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Genetic toxicity in vitro

Description of key information

The capacity of glutaric acid to induce gene mutation in bacteria was determined using a GLP-compliant study performed according to OECD Testing Guideline 471. Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2) methods were performed at 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). There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2. It was concluded that the substance was not mutagenic under the conditions of the test.

The capacity of glutaric acid to induce structural chromosomal aberrations in mammalian cells was assessed using unpublished literature data on the substance, QSAR predictions, and literature data on the analogous substance adipic acid. QSAR Toolbox predictions of the in vitro mammalian chromosome aberration test and in vivo micronucleus test were performed by grouping of chemicals and were negative with moderate to strong confidence. The endpoint predictions on glutaric acid are supported by the consistent results obtained during an in vivo mouse micronucleus assay on the substance. The absence of mutagenic effects was further supported by studies on adipic acid that returned negative results. It was concluded that the substance does not have the potential to induce structural chromosomal aberrations in mammalian cells.

The capacity of glutaric acid to induce gene mutation in mammalian cells was assessed using unpublished literature data on the substance and QSAR predictions. QSAR Toolbox prediction of the in vitro mammalian cell gene mutation assay was performed by grouping of chemicals and were negative with moderate confidence. The endpoint predictions on glutaric acid is supported by the consistent results obtained during an in vitro mouse lymphoma forward mutation assay performed on the substance. It was concluded that the substance does not have the potential to induce gene mutation in mammalian cells.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 16 August 2016 to 05 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine for Salmonella.Tryptophan for E.Coli
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (highest dose recommended)
Experiment 2:15, 50, 150, 500, 1500 and 5000 μg/plate (based on results of Experiment 1)
Vehicle:
- Vehicle(s)/solvent(s) used: sterile distilled water
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and conditions:
Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
- Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- Without Metabolic Activation
0.1 mL of the appropriate concentration of test item or solvent vehicle or 0.1 mL of appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
- With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
- Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
- Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
- Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
- With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
- Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.
Conclusions:
The test item was considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.
Executive summary:

The in vitro genotoxicity in bacteria of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. Ames plate incorporation (Experiment 1) and pre-incubation (Experiment 2) methods were performed at 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 for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four nontoxic dose levels and the potential toxic limit of the test item following the change in test methodology.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

It was concluded that the substance was not mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: expert assessment
Adequacy of study:
weight of evidence
Study period:
09 December 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: An assessment was performed based on literature data on the substance and QSAR predictions.
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
An assessment was performed based on literature data on the substance and QSAR predictions.
GLP compliance:
no
Type of assay:
other: An assessment was performed based on literature data on the substance and QSAR predictions.
Test material information:
Composition 1
Key result
Genotoxicity:
negative
Remarks on result:
other: An assessment was performed based on literature data on the substance and QSAR predictions.
Conclusions:
It is concluded that glutaric acid does not have the potential to induce gene mutation in mammalian cells. No additional testing is required.
Executive summary:

The capacity of glutaric acid to induce gene mutation in mammalian cells was assessed using an endpoint prediction from the OECD QSAR Toolbox. This prediction is supported by the existing result from an in vitro mouse lymphoma forward mutation assay performed on the substance.

QSAR Toolbox prediction of the in vitro mammalian cell gene mutation assay was performed by grouping of chemicals. The profilers did not identify alerts regarding the mutagenicity of the substance. Prediction of the in vitro mammalian cell gene mutation assay was negative and statistically significant with moderate confidence level (p=0.0453). The endpoint predictions on glutaric acid are supported by the consistent results obtained during an in vitro mouse lymphoma forward mutation assay on the substance performed by Sterling-Winthrop Research Institute (1981) according to a method similar to OECD Testing Guideline 476.

It is therefore concluded that glutaric acid does not have the potential to induce gene mutation in mammalian cells. No additional testing is required.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
other: expert assessment
Adequacy of study:
weight of evidence
Study period:
09 December 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: An assessment was performed based on literature data on the substance, QSAR predictions, and literature data on an analogous substance.
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
An assessment was performed based on literature data on the substance, QSAR predictions, and literature data on an analogous substance.
GLP compliance:
no
Type of assay:
other: An assessment was performed based on literature data on the substance, QSAR predictions, and literature data on an analogous substance.
Test material information:
Composition 1
Key result
Genotoxicity:
negative
Remarks on result:
other: An assessment was performed based on literature data on the substance, QSAR predictions, and literature data on an analogous substance.
Conclusions:
It is concluded that glutaric acid does not have the potential to induce structural chromosomal aberrations in mammalian cells. An experimental study to determine the potential of glutaric acid to induce structural chromosomal aberration in mammalian cells is not considered necessary.
Executive summary:

The capacity of glutaric acid to induce structural chromosomal aberration in mammalian cells was assessed using an endpoint prediction from the OECD QSAR ToolBox. This prediction was supported by available data from the analogous substance adipic acid and existing result from an in vivo mouse micronucleus assay performed on glutaric acid.

QSAR Toolbox predictions of the in vitro mammalian chromosome aberration test and in vivo micronucleus test were performed by grouping of chemicals. The QSAR toolbox profiler did not identify any alerts regarding the mutagenicity of the substance. Prediction of the in vitro mammalian chromosome aberration test was negative and statistically significant with moderate confidence level (p=0.0312). Predictions of the in vivo micronucleus assay was negative and statistically significant with strong confidence level (p=4.12*10-3). The endpoint predictions on glutaric acid are supported by the consistent results obtained during an in vivo mouse micronucleus assay on the substance performed by Sterling-Winthrop Research Institute (1983) according to a method similar to OECD Testing Guideline 474.

The absence of mutagenic effects was further supported by studies on adipic acid by Litton Bionetics, Inc. (1974) investigated the potential of the substance to induce structural chromosomal aberration. Studies were conducted in accordance to two different OECD guidelines (OECD Testing Guideline 473 and OECD Testing Guideline 475) and produced no detectable mutagenic effects. These results are also supported by the historical uses of adipic acid in foodstuff, feed and industrial processes.

It is concluded that glutaric acid does not have the potential to induce structural chromosomal aberrations in mammalian cells. An experimental study to determine the potential of glutaric acid to induce structural chromosomal aberration in mammalian cells is not considered necessary.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In accordance with Annex VIII of REACH, in vivo genotoxicity studies shall only be considered in case of positive results obtained during the in vitro genotoxicity evaluation of a substance.

Additional information

Justification for classification or non-classification

The in vitro genetic toxicity of glutaric acid was investigated in accordance with Annex VIII of REACH and returned negative results. It is therefore concluded that the substance does not meet the criteria for classification as genotoxic according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.