Carboxylic acids, C5-9

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017 - 2018

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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 gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, C5-9

EC Number:

271-676-4

EC Name:

Carboxylic acids, C5-9

Cas Number:

68603-84-9

Molecular formula:

UVCB

IUPAC Name:

Carboxylic acids, C5-9

Test material form:

liquid

Details on test material:

Standard production of Matrica

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rat

Test concentrations with justification for top dose:

Toxicity test:

50, 158, 500, 1580 and 5000 µg/plate

Main Assay I:

Tester strain S9 Dose level (µL/plate)
TA1535, WP2 uvrA, TA98 ± 5.00, 2.50, 1.25, 0.625, 0.313
TA1537 ± 2.50, 1.25, 0.625, 0.313, 0.156
TA100 ± 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781

In Main Assay II, the test item was assayed at the same dose levels used in Main Assay I.

Dose related toxicity was observed at the two highest dose levels with TA1537 and TA100 tester strains, both in the absence and presence of S9 metabolic activation. Test item treatments did not induce any increase in revertant colonies with any tester strain/activation condition combination. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in theabsence or presence of S9 metabolism.
The maximum concentration of the test item to be used in the main experiments was selected as the concentration which elicits a moderate toxicity.

Vehicle / solvent:

The test item was used as a solution in DMSO.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

water, DMSO

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work PRELIMINARY TOXICITY TEST A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. MAIN EXPERIMENTS Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures. The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube. The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. INCUBATION AND SCORING The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were scored by counting the number of revertant colonies on each plate.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY TEST
The test item Carboxylic Acids, C5-9 was assayed in the toxicity test at a maximum dose level of 5.00 µL/plate and at four lower concentrations spaced at approximately half-log intervals:
1.58, 0.500, 0.158 and 0.0500 µL/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration.
Dose related toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, was observed at the two highest dose levels with TA1537 and TA100 tester strains, both in the absence and presence of S9 metabolic activation. No increase in revertant numbers was noted with any tester strain/activation condition combination.
ASSAY FOR REVERSE MUTATION
Two experiments were performed. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate (-S9) and 1600, 800, 400, 200, 100 μg/plate (+S9). Toxicity, as indicated by reduction in revertant numbers and/or thinning of the background lawn, was observed with TA1537 and TA100 tester strains at the highest dose level, both in the absence and presence of S9 metabolic activation. As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments.
Dose related toxicity, as indicated by microcolony formation, reduction in revertant numbers and/or thinning of the background lawn, was observed at higherconcentrations with all tester strains, both in the absence and presence of S9 metabolism. No increase in the revertant colonies was noted with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
No precipitation of the test item was observed at the end of the incubation period, at any concentration in any experiment. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Applicant's summary and conclusion

Conclusions:

negative

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level,
in any tester strain, in the absence or presence of S9 metabolism.
It is concluded that the test item Carboxylic Acids, C5-9 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions

Executive summary:

The test item Carboxylic Acids, C5 -9 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in DMSO.

Toxicity was observed only with TA1537 and TA100 tester strains at the highest dose level, both in the absence and presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments.

Both in the absence and presence of S9 metabolism, toxicity, from severe to slight, was observed with all tester strains at higher dose levels. No increase in the revertant colonies was noted with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration in any experiment.

It is concluded that the test item CArboxylic Acids, C5 -9 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.