Carboxylic acids, C5-9

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All the available studies both on the target substance (Ames and Micronucleous studies in vitro) and on the substance constituents (ames test on octanoic acid, Chromosome aberration in mammalian cells on octanoic and decanoic acid, in vitro gene mutation study in mammalian cells on nonanoic acid, do not indicate any potential adverse effects of Carboxylic Acids.

Link to relevant study records

Reference

Reference 1

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

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 gene mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

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.

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.

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: Test Methods for New Chemical Substances and the Like

Version / remarks:

partially revised by Medicines and Foodstuffs Ordinance 0331 No. 7, Production Agency March 29, 2011, No. 5, Environmental Planning Agency Ordinance No. 110331009, March 31, 2011, Medicines and Foodstuffs Ordinance 1221 No. 1, 20151209 Bureau No. 1, Environmental Planning Agency Ordinance No. 1512211, December 21, 2015.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name：Decanoic acid
CAS number：334-48-5
Chemical Evaluation Law, Ministerial Circular Publication, Adjustment No: (2) -608
Rational formula：CH3(CH2)8COOH
Amount used: ：849.0 mg
Purity：99.5%
Name of impurity and concentration：0.08% (moisture)
Molecular weight：172.26
Characteristics at normal temperature: white to faint yellow crystal, when dissolved: colorless to faint yellow liquid
Stability: stable when hot
Solubility: insoluble in water, dissolves in dimethyl sulfoxide1)
Stability in solvents: stable in water and dimethyl sulfoxide1)
Caution when handling: irritative
Storage conditions: keep away from light, sealed, at normal temperature （15-25°C）
Storage location: 3rd animal experimental wing, specimen preparation room, storage cabinet at normal temperature permissible range: 15-25°C）
Storage period: October 26, 2017 to December 25, 2017
Storage temperature (actual measured values) 16-20°C
Control of remainder of test substance: after test operations have been completed, transferred to party responsible for management of test substance
1) Based on results of solubility test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

Animals Used: Rats: Sprague-Dawley strain
Sex, Age (weeks)/weight range: Male, 7 weeks/204.4 ± 8.2 g
Lot Number: 17080405
Date of manufacture: August 4, 2017
Derivative substance: Phenobarbitol (PB) and 5,6-Benzoflavone (BF)

Dose administered and number of administrations:
PB:30 mg/kg once（first day), 60 mg/kg 3 times（2nd to 4th days)
BF:80 mg/kg once（3rd day)
Method of Administration: Peritoneal administration
Amount of protein:19.8 mg/mL S9

Vehicle / solvent:

the injection water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

In these tests, the incidence of naturally occurring chromosomal aberrations was low and the stability and reproduceability of the cells was good. There were few chromosomes and these were comparatively large (25 chromosomal number modes) and were highly sensitive to many chemical substances. We used the following cells which are generally used in chromosomal aberration tests using mammalian cultured cells.
Cells: CLU/UL cells originating in female Chinese hamster lungs
Supplier: DS Pharmabio Medical Ltd.
Date received: August 22, 2017
Cell cycle: 11.2 hours
Mycoplasma: negative
Cell number mode: 25 units
Number of passages: 21

Rationale for test conditions:

Conditions for Completion of Test
The test was completed when the following conditions were satisfied.
1) The occurrence frequency of the cells and the polyploids in which the negative control had a structural aberration was within the fluctuation range of the negative control (attached materials) of the background data.
2) The occurrence frequency of the cells in which the positive control had structural aberrations were within the fluctuation range of the positive control (attached materials) in the background data.
3) The occurrence frequency of cells in the positive control with structural aberrations increased significantly statistically compared to the negative control.
4) There were at least three doses amount the doses observed with 300 metaphase cells when the test substance being processed was observed.

Evaluation criteria:

The structural aberrations and numeric aberrations were characteristic in that the occurrence frequency of the cells or the polyploids with structural aberrations exceeded the fluctuation range of the negative control in the background data, there was a statistically significant increase compared to the negative control and it was determined to be negative when a dose dependency was confirmed in that increase. The occurrence frequency of the cells or the polyploids of cells with structural aberrations were characteristic in that no statistically significant increase was confirmed compared to the negative control and was determined to be negative when no dose reactivity was confirmed.

Statistics:

We used an χ2 test accompanying Yates compensation for each dose for the occurrence frequency of the cells and the polyploidy with structural aberrations for the substance processing and the positive control and analyzed the significance of the difference with the negative control at a significance level on one side of 5 %. For the test substance processing, we analyzed the dose reactivity using the Cochran-Armitage trend test (significance level on one side of 5 %, including negative control).

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The chromosomal aberration inducibility of decanoic acid relative to the CHL/IU cells originating in the female Chinese hamster lungs was determined to be negative.

Executive summary:

We studied the chromosomal aberration inducibility of decanoic acid without metabolic activation and with metabolic activation using the short-term processing method using CHL/IU cells originating in female Chinese hamster lungs and using the continuous processing method (24-hour processing).

Proliferation suppression was confirmed wherein the test substance indicated an RPD of 45.4 % (216 µg/mL) when not metabolically activated using the short-term processing method in cell proliferation suppression tests; an RPD of 45.0 % (216 µg/mL) when metabolically activated and had a strong cell toxicity in doses of 216 µg/mL and above. The RPD when not metabolically activated using the short-term processing method in chromosomal aberration tests was 53.4 % (216 µg/mL), when metabolically activated, the RPD was 51.3 % (216 µg/mL) and the RPD in the continuous processing method (24-hour processing) was 47.8 % (90.0 µg/mL.  Although no dose was observed in which a PRD of 50 % and under was obtained in short-term processing method in chromosomal aberration tests, at the highest doses, a sufficiently high proliferation suppression of approximately 50 % was indicated so that it was determined that there was no effect on the evaluation of the chromosomal aberration tests.

Observation of the chromosomes based on these results was carried out using 4 continuous doses   (27.0, 54.1, 108, 216 µg/mL) starting from 216 µg/mL in the short-term processing method using as reference a dose in which the RPD was close to 50 %; and 4 continuous doses (11.3, 22.5, 45.0, 90.0 µg/mL) using the continuous processing method (24-hour processing). A negative control and positive control were also set up under all of the processing conditions. The cell proliferation rate of the chromosomal aberration tests is indicated in Table 2 and the results of the chromosomal aberration tests are indicated in Tables 3 and 4 and in Figure 1.

Observation of the chromosomes showed that the occurrence frequency of the cells and the polyploids whose chromosomes had structural aberrations in all of the processing conditions was within the fluctuation parameters of the negative control calculated from the background data. In a dose of 54.1 µg/mL not metabolically activated in the short-term processing method, the occurrence frequency of the cells with structural aberrations increased significantly statistically compared to the