Aluminate(2),[[N,N'-1,2-ethanediylbis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON]hydroxy-,disodium

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

9th addendum

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material:040801#
- Expiration date of the lot/batch: 05.08.2016
- Purity test date:07/01/2015
- Purity: 91.61%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item is dissolved in Aqua destillata and diluted prior to treatment. Aqua distilla is compatible with bacteria and the S9 activity.
- Final dilution of a dissolved solid, stock liquid or gel: different concentration tested (see below)

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: His D 3052; rfa-; uvrB-; R-factor

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: His G 46; rfa-; uvrB-; R-factor

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: His G428 (pAQ1); rfa-; uvrB-; R-factor

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: His G 46; rfa-; uvrB-

Species / strain 6

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: His C 3076; rfa-; uvrB-

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate and 1.00 µg/plate only for experiment II TA1537 (with metabolic activation)

Justification of top dose: for soluble non toxic test compounds the recommended maximum test concentration is 5 µl/plate and a pre-experiment test was performed for toxicity evaluation showing no background lawn from 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle:compatible with the survival of the bacteria and the S9 activity

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Serves as negative control also

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (TA98, TA 1537, without metabolic activation) / 2-aminoanthracene

Remarks:

Positive control varies depending on tester strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for preliminary test and experiment I; pre-incubation method into the experiment II
Cell density at seeding: approx. 109 cells/m
DURATION
- Preincubation period:60 min
- Exposure duration (for both experiment):48 h for both experiments

NUMBER OF REPLICATIONS:3
NUMBER OF EXPERIMENTS: 2


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:detected by a clearing / diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of 0.5 or less in relation to the solvent control
OTHER EXAMINATIONS: colonies counted using a ProtoCOL counter (Meintrup DWS Laborgäte GmbH) or manually (for TA 1535 and 1537 or if precipitation occurred)

- OTHER:- preparation of bacteria: each strain was grown by culturing for 12h at 37°C in Nutrient Broth (8g/l Nutrient Broth + 5 g/l NaCl). Ampicillin (10 mg/ml) was added to TA 98, TA 100 and TA 102.
- Agar plates: Vogel-Bonner Medium E agar plates with 2% glucose, sterilized 20 min at 121°C in autoclave.
- Overlay agar: contains agar-agar, NaCl, LhistidinexHClxH2O, biotin, sterilized 20 min at 121°C in autoclave
- Metabolic activation system: S9 liver microsomal fraction (from Male Wistar rats and Sprague Dawley rats (phenobarbital/Bêta-naphthoflavone)) in a S9 mix preparation with MgCl2, KCl, glucose-6-phosphate and NADP, stored on ice.
- For the test without metabolic activation, S9 mix is replaced by a sterilized phosphate buffer solution stored at 4°C

Evaluation criteria:

Mutation factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control.

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control

Statistics:

Not necessary because the biological relevance of the results is the criterion for the interpretation of the results

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from dose 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from dose 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from dose 316 µg/plate with the direct plate incorporation method, from 1000µg/plate with the preincubation method

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from dose 1000 µg/l

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 316 µg/plate (direct plate incorporation) from 1000 µg/plate (pre incubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1000 µg/plate (direct plate incorporation), at 2500µg/plate (pre-incubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2500 µg/plate (direct plate incorporation) and from 100 µg/plate (preincubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)

- Negative (solvent/vehicle) historical control data:
mean values of the spontaneous reversion frequency are within the historical control data range
(2012 -2014):
- S9
+ S9
min max min max
TA 98 13 48 13 61
TA 100 61 182 68 194
TA 1535 4 35 4 34
TA 1537 2 27 3 31
TA 102 136 415 91 495

Applicant's summary and conclusion

Conclusions:

Under these experimental conditions and during the described mutagenicity test, X300 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore X300 is considered to be non mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of X300 for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation.

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with X300 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, under these experimental conditions and during the described mutagenicity test, X300 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore X300 is considered to be non mutagenic in this bacterial reverse mutation assay.