Sodium 5-oxo-DL-prolinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Link to relevant study records

Reference

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

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

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.

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

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.

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.

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.

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

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.

Additional information

Four studies are available to address this endpoint.

The first three studies were performed using read across substance, sodium 5 -oxo-L-prolinate which is the L- isomeric form of the substance. Read-across is considered to be suitable based on the structural similarities between the read across substance (sodium 5-oxo-L-prolinate) and the substance to be registered (sodium 5-oxo-DL-prolinate). As such the difference in isomeric forms of the substance is unlikely to affect the genetic toxicity of the substance. All three studies have been assigned a reliability score of 2 in line with the principles for assessing data quality as defined by Klimisch (1997). 

 

IN VITRO BACTERIAL GENE MUTATION

In the key study (Hargitai, 2012) the mutagenic potential of the test material was assessed in an Ames test conducted under GLP conditions and in line with standardised guidelines.

S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2uvrA were exposed to the test material at concentrations of 5000, 2500, 1250, 625, 312.5, 156.25 and 78.125 µg/plate using the pre-incubation method. Positive and solvent controls were run concurrently for comparison.

Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), both in the presence and in the absence of metabolic activation.

 

IN VITRO MAMMALIAN CELL CYTOGENICITY

In the key study (Hargitai, 2013a) the clastogenic potential of the test material was determined in an in vitro mammalian cell chromosome aberration assay performed under GLP conditions and in line with standardised guidelines.

Cultures of Chinese hamster lung fibroblasts (V79) cells were exposed to the test material at concentrations of 5000, 2500, 1250, 625 and 312.5 μg/mL, in two assays, with and without metabolic activation. Assay 1 was performed with two experiments both with a three hour exposure period and 20 hours harvesting time. The experiments were performed in the presence or absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Assay 2 was performed either in the presence of S9-mix with a three hour exposure period or in the absence of S9-mix with a 20 hour exposure period. Both experiments were conducted with a harvesting time of 28 hours.

Treatment with the test material did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system. Additionally there was no indication of polyploidy or endoreplication.

The test material is therefore considered not to be clastogenic in this test system.

IN VITRO MAMMALIAN CELL GENE MUTATION

In the key study (Hargitai, 2013b) the mutagenic potential of the test material was determined in an in vitro mouse lymphoma assay, conducted under GLP conditions and in line with standardised guidelines.

Cultures of mouse lymphoma L5178Y cells were exposed to the test material at concentrations of 5000, 1666.7, 555.6, 185.2, 61.73 and 20.58 μg/mL, in two assays, both with and without the addition of a rat metabolic activation system (S9 fraction). Assay 1 was performed as two experiments, both with a three hour exposure period; however the first was performed in the presence of metabolic activation and the second without. Assay 2 was again performed as two experiments. The first was performed with metabolic activation and an exposure period of 3 hours, whereas the second experiment was performed without metabolic activation with a 24 hour exposure period. All experiments were conducted with an expression time of 3 days and a selection time of 2 weeks, with trifuorothymidine used as the selection agent.

Treatment with the test material did not result in a statistically and biologically significant increase in mutation frequencies either in the presence or absence of metabolic activation system in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential under the conditions of the study.

Justification for selection of genetic toxicity endpoint
One single study could not be selected at key, since all three studies are necessary to address this endpoint. The three studies assess different types of in vitro genetic toxicity; bacterial gene mutation, mammalian chromosome aberrations and mammalian gene mutation. All three studies were performed under GLP conditions and in accordance with standardised guidelines. The studies have been assigned a reliability score of 2 in line with Klimisch (1997) as they have been performed on the read across substance, sodium 5 -oxo-L-prolinate which is the L- isomeric form of the substance. Read-across is considered to be suitable based on the structural similarities between the read across substance (sodium 5-oxo-L-prolinate) and the substance to be registered (sodium 5-oxo-DL-prolinate). As such the difference in isomeric forms of the substance is unlikely to affect the genetic toxicity of the substance.

Short description of key information:
Ames test: Negative, OECD 471, Hargitai (2012) (read-across)
Chromosome aberration: Negative, OECD 473, Hargitai (2013) (read-across)
Mouse lymphoma assay: Negative, OECD 476, Hargitai (2013) (read-across)

A fourth experimental study was perfomed to further support the read across data provided in adressing the genetic toxicity endpoint.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

IN VITROBACTERIAL GENE MUTATION

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.

Short description of key information:

Ames test: Negative, OECD 471, EU Method B.13/14., and USA EPA 870.5100, Orovecz (2016) (experimental study)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the read-across in vitro data and the test report data, testing does not indicate any evidence of genetic toxicity of the test material. In accordance with criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the test material does not require classification for genetic toxicity.