Dodecanedioic acid, compound with 2,2',2''-nitrilotriethanol (1:2)

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:

20 March 2017 - 13 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dodecanedioic acid, compound with 2,2',2''-nitrilotriethanol

EC Number:

285-271-5

EC Name:

Dodecanedioic acid, compound with 2,2',2''-nitrilotriethanol

Cas Number:

85049-97-4

Molecular formula:

C12H22O4.xC6H15NO3

IUPAC Name:

dodecanedioic acid - 2,2',2''-nitrilotriethanol (1:1)

1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid

Remarks:

paste

Details on test material:

Name as cited in report: DDDA TEA salt
Physical appearance: beige to pale yellow paste
Storage conditions: at room temperature

Specific details on test material used for the study:

Purity correction factor: 1.25
Solubility in vehicle: not indicated
Stability in vehicle: not indicated
Test substance is irritant or corrosive

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254 (5 and 10%)

Test concentrations with justification for top dose:

Dose-range finding test (TA100 and WP2 uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of experiment 1)
Experiment 1 (TA1535, TA1537 and TA98): 52, 164, 512, 1600 and 5000 μg/plate
Experiment 2 (all tester strains): 492, 878, 1568, 2800 and 5000 μg/plate

The top dose used is according to OECD guideline 471.

Vehicle / solvent:

- Vehicle used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the vehicle has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9

Controls 6

Positive control substance:

other: 2-aminoanthracene

Remarks:

With 5 and 10% S9

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With 5% S9

Controls 8

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With 5 and 10% S9

Controls 9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With 5% S9

Controls 10

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With 10% S9

Controls 11

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with 5 and 10% S9

Controls 12

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

with 10% S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted: in the first experiment 5% S9 was used and in the second experiment 10% S9 was used.

METHODS OF SLIDE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. Fresh bacterial culture, dilution of the test item in DMSO and either S9-mix or 0.1 M phosphate buffer were added to the molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

DECISION CRITERIA:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

ACCEPTABILITY CRITERIA:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At all concentrations tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance on the plates was observed at the start or at the end of the incubation periods for both experiments.

RANGE-FINDING/SCREENING STUDIES: a range-finding test was conducted as part of the first experiment in tester strains TA100 and WP2 uvr A. No cytotoxicity or mutagenicity was observed.

- Cytotoxicity: in the first experiment, cytotoxicity was only observed in tester strain TA98 in the absence of S9-mix; in all other tester strain, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
- In strains TA1535 (absence of S9-mix) and TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions
are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

HISTORICAL CONTROL DATA: see table 1 and 2 in 'any other information on results'.
- The negative control values were within the laboratory historical control data ranges, except the responses for TA1535 and TA1537 in the absence of S9-mix in the first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (4 and 2 revertant colonies in the tester strains TA1535 and TA1537, respectively) when compared against relevant historical control data (5 and 3 revertant colonies in the tester strains TA1535 and TA1537, respectively), the validity of the test was considered to be not affected.
- The 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.

Any other information on results incl. tables

Table 1 Historical data of the solvent control

		TA1535		TA1537		TA98		TA100		WP2uvrA
	S9-mix	-	+	-	+	-	+	-	+	-	+
	Range	5 - 36	3 - 32	3 – 23	3 – 23	8 - 41	9 - 52	66 - 156	65 - 154	10 – 56	9 - 69
	Mean	12	12	6	8	16	23	100	100	25	31
	SD	5	4	3	4	5	7	15	16	6	7
	n	1865	1862	1740	1715	1852	1912	1853	1877	1571	1583

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between November 2014 and November 2016.

Table 2 Historical data of the positive control items

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	125 - 1381	78 - 1058	55 – 1311	55 – 1051	410 – 1995	250 - 1907	554 – 1848	408 - 2651	112 – 1951	85 - 1359
Mean	828	218	686	376	1270	883	892	1352	1165	388
SD	151	109	320	142	338	340	174	342	488	152
n	1875	1829	1560	1716	1766	1851	1820	1857	1506	1557

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between November 2014 and November 2016.

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD guideline 471 and GLP principles, DDDA TEA salt was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with or without metabolic activation.