Phenylsuccinic acid

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:

2018-04-30 - 2018-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenylsuccinic acid

EC Number:

211-238-1

EC Name:

Phenylsuccinic acid

Cas Number:

635-51-8

Molecular formula:

C10H10O4

IUPAC Name:

2-phenylbutanedioic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Molecular formula:
C10H10O4
Molecular Mass:
194.18
Characteristics (Physical Appearance):
White Crystalline powder
CAS No.:
635-51-8
Batch Number:
123
Purity:
100%

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Test concentrations with justification for top dose:

5000, 1500, 500, 150 and 50 μg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: ICR 191, 3-Methylmethane sulphonate, 2-Aminoanthracene, 2-Aminofluorene,Danthron

Details on test system and experimental conditions:

PREPARATION OF TEST CULTURES
Fresh cultures for mutagenicity testing were prepared by transferring 40 μl of frozen permanent of Salmonella typhimurium to sterile tubes containing 10 ml of Oxoid Nutrient Broth No 2. Inoculated cultures were incubated in orbital shaker incubator for about 16 hours at 37 ºC to achieve cell density of about 1-2×109 cells / ml.
Bacterial Cell Count
The bacterial cell count was taken by hemocytometer. All the 25 small squares of RBC chamber were counted under high power (40X). Number of bacterial cells / ml of the culture was calculated by formula:
Total number of bacterial cells / ml of the culture = Sum of the cells counted in 25 small squares in the central ruled area of hemocytometer x 104 x dilution factor (102).
MEDIUM
The bacterial strains were cultured in Oxoid Nutrient Broth No. 2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 2% glucose. The top agar contained 0.6% agar, 0.5% NaCl and 0.05 mM histidine - biotin (Maron and Ames, 1983).
METABOLIC ACTIVATION
The cofactor supplemented post-mitochondrial fraction (S9) was employed as the metabolic activation system.
Suspensions of bacterial cells were exposed to the test item in the presence and absence of an exogenous metabolic activation system. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ºC for 20 minutes. These suspensions were mixed with an top agar and plated immediately onto minimal medium. After about 72 hours for Experiment No. 1 and 48 hours for Experiment No. 2 incubation period revertant colonies were counted and compared to the number of spontaneous revertant colonies on control plates. In order to confirm the reproducibility of the results, the entire study was carried out twice as Experiment No.1 and Experiment No. 2.
EXPOSURE CONCENTRATIONS
Test concentrations for this study were selected from the results obtained with solubility / precipitation test and preliminary cytotoxicity test of the test item employing only TA100 with and without metabolic activation system.
Solubility / Precipitation Test
2-Phenylsuccinic Acid was found completely soluble in DMSO at a concentration of 50 mg/ml. No precipitation was observed in the final reaction mixture at the concentrations from 2.38 to 0.0595 mg/ml corresponding to final test concentrations from 5000 to 125 g/plate respectively. Hence 5000 μg/plate showing no precipitation was the highest concentration selected for the preliminary cytotoxicity study.
Preliminary Cytotoxicity Test
Before commencing the study, the test item was assessed for cytotoxicity to the tester bacteria using tester strain TA100. Eight concentrations viz. 5000, 4000, 3000, 2000, 1000, 500, 250 and 125 μg/plate of the test item, formulated in DMSO, were tested for toxicity to bacterial cells.
No cytotoxicity was observed to revertant colonies and bacterial background lawn at the concentrations from 5000 to 125 μg/plate both in the absence and presence of metabolic activation system. Hence, 5000 μg/plate was selected as the maximum test concentration for main study.
CONTROLS
Concurrent vehicle control and strain specific positive controls, both with and without metabolic activation, were included in each assay.
Vehicle Control
DMSO was employed as a vehicle. Plates containing the reaction mixture comprising of vehicle and top agar supplemented with histidine but not added with 2-Phenylsuccinic Acid served as a control to check spontaneous revertants.
Maximum concentration of 5000 μg/plate, which was found non cytotoxic to TA100 in the preliminary cytotoxicity, was selected as the highest concentration for main study, with four lower concentrations viz. 1500, 500, 150 and 50 μg/plate. Triplicate plating was performed at each dose level for an adequate evaluation of the variation.
PRE-INCUBATION ASSAY WITHOUT METABOLIC ACTIVATION
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. Sterile phosphate buffer (0.5 ml) and freshly prepared test item / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ºC for 20 minutes in orbital shaker incubator.
Finally 2 ml of top agar with L-histidine and D-biotin solution was added. The contents were mixed and poured over the surface of the petri plates containing minimal glucose VB agar and allow them to solidify.
PRE-INCUBATION ASSAY WITH METABOLIC ACTIVATION
Aliquots of 0.1 ml cell suspension of the tester strains, containing about 108 viable cells, were added to sets of sterile tubes. The S9 mixture (0.5 ml) and freshly prepared test item / control sample / positive control (0.1 ml) were added to the tube. All the constituents of reaction mixture were introduced into sterile test tubes on an ice bath. The tubes with reaction mixture were mixed by shaking gently and were incubated at 37 ºC for 20 minutes in orbital shaker incubator.
Finally 2 ml of top agar with L-histidine and D-biotin solution was added. The contents were mixed and poured over the surface of the petri plates containing minimal glucose VB agar and allow them to solidify.

