3,3'-ethylenedioxybis(propylamine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: This study was performed to evaluate the mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. The test substance provided negative results to induce reverse mutation in the presence and absence of an exogenous metabolic activation system.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-11 - 2017-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 903-2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Solubility of the test substance in the solvent/vehicle: The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance.

Target gene:

The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. The top dose is determined per the guidelines and solubility of the test substance.Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate. In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water for the test substance; all positive controls were diluted indimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water.
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 3 in the mutagenicity assay

NUMBER OF CELLS EVALUATED: 1.6 to 3.9 x10E8 cells per plate.

DETERMINATION OF CYTOTOXICITY
- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.

Evaluation criteria:

The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter).
The mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:Strains TA1535 and TA1537Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.Strains TA98, TA100 and WP2 uvrAData sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive.
This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Sterility Results
No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Preliminary Toxicity Assay
The maximum dose of 5000 μg per plate was achieved using a concentration of 50.0 mg/mL and a 100 μL plating aliquot. Neither precipitate nor toxicity was observed.

Mutagenicity Assay
Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Historical control Data:

Historical Negative and positive control values 2015 revertants per plate
		Activation
		None					Rat Liver
Strain	Control	Mean	SD	Min	Max	95% CL	Mean	SD	Min	Max	95% CL
TA 98 (2015)	Neg	16	5	6	43	6 -26	23	7	5	53	9 -37
TA 98 (2015)	Pos	190	191	42	2468		329	176	51	1786
TA 100 (2015)	Neg	90	12	62	233	66 -114	98	15	63	157	68 -128
TA 100 (2015)	Pos	697	172	239	1767		671	284	138	2692
TA 1535 (2015)	Neg	13	5	2	35	3 -23	13	5	3	33	3 -23
TA 1535 (2015)	Pos	624	196	50	2509		137	110	24	1060
TA 1537 (2015)	Neg	7	3	1	20	1 -13	9	3	2	23	3 -15
TA 1537 (2015)	Pos	392	292	24	2887		73	53	19	574
WP2 uvrA (2015)	Neg	25	8	7	73	9 -41	28	8	10	96	12 -44
WP2 uvrA (2015)	Pos	336	112	89	10126		352	117	78	1409
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Metabolic activation	Test substance	Dose level  (μg/plate)	Revertant Colony Counts (mean +/- SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without metabolic activation	water	100 μL/plate	16 ± 4	87 ± 4	14 ± 0	10 ± 3	25 ± 3
Without metabolic activation	Jeffamine EDR 176	15.0	19 ± 2	86 ± 12	15 ± 1	9 ± 5	28 ± 3
	Jeffamine EDR 176	50.0	18 ± 5	89 ± 11	12 ± 3	7 ± 1	26 ± 8
	Jeffamine EDR 176	150	14 ± 2	83 ± 7	11 ± 6	6 ± 1	25 ± 6
	Jeffamine EDR 176	500	20 ± 4	92 ± 2	17 ± 2	9 ± 5	25 ± 2
	Jeffamine EDR 176	1500	16 ±2	85 ± 6	12 ± 2	8 ± 1	20 ± 5
	Jeffamine EDR 176	5000	16 ±2	86 ± 7	15 ± 3	6 ± 2	29 ± 4
	2NF	1.00	131 ±33
	SA	1.00		644 ±45	686 ± 47
	9AAD	75.0				609 ±96
	MMS	1000					324 ± 28
With metabolic activation	water	100 μL/plate	19 ± 4	98 ± 6	12 ± 5	9 ± 4	27 ± 8
With metabolic activation	Jeffamine EDR 176	15.0	17 ±6	89 ± 7	14 ± 2	10 ± 4	30 ± 6
With metabolic activation		50.0	19 ± 5	108 ± 13	15 ± 7	9 ± 2	27 ± 3
		150	23 ± 7	114 ± 27	11 ± 2	8 ± 8	28 ± 8
		500	21 ± 2	112 ± 17	15 ± 6	8 ± 2	32 ± 9
		1500	16 ±1	96 ± 5	11 ± 5	9 ± 4	27 ± 6
		5000	17 ± 1	91 ± 7	13 ± 3	7 ± 6	31 ± 6
		1.00	248 ±85		77 ± 14
		2.00		612 ±79		64 ±33
		15.0					288 ± 26

Key to Positive Controls

SA: sodium azide

2AA: 2-aminoanthracene

9AAD: 9-Aminoacridine

2NF: 2-nitrofluorene

MMS: methyl methanesulfonate

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay

The test substance was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Water was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Neither precipitate nor toxicity was observed. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate the test substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008).