500 μL S9-mix (see chapter 6.4.13, page 18 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
3. 100 μL bacteria suspension (see chapter 6.2.2, page 13, test system, culture of the strains)
4. 2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

Pre-incubation method:
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
1. 100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 μL of the stock solu-tion were applied to achieve a final concentration of 0.5 μL/plate.
2. 500 μL S9-mix (see chapter 6.4.13, page 18 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
3. 100 μL bacteria suspension (see chapter 6.2.2, page 13, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.

Evaluation criteria:

Five different analysable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test item.
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, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spon-taneous revertants) of the test item solutions and the positive controls. Additionally, the ab-solute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A result is considered as positive if a clear and dose-related increase in the number of re-vertants occurs and/or a biologically relevant positive response for at least one of the con-centrations occurs in at least one tested strain with or without metabolic activation.

A biologically relevant increase is described as follows:
1. if in the bacteria strains S. typhimurium TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
2. if in the bacteria strains S. typhimurium TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A substance is not mutagenic if it does not meet the criteria above.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

All strains met the criterion of at least 109 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. all determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range (including mean ± 3 standard deviations) of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and all were within the historical control data ranges.

In the first experiment, the test item showed no precipitateson the plates in all tested con-centrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not re-duced.
No significant or dose-related increase of the number of revertant colonies in the treat-ments with and without metabolic activation could be observed.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment was performed.
The mean revertant values of experiment 1 are shown in Table 8.1-a.

In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
Signs of toxicity towards the bacteria strains TA100, TA102, TA1535 and TA1537 could be observed in the highest concentration (5µL/plate) with and without metabolic activation, as the number of revertants was clearly decreased. For TA98, the number of revertants was only slightly reduced.
In the lower concentrations the number of revertant colonies was not relevantly reduced for the strains TA98, TA100, TA102 with and without metabolic activation and for TA1537 with metabolic activation.
For TA1535 and TA1537 (-S9) the number of revertants was slightly decreased at several concentrations, but as the bacterial background lawn was not affected in any of the tested concentrations, and additionally, no concentration-dependent toxicity could be observed, these observations were assessed as variation in bacterial growth.
No significant or dose-related increase of the number of revertant colonies in the treat-ments with and without metabolic activation could be observed.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
The mean revertant values of experiment 2 are shown in Table 8.2-a.

Any other information on results incl. tables

Table 8.1‑a       Mean Revertants Experiment 1

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	16	21	100	101	233	239	9	9	4	4
	sd	1.5	0.6	6.7	3.1	12.9	19.7	0.6	1.0	1.0	1.2
DMSO	Mean	17	21	101	102	235	236	11	9	3	4
	sd	1.5	0.6	5.1	3.6	6.1	10.6	0.6	0.6	0.6	1.0
0.9% NaCl	Mean	-	-	-	-	237	-	-	-	-	-
	sd	-	-	-	-	8.3	-	-	-	-	-
Positive Controls*	Mean	695	135	500	1197	545	745	189	152	81	59
	sd	22.0	14.0	25.0	69.9	34.9	16.2	12.9	14.4	2.6	4.4
	f(I)	40.88	6.43	5.00	11.74	2.30	3.16	21.00	16.89	27.00	14.75
5 µL/plate	Mean	14	13	51	81	211	205	6	6	4	4
	sd	0.0	1.5	15.6	12.0	8.3	20.1	1.2	1.0	1.0	1.0
	f(I)	0.82	0.62	0.50	0.79	0.90	0.87	0.55	0.67	1.33	1.00
1.5 µL/plate	Mean	14	15	108	96*^2	223	223	7	10	3	5
	sd	1.5	2.1	19.1	0.0	6.1	4.6	1.2	2.5	0.6	1.2
	f(I)	0.82	0.71	1.07	0.94	0.95	0.94	0.64	1.11	1.00	1.25
0.5 µL/plate	Mean	14	17	98	74	227	215	8	10	4	4
	sd	1.5	1.5	19.7	19.7	6.1	12.2	2.0	2.6	0.6	0.6
	f(I)	0.82	0.81	0.97	0.73	0.97	0.91	0.73	1.11	1.33	1.00
0.15 µL/plate	Mean	16	16	95	80	228	217	9	7	4	5
	sd	1.2	0.6	20.0	14.0	14.4	6.1	2.0	1.2	0.0	0.6
	f(I)	0.94	0.76	0.94	0.78	0.97	0.92	0.82	0.78	1.33	1.25
0.05 µL/plate	Mean	16	19	108	107	217	220	9	8	4	5
	sd	0.6	2.6	24.0	7.0	4.6	4.0	0.6	1.5	0.6	0.6
	f(I)	0.94	0.90	1.07	1.05	0.92	0.93	0.82	0.89	1.33	1.25

