Bis[2-(diethylamino)ethyl] adipate

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)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

Bis[2-(diethylamino)ethyl] adipate

EC Number:

240-610-6

EC Name:

Bis[2-(diethylamino)ethyl] adipate

Cas Number:

16545-00-9

Molecular formula:

C18H36N2O4

IUPAC Name:

1,6-bis[2-(diethylamino)ethyl] hexanedioate

impurity 1

Reference substance name:

2-diethylaminoethanol

EC Number:

202-845-2

EC Name:

2-diethylaminoethanol

Cas Number:

100-37-8

Molecular formula:

C6H15NO

IUPAC Name:

2-(diethylamino)ethanol

Test material form:

liquid

Method

Target gene:

hisD6610
hisD3052
hisG46
hisG428
uvrB
rfa
pKM101
pAQ1

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

5000 µg/plate, 2500 µg/plate, 1250 µg/plate, 625 µg/plate, 313 µg/plate and 156 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: Amino-Anthracene, 4-Nitro-1,2-phenylene Diamine

Details on test system and experimental conditions:

Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem GmbH (batch of the bacteria strains: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the fridge at 2-8 °C.
The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.

Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation, overnight 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.

Statistics:

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 minus mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant 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

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

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:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table8.1‑a      Mean Revertants First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	100	98	14	14	79	97	225	289	13	15
	sd	21.9	4.4	4.6	4.0	3.6	4.6	13.3	12.9	3.1	3.5
DMSO	Mean	79	94	13	16	85	89	245	341	14	12
	sd	4.6	12.2	2.1	4.4	10.1	15.7	16.3	20.5	1.0	2.5
Positive Controls*	Mean	481	431	397	50	339	984	851	1040	240	90
	sd	117.1	98.7	63.8	10.5	44.6	110.0	110.9	224.4	28.8	13.6
	f(I)	6.09	4.59	30.54	3.13	4.29	11.06	3.47	3.05	18.46	7.50
5000 µg/plate	Mean	95	102	10	15	81	80	248	272	14	13
	sd	14.8	0.6	1.7	2.1	4.6	4.7	25.0	73.2	3.1	2.0
	f(I)	1.20	1.09	0.77	0.94	0.95	0.90	1.01	0.80	1.00	1.08
1500 µg/plate	Mean	94	85	12	13	76	75	304	264	11	12
	sd	9.3	9.6	1.5	4.0	7.2	6.1	38.2	45.1	1.5	1.0
	f(I)	1.19	0.90	0.92	0.81	0.89	0.84	1.24	0.77	0.79	1.00
500 µg/plate	Mean	75	108	13	15	88	86	233	257	13	15
	sd	13.0	22.2	4.7	1.2	6.7	0.6	28.1	45.4	2.1	3.0
	f(I)	0.95	1.15	1.00	0.94	1.04	0.97	0.95	0.75	0.93	1.25
150 µg/plate	Mean	102	91	16	14	82	80	272	224	18	11
	sd	10.6	4.5	1.5	3.1	6.7	6.4	40.6	17.4	2.3	1.2
	f(I)	1.29	0.97	1.23	0.88	0.96	0.90	1.11	0.66	1.29	0.92
50 µg/plate	Mean	87	107	14	12	84	86	380	344	12	12
	sd	11.9	20.8	3.2	4.0	9.0	0.6	69.7	18.3	3.8	1.2
	f(I)	1.10	1.14	1.08	0.75	0.99	0.97	1.55	1.01	0.86	1.00

Mean Revertants Second Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	63	84	13	18	75	93	236	280	14	12
	sd	6.2	11.2	0.6	3.1	2.1	3.5	14.4	21.2	4.5	3.2
DMSO	Mean	62	90	11	13	73	79	256	237	11	10
	sd	12.5	16.9	2.3	1.7	4.6	5.6	24.3	6.1	3.0	1.5
Positive Controls*	Mean	587	368	396	66	555	1523	2176	1485	284	82
	sd	237.4	39.4	86.8	7.0	92.7	69.0	180.1	166.5	83.2	20.4
	f(I)	9.47	4.09	36.00	5.08	7.40	19.28	8.50	6.27	20.29	8.20
5000 µg/plate	Mean	73	87	14	13	85	85	239	271	13	12
	sd	9.2	18.6	2.3	1.5	13.0	11.9	12.1	40.3	3.2	4.6
	f(I)	1.18	0.97	1.27	1.00	1.16	1.08	0.93	1.14	1.18	1.20
2500 µg/plate	Mean	60	81	8	14	74	78	252	296	14	13
	sd	15.5	10.6	1.2	2.5	8.7	8.7	21.2	52.3	2.5	3.2
	f(I)	0.97	0.90	0.73	1.08	1.01	0.99	0.98	1.25	1.27	1.30
1250 µg/plate	Mean	59	74	9	12	70	86	268	257	12	12
	sd	10.1	3.1	4.0	1.0	5.9	9.0	52.0	16.7	3.2	2.5
	f(I)	0.95	0.82	0.82	0.92	0.96	1.09	1.05	1.08	1.09	1.20
625 µg/plate	Mean	60	81	9	13	79	90	296	296	12	17
	sd	4.9	11.0	0.6	6.0	4.7	8.1	52.3	40.6	3.0	3.1
	f(I)	0.97	0.90	0.82	1.00	1.08	1.14	1.16	1.25	1.09	1.70
312 µg/plate	Mean	64	104	10	12	69	85	251	247	12	12
	sd	6.5	7.1	3.5	2.9	7.0	10.6	41.6	31.1	3.5	5.5
	f(I)	1.03	1.16	0.91	0.92	0.95	1.08	0.98	1.04	1.09	1.20
156 µg/plate	Mean	71	85	10	11	81	85	253	259	9	10
	sd	3.5	9.5	2.3	4.2	4.7	4.4	42.8	16.2	1.7	2.1
	f(I)	1.15	0.94	0.91	0.85	1.11	1.08	0.99	1.09	0.82	1.00

Applicant's summary and conclusion

Conclusions:

In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 µg/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. 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 re-vertant colonies of the positive controls were within the range of the historical data of the laboratory (historical data of the laboratory see chapter 15, page 40) 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 Bis[2-(diethylamino)ethyl]adipate 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 S9-mix (rat liver S9-mix induced by Aroclor 1254). In the first experiment, Bis[2-(diethylamino)ethyl]adipate (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment)in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. Bis[2-(diethylamino)ethyl]adipate 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 item Bis[2-(diethylamino)ethyl]adipate showed 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. On the base of the first experiment, Bis[2-(diethylamino)ethyl]adipate was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strain using the pre-incubation method. Bis[2-(diethylamino)ethyl]adipate 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 item Bis[2-(diethylamino)ethyl]adipate showed 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 Bis[2-(diethylamino)ethyl]adipate 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 Bis[2-(diethylamino)ethyl]adipate 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 Bis[2-(diethylamino)ethyl]adipate 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.