Dimethyl itaconate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2011-08-30 to 2011-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according guideline (OECD 471) under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

DMI Proviron

IUPAC Name:

DMI Proviron

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): DMI Proviron or test item
- Description: Pale yellow liquid
- Analytical purity: > 97%
- Lot/batch No.: Provichem 0911
- Expiration date of the lot/batch: 2013-08-01
- Storage condition of test material: in the refrigerator at 2-8 °C
- Stability in solvent: not indicated by the sponsor

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: frame shift mutations: his C 3076; rfa-minus; uvrB-minus

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: frame shift mutations: his D 3052; rfa-minus; uvrB-minus; R-factor

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: base-pair substitutions: his G 46; rfa-minus; uvrB-minus

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: base-pair substitutions: his G 46; rfa-minus; uvrB-minus; R-factor

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: base-pair substitutions and others: trp-minus; uvrA-minus

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since a positive response was observed a second experiment was not performed. The concentration range included two logarithmic decades. The following concentrations were tested in experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item DMI Proviron was dissolved in DMSO (MERCK, 64293 Darmstadt/Germany; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria. No precipitation of the test item occurred up to the highest investigated dose (5000 µg/plate) .

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:

Strains: TA 1535, TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Strains: TA 1537, TA 98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strains: WP2 uvrA

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Details on test system and experimental conditions:

Negative Controls: Concurrent untreated and solvent controls were performed.

Positive Control Substances:

Without metabolic activation

Strains: TA 1535, TA 100
Name: sodium azide, NaN3
Supplier: SERVA, 69042 Heidelberg/Germany
Catalogue No.: 30175
Purity: at least 99%
Dissolved in: deionised water
Concentration: 10 µg/plate

Strains: TA 1537, TA 98
Name: 4-nitro-o-phenylene-diamine, 4-NOPD
Supplier: Fluka (Sigma Aldrich), 82024 Taufkirchen/Germany
Catalogue No.: 73630
Purity: > 99.9%
Dissolved in: DMSO (MERCK, 64293 Darmstadt/Germany, purity > 99%)
Concentration: 10 µg/plate in TA 98, 50 µg/plate in TA 1537

Strain: WP2 uvrA
Name: methyl methane sulfonate, MMS
Supplier: Sigma Aldrich, 82024 Taufkirchen/Germany
Catalogue No.: 129925
Purity: > 99.0%
Dissolved in: deionised water
Concentration: 3 µL/plate

With metabolic activation

Strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Name: 2-aminoanthracene, 2-AA
Supplier: Sigma Aldrich, 82024 Taufkirchen/Germany
Catalogue No.: A3,880 - 0
Purity: 97.5%
Dissolved in: DMSO (MERCK, 64293 Darmstadt/Germany, purity > 99%)
Concentration: 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100),
10 µg/plate (WP2 uvrA)
The stability of the positive control substances in solution is unknown but a mutagenic
response in the expected range is sufficient evidence of biological stability.

Test System:

Characterisation of the Salmonella typhimurium Strains and Escherichia coli Strain

The histidine dependent strains are derived from Salmonella typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitratereductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base changeelsewhere 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 mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Storage:

The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO (MERCK, 64293 Darmstadt/Germany) in liquid nitrogen.

Precultures:

From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 OL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per
litre:
8 g Nutrient Broth (MERCK, 64293 Darmstadt/Germany)
5 g NaCl (MERCK, 64293 Darmstadt/Germany)

The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (108-109 cells/mL).

Selective Agar:

The plates with the selective agar were obtained from E. Merck, 64293 Darmstadt/Germany.
Overlay Agar:

The overlay agar contains per litre:

Sterilisations were performed at 121 °C in an autoclave.

for Salmonella typhimurium
7.0 g Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine×HCl×H2O*
12.2 mg Biotin*

for Escherichia coli
7.0 g Agar Agar*
6.0 g NaCl*
10.2 mg Tryptophan*

* (MERCK, 64293 Darmstadt/Germany).

Mammalian Microsomal Fraction S9 Mix:

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

S9 (Preparation by Harlan C C R):

Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of -naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene. The protein concentration in the S9 preparation was 25.7 mg/mL (lot no. R 290711).

S9 Mix:

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed
with S9 cofactor solution. The amount of S9 supernatant was 10% (v/v) in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:

8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Pre-Experiment for Toxicity:

To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (> 0 colonies) at five concentrations or more in all strains used.

Experimental Performance:

For each strain and dose level including the controls, three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:

100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test
without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in
the dark.

Evaluation criteria:

Data Recording:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. The counter was connected to a PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

Acceptability of the Assay:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Biometry:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item DMI Proviron was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Substantial and dose dependent increases in revertant colony numbers were observed following treatment with DMI Proviron in strain WP2 uvrA in the presence and absence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at 2500 and 5000 µg/plate. A minor increase in the number of revertant colony numbers was also observed in strain TA 100 with and without S9 mix, but the threshold of was not quite reached. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes in the genome of strain WP2 uvrA in the absence and presence of metabolic activation.

Any other information on results incl. tables

Please see attached background material.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by base pair changes in the genome of strain WP2 uvrA. Therefore, DMI Proviron is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The experiment was performed to assess the potential of the test item to induce gene mutations by the Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a positive result was obtained in this experiment, experiment II was not performed.

This study was performed to investigate the potential of DMI Proviron to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Substantial and dose dependent increases in revertant colony numbers were observed following treatment with DMI Proviron in strain WP2 uvrA in the presence and absence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at 2500 and 5000 µg/plate.