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Diss Factsheets

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:
01 June 2016 to 03 October 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Reference substance name:
Sodium DL-Pyrrolidonecarboxylate
Cas Number:
54571-67-4
IUPAC Name:
Sodium DL-Pyrrolidonecarboxylate
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White to pale yellow powder
- Storage Conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: provided by sponsor
- Lot/batch No.of test material: 160224
- Expiration date of the lot/batch: 24 February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light.

Method

Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Molecular Toxicology Inc., Boone, North Carolina, USA

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 ºC in a Gyrotory Water Bath Shaker.
- Properly maintained: Yes. The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes. The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly.
- Periodically 'cleansed' against high spontaneous background: Yes. Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011 to 2014 were (as guide) as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82.


Additional strain / cell type characteristics:
other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation
Metabolic activation:
with and without
Metabolic activation system:
Cofactor-supplemented post-mitochondrial S9 fraction.
Test concentrations with justification for top dose:
Preliminary test: 10, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Initial Mutation Test and Confirmatory Mutation Test: 15.81, 50, 158.1, 500, 1581, and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide
(DMSO) and N,N-Dimethylformamide (DMF). The test material was insoluble in DMF at 100 mg/mL concentrations. Partial dissolution was observed at 100 mg/mL concentration using DMSO. However, the formulation at 100 mg/mL concentration using Distilled water as vehicle (solvent) was a clear solution. Therefore, Distilled water was selected as vehicle (solvent) for the study.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation; in suspension

In the pre-incubation procedure bacteria were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The test material and other components were prepared freshly. Before the overlaying, 50 μL of test material formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells
and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 ºC in a shaking incubator.

After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hour.

DURATION
- Preincubation period: 20 min at 37 ºC in a shaking incubator
- Exposure duration: 37 °C for 48 ± 1 hour


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; growth inhibition

Evaluation criteria:
EVALUATION OF EXPERIMENTAL DATA
The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Criteria for Validity:
The study was considered valid if:
- The number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the negative (solvent) control.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test), the pre-incubation method was used. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
In the Preliminary Concentration Range Finding Test, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). Precipitate was not detected in the Preliminary Concentration Range Finding Test. No inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test.


The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests, each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate with and without metabolic activation.
In the Initial Mutation Test (using the pre incubation method), higher numbers of revertant colonies compared to the solvent control plates were observed in Salmonella typhimurium TA1535 bacterial strain at 5000 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF:2.07). Although this value was above the respective threshold value (MF=2) in this strain, there was no clear dose-dependent relationship and the positive effect was not reproducible. Thus, the observed values were considered as having no real biological relevance, just indicating higher than usual variability in this case.
In the Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 5000 μg/plate concentration with metabolic activation (the observed mutation factor values were: MF: 1.67). Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain with metabolic activation. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were within the historical control range in all cases, thus they were considered as biological variability of the test system. Precipitate was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.
No inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation


TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was not detected in the Initial Mutation Test and Confirmatory Mutation Tests.

Any other information on results incl. tables

Table 2. Summary Table of the Initial Mutation Test (Pre-Incubation Method)

+/- S9 Mix

Concentration

(µg/plate)

Mean number of revertants

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Untreated control

DMSO control

Distilled water control

5000

1581

500

158.1

50

15.81

99.0

-

99.0

77.3

86.7

91.0

87.0

83.3

89.0

8.3

-

9.7

20.0

17.3

17.3

18.7

13.0

16.7

28.0

-

28.0

29.3

27.3

27.7

27.0

28.3

23.7

28.3

21.7

24.3

20.7

20.3

24.0

21.3

20.7

19.0

 11.0

8.7

8.0

11.0

11.7

13.3

10.0

10.7

13.3

+

Untreated control

DMSO control

Distilled water control

5000

1581

500

158.1

50

15.8

108.7

96.7

95.7

91.7

84.0

88.7

92.3

107.0

96.7

10.7

10.7

12.3

11.0

15.7

11.3

11.0

10.7

12.0

30.0

34.0

34.0

37.3

25.3

32.7

36.0

31.0

32.7

25.7

26.3

32.7

22.3

23.3

24.7

26.7

24.0

22.0

11.7

8.3

9.7

8.7

11.3

9.3

11.3

11.0

8.3

Positive Controls

-

Name

SAZ

 SAZ

MMS

NPD

9AA

Concentration (µg/plate)

2

2

2

4

80

Mean no. colonies/plate

994.7

1000

932.0

408.7

1099

+

Name

2AA

2AA

2AA

 2AA

2AA

Concentration (µg/plate)

2

2

50

2

2

Mean no. colonies/plate

2232.0

225.3

234.7

346

2144.0

Table 3. Summary Table of the Confirmatory Mutation Test (Pre-Incubation Method)

+/- S9 Mix

Concentration

(µg/plate)

Mean number of revertants

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

Untreated control

DMSO control

Distilled water control

5000

1581

500

158.1

50

15.81

107.3

-

109.3

119.0

122.0

122.3

116.3

125.7

117.3

14.0

-

13.3

11.7

11.7

12.3

13.7

8.3

11.0

29.0

-

32.7

33.3

36.7

41.3

28.7

32.3

38.7

31.7

23.7

26.3

24.3

30.7

31.3

30.7

34.3

33.3

 8.3

8.7

9.0

15.0

10.0

11.3

11.3

11.0

12.3

+

Untreated control

DMSO control

Distilled water control

5000

1581

500

158.1

50

15.8

120.0

115.7

119.0

140.7

139.0

135.7

144.0

128.3

136.7

10.3

12.7

11.7

12.0

11.7

10.0

9.3

11.0

10.3

33.0

35.7

42.3

40.3

41.3

40.7

39.3

41.3

43.3

38.0

31.0

34.7

28.7

36.0

34.7

32.0

30.3

36.3

10.3

9.3

10.7

12.7

12.0

14.3

12.7

13.3

10.0

Positive Controls

-

Name

SAZ

 SAZ

MMS

NPD

9AA

Concentration (µg/plate)

2

2

2

4

80

Mean no. colonies/plate

110.7

1196.0

865.3

369.7

1099

+

Name

2AA

2AA

2AA

 2AA

2AA

Concentration (µg/plate)

2

2

50

2

2

Mean no. colonies/plate

2521.3

203.7

214.0

346

2542.7

Key for Positive Controls

Sodium azide (SAZ)

Methyl-methanesulfonate (MMS)

4-nitro-1,2-phenylenediamine (NPD)

9-aminoacridine (9AA)

2-aminoanthracene (2AA)

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was considered to be non-mutagenic.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471, EU Method B.13/14. and USA EPA 870.5100 under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the pre-incubation method with and without metabolic activation. The dose levels assessed were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate based the results of a preliminary test. Based on the results of a solubility test, the test material was formulated in distilled water. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test material treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system. Precipitate was not detected in the Initial Mutation Test and Confirmatory Mutation Tests. Inhibitory, cytotoxic effect of the test material was not detected in the Initial Mutation Test and Confirmatory Mutation Tests. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

Under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.