Amides, adipic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2019 to 27 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

15, 50, 150, 500, 1500, 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains

Vehicle / solvent:

Demineralised water, prepared by LAUS GmbH, from an ion-exchanger, batch: T20190510

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

methylmethanesulfonate

other: 2-Amino-anthracene, 4-Nitro-1,2-phenylene diamine

Details on test system and experimental conditions:

Test System:
Species: Salmonella typhimurium LT2, Escherichia coli
Strains: Salmonella typhimurium LT2: TA97a, TA98, TA100, TA1535; E. coli: WP2

Experimental Parameters:
Concentrations tested - 5000 / 1500 / 500 / 150 / 50 / 15 μg/plate
Incubation time - 48 h Incubation temperature 37 ±1°C
Tested strains- TA97a, TA98, TA100, TA1535, Escherichia coli WP2
Method - pre-incubation method
Incubation time- 20 min
No. of replicates - 3

Description of the Method:

General preparation:
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM resp. 10 mL of the tryptophan-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1°C.

Pre-incubation method:
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
• 100 μL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
• 500 μL S9 mix , for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate. The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1°C.

Sterility Control:
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37 ±1°C, four replicates.

Solubility:
Plates were checked for precipitation of test item at the end of the incubation by visual in-spection.

Positive Controls:
Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.

Evaluation criteria:

Evaluation:
Six different analysable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test substance.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each three fold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spon-taneous revertants) of the test substance solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants less mean spontaneous revertants) was given.
A substance is considered to be mutagenic, if a reproducible increase with or without meta-bolic activation of revertant colonies per plate exceeding an increasefactor of 2 for the bac-teria strains TA97a, TA98, TA100, TA1535 and E.coli compared to vehicle controls in at least one strain can be observed and there is a concentration-related increase. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
A substance is not mutagenic if it does not meet these criteria. If the criteria listed above are not clearly met, the results are assessed as equivocal and are discussed.

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

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All of the means of all replicates of the spontaneous revertants (in vehicle and negative controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.

In the experiment, no precipitation of the test substance was observed at any of the tested concentrations up to 5000 μg/plate.

In the experiment using the pre-incubation method, the test item caused no cytotoxicity towards all bacteria strains. The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 10E-9 bacteria/mL.The test item substance showed no increase in the number of revertants in all bacteria strains in the experiment with the pre- incubation method. No concentration-related increase over the tested range was found.

Conclusions:

Under the study conditions, the test substance was not found to be mutagenic.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA in the Ames plate incorporation and pre-incubation methods up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range study was performed in range of 15 to 5000 μg/plate. The test substance showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test substance showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Under the study conditions, the test substance was not found to be mutagenic (Andres, 2019).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2019 to 08 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Five concentrations (5000, 1000, 500, 100 and 50 μg/plate).

5000 μg/plate was selected as a top concentration, no reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity. The test substance did not cause a significant increase of the number of revertants of any bacterial tester strain.

Vehicle / solvent:

Tissue culture water (TCH2O), Lot No. RNBG4321

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2- Aminoanthracene and Daunomycin

Remarks:

Vehicle for positive controls: Tissue culture water (TCH2O), Lot No. RNBG4321 and Dimethyl sulfoxide (DMSO), Lot No. 168315

Details on test system and experimental conditions:

Test system: E. coli WP2 trp uvrA (cat No.72-188L), S. typh. TA-97a (cat No.71-097L), S. typh. TA-1535 (cat No.71-1535L), S. typh. TA-98 (cat No.71-098L) and S. typh. TA-100
(cat No.71-100L)

DURATION - incubation period: 48 - 72 h at 37°C
NUMBER OF REPLICATS: 3
DETERMINATION OF CYTOTOXICITY: - Method: counting the colonies per plate using an AlphaImager™ imaging system.

Preparation of the Tester Strains:
Bacterial cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth (Molecular Toxicology, Inc. (Moltox) Boone, NC, cat. No.26-505). Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37±2°C with agitation. The cultures were used after they reached the appropriate density as determined by absorbance readings at 600 nm as determined by a Spectronic 20D Colorimeter (Milton Roy, Rochester, NY).

Treatment of the Test System:
Top agar supplemented with appropriate amino acids was prepared, as 2 ml aliquots, and maintained at 45-50°C in sterile culture tubes. A 0.5 ml volume of Dulbecco’s phosphate-buffered saline (DPBS) was added to the tubes not undergoing S9 activation (i.e., without S9, or –S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, vehicle control or positive control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were gently vortexed and overlaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37±2°C for 48 to 72 hours.

