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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21 1997
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

Constituent 1
Chemical structure
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:
2,2',2''-nitrilotriethanol citrate
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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
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 resultsopen allclose all
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
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
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
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

Applicant's summary and conclusion