sd = standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 15

f(I) = increase factor, calculation see chapter 7.4, page 22

- = not tested

*² = in one replicate, no colony growth could be observed, whereas in the other two replicates 96 colonies were found. The replicate with no colony was declared as outlier and not used for evaluation.

 

Table 8.2‑a       Mean RevertantsExperiment 2

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	17	18	85	85	223	224	10	10	4	5
	sd	1.5	1.0	5.3	4.7	4.6	12.0	1.5	1.0	1.2	1.5
DMSO	Mean	17	18	75	86	217	231	10	9	6	6
	sd	0.6	2.0	3.5	1.5	9.2	2.3	1.5	1.2	1.2	0.6
0.9% NaCl	Mean	-	-	-	-	229	-	-	-	-	-
	sd	-	-	-	-	6.1	-	-	-	-	-
Positive Controls*	Mean	564	169	436	1504	656	716	239	161	92	42
	sd	30.2	10.1	30.3	48.0	36.7	20.0	11.5	6.1	2.6	4.0
	f(I)	33.18	9.39	5.13	17.49	2.86	3.10	23.90	17.89	15.33	7.00
5 µL/plate	Mean	11	13	23	33	96	84	1	0	2	1
	sd	1.0	2.6	9.2	20.8	17.4	10.6	1.0	0.6	0.6	0.6
	f(I)	0.65	0.72	0.31	0.38	0.44	0.36	0.10	0.00	0.33	0.17
2.5 µL/plate	Mean	14	18	40	54	124	221	3	3	3	5
	sd	2.1	2.1	6.0	18.2	14.4	8.3	2.5	1.0	2.0	2.1
	f(I)	0.82	1.00	0.53	0.63	0.57	0.96	0.30	0.33	0.50	0.83
1.25 µL/plate	Mean	14	17	54	62	216	220	4	5	4	4
	sd	1.2	2.1	7.0	3.0	10.6	12.0	1.7	2.5	1.0	2.1
	f(I)	0.82	0.94	0.72	0.72	1.00	0.95	0.40	0.56	0.67	0.67
0.63 µL/plate	Mean	15	17	60	63	216	224	6	4	3	5
	sd	3.1	1.5	4.7	12.5	14.4	14.4	6.4	2.3	1.5	2.0
	f(I)	0.88	0.94	0.80	0.73	1.00	0.97	0.60	0.44	0.50	0.83
0.31 µL/plate	Mean	15	13	62	65	209	215	7	5	3	4
	sd	2.5	1.2	3.1	22.2	8.3	12.2	2.3	2.0	1.0	1.2
	f(I)	0.88	0.72	0.83	0.76	0.96	0.93	0.70	0.56	0.50	0.67
0.16 µg/plate	Mean	13	15	57	63	207	220	5	5	3	4
	sd	3.2	2.5	1.5	9.1	8.3	10.6	2.6	2.1	1.5	1.5
	f(I)	0.76	0.83	0.76	0.73	0.95	0.95	0.50	0.56	0.50	0.67
0.08µg/plate	Mean	14	16	76	48	211	225	4	6	3	5
	sd	0.6	4.2	8.4	13.3	6.1	18.9	1.2	0.6	0.6	2.0
	f(I)	0.82	0.89	1.01	0.56	0.97	0.97	0.40	0.67	0.50	0.83

sd = standard deviation ±

* Different positive controls were used, see chapter 6.2.4, page 15

f(I) = increase factor, calculation see chapter 7.4, page 22

- = not tested

Applicant's summary and conclusion

Conclusions:

3,4-dihydrocoumarin is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.