Evaluation criteria:

EVALUATION OF RESULTS
The mean number of histidine revertant colonies for all the treatment groups was compared with the number of revertants in the respective vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:
Criteria for Evaluation
1. Criteria for a Positive Response
A test item is considered to be positive (mutagenic), if it induces a concentration dependent increase and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate, in at least one strain with or without metabolic activation system, which is at least 2-fold (3-fold for TA1535) of that observed in the corresponding concurrent vehicle control.
2. Criteria for a Negative Response
A test item for which the results do not meet the above criteria is considered non-mutagenic in this test. In order a substance considered to be negative if the revertant colonies cannot be greater than 2 (or 3 for strain TA 1535), or less than 0.5 in at least 5 doses for all strains tested.
However, reproducibility of negative results was confirmed by repeat experimentation.
3. Criteria for an Equivocal Response
Occasionally, a test item cannot be judged to be positive or negative [e.g., concentration dependent increases that fail to reach 2-fold (3-fold for TA1535) control values, or  2-fold (3-fold for TA1535) increases that do not appear to be concentration dependent]. In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified study plan such as appropriate spacing of dose levels.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Salmonella typhimurium, Reverse Mutation Assay of 2-Phenylsuccinic Acid was carried out in compliance with the Organization for Economic co-operation and Development (OECD) Guidelines for Testing of Chemicals (Guideline No. 471, Section 4: Health Effects) on conduct of "Bacterial Reverse Mutation Test", adopted by the council on 21 July 1997.
Under the conditions described for this study, it is concluded that, 2-Phenylsuccinic Acid is non-mutagenic in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535.

Executive summary:

2-Phenylsuccinic Acid was evaluated in Ames test to determine its ability to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation system (S9). Based upon the preliminary tests conducted to assess the solubility / precipitation and cytotoxicity of 2-Phenylsuccinic Acid, the tester strains were exposed to the test item in triplicate cultures at the concentrations of 5000, 1500, 500, 150 and 50 μg/plate, both in the presence and absence of metabolic activation system (S9). Liver S9, induced in Wistar rats with sodium phenobarbitone and  - naphthoflavone, was used for this purpose.

DMSO was used as a vehicle. The exposed bacteria were plated onto minimal glucose agar medium supplemented with L-histidine and D-Biotin solution. The plates were incubated at 37 ºC for about 72 hours for Experiment No. 1 and 48 hours for Experiment No. 2 after which the histidine revertant colonies were counted and their frequency was compared with that in the vehicle control group. Concurrent positive control groups were also included in the experiment, as specified by the test guideline.

Results of this test indicated that the frequencies of histidine revertant colonies at all concentrations of 2-Phenylsuccinic Acid in strains TA1535, TA97a, TA98, TA100 and TA102, in the presence and absence of a metabolic activation system, were comparable to those observed in the vehicle control group, as per the criteria employed for evaluation of mutagenic potential, and this observation was confirmed by repetition of the experiments.

Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX PVT. LTD. They also compared well with the range reported in the literature. Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation

Analysis of the samples formulated for experiment No. 1 revealed that the average measured concentrations of 2-Phenylsuccinic Acid were 0.50 and 50.02 mg/ml against the nominal concentrations of 0.50 and 50 mg/ml respectively. The small observed difference of 0.21% and 0.04% respectively for above mentioned doses confirmed that the test system received an adequate dose of the Test Item.

It is concluded that, under the conditions of this study, 2-Phenylsuccinic Acid is non-mutagenic in Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102.