2,2',2''-nitrilotriethanol citrate

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:

13 Apr 2017 to 01 May 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:

July 21 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol citrate

EC Number:

249-576-7

EC Name:

2,2',2''-nitrilotriethanol citrate

Cas Number:

29340-81-6

Molecular formula:

C6H15NO3.xC6H8O7

IUPAC Name:

tris(2-hydroxyethyl)azanium 3,4-dicarboxy-3-hydroxybutanoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: 1184 ZITRONENSÄURE, TEA-SALZ
- Batch: SEALS 2016-198-001
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 03 January 2018 (expiry date)
- Solubility in vehicle: Water: >770 g/L
- Stability in vehicle: Water: Stable
- Appearance: Clear colourless liquid (determined by Charles River Den Bosch)

OTHER
- Purity/Composition correction factor: Yes, according to the purity: 1.28

Method

Target gene:

- S. typhimurium: his
- E. coli: trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 (i.p.) induced rats livers

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST
Strains TA100 and the WP2uvrA, with and without S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

EXPERIMENT 1 (DIRECT PLATE ASSAY)
- Strains TA98, TA1535 and TA1537, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate

EXPERIMENT 2 (PRE-INCUBATION ASSAY)
- Strains TA98, TA100, TA1535, TA1537 and WP2 uvrA, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate

Vehicle / solvent:

- Vehicle used: Milli-Q water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION
- DOSE-RANGE FINDING TEST and EXPERIMENT 1: in agar (plate incorporation)
- EXPERIMENT 2: preincubation

DURATION
- Preincubation period (EXPERIMENT 2 only): 30 minutes by 70 rpm at 37°C
- Exposure duration: 48 ± 4 h in the dark at 37.0 ± 1.0°C

NUMBER OF REPLICATIONS
3 plates/dose

COLONY COUNTING
Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

see section "Any other information on materials and methods incl. tables"

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA1535, TA1537 and TA98

Remarks:

Dose range Experiment and Experiment 1: Direct Plate Assay

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

Test results 2

Species / strain:

E. coli WP2 uvr A

Remarks:

Dose range Experiment: Direct Plate Assay

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

Test results 3

Key result

Species / strain:

other: TA 1535, TA 100, TA 1537, TA 98

Remarks:

Experiment 2: Pre-Incubation Assay

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

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 2: Pre-Incubation Assay

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

HISTORICAL CONTROL DATA
- Positive historical control data: see Table 1 in ‘Any other information on results inc. tables’.
- Negative (solvent) historical control data: see Table 2 in ‘Any other information on results inc. tables’.

Any other information on results incl. tables

Table 1 Negative (solvent) historical control data

	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

Table 2 Positive historical control data

	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