Solubility Testing:
The test substance was ground to a fine powder with a mortar and pestle. Prior to the cytotoxicity screen, the solubility of the test substance was checked in TCH2O. The test substance was freely soluble at a concentration of 50 mg/ml. The Study Director, in consultation with the Sponsor, chose TCH2O as the vehicle for the study.

Screen:
A cytotoxicity screen was conducted in the TA-100 tester strain using eight concentrations (5000, 1000, 500, 100, 50, 10, 5, and 1 μg/plate) of the test article, two plates per concentration, with and without S9. A vehicle control (TCH2O) was tested concurrently, with and without S9. The plates were incubated at 37±2°C for 48 to 72 hours.

Main Assay:
Five concentrations (5000, 1000, 500, 100 and 50 μg/plate) of the test substance were tested in each of five bacterial tester strains (Escherichia coli WP2 trp uvrA, and Salmonella typhimurium strains TA-97a, TA-1535, TA-98, and TA-100). Two sets of culture plates were dosed per concentration (+S9 and –S9). A vehicle control and positive controls specific to each bacterial strain were treated in a similar manner as the test substance concentrations. The plates were incubated at 37±2°C for 48 to 72 hours. Plates that were
not scored immediately following the incubation period were stored at 2-8°C, to stop bacterial growth, until scoring.

Revertant Colony Count:
Counting of the revertants per plate was performed using an AlphaImager™ 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g., high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test substance. The plates were also examined visually for test substance precipitate.

Quality Check of the Assay:
Sterility Test:
Sterile technique is required throughout the course of the study. To ascertain each component’s sterility, a small amount (1 ml) was placed on agar plates and incubated to encourage growth of any contaminating microorganisms. The plates were incubated for 48 to 72 hours at 37±2°C and visually observed for microbial growth.
Vehicle Control:
The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain of bacteria was calculated and compared to in-house historical ranges.
Positive Control:
Positive control treatment for each tester strain of bacteria must result in at least a 2-fold increase of revertants over the mean vehicle control value.

Analysis of Data
Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test substance plate were averaged and plotted versus concentration of the test substance. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test substance as well as quality checks of the assay were taken into account.
In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose related increases approaching a 2-fold increase are deemed equivocal.
A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test substance as well as the quality checks of the assay.
Positive results from the bacterial reverse mutation test indicate that the material induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

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

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

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

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Cytotoxicity test:

The test article did not show obvious cytotoxicity to tester strain TA-100 at a dose range of 1, 5, 10, 50, 100, 500, 1000, and 5000 μg/plate, with or without S9.

Main Test:

No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test article, with or without S9. All positive and negative control values were within acceptable ranges, and all criteria for a valid study were met.

Conclusions:

Under the study conditions, the test substance was not found to be mutagenic

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains of Salmonella typhimurium (TA1535, TA97a, TA98 and TA 100) and Escherichia coli strain WP2uvrA. The test substance did not show obvious cytotoxicity to tester strain TA-100 at a dose range of 1, 5, 10, 50, 100, 500, 1000, and 5000 μg/plate in an initial cytotoxicity assay. The main plate incorporation assay was conducted with all the five strains in triplicate. No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test substance under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test substance, with or without S9. It is to be noted that, the negative results were not confirmed with an independent repeat assay. Under the study conditions, the test substance was not found to be mutagenic (Troese, 2019).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro - Ames test (plate incorporation)

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains of Salmonella typhimurium (TA1535, TA97a, TA98 and TA 100) and Escherichia coli strain WP2uvrA. The test substance did not show obvious cytotoxicity to tester strain TA-100 at a dose range of 1, 5, 10, 50, 100, 500, 1000, and 5000 μg/plate in an initial cytotoxicity assay. The main plate incorporation assay was conducted with all the five strains in triplicate. No reduction or clearing of the bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test substance under test conditions. There was no significant increase or dose-dependent increase of the number of revertants in any tester strain treated with the test substance, with or without S9. It is to be noted that, the negative results were not confirmed with an independent repeat assay. Under the study conditions, the test substance was not found to be mutagenic (Troese, 2019).

 

Genetic toxicity in vitro - Ames test (Andres)

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, in compliance with GLP. The test substance was examined using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA in the Ames plate incorporation and pre-incubation methods up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range study was performed in range of 15 to 5000 μg/plate. The test substance showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test substance showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Under the study conditions, the test substance was not found to be mutagenic (Andres, 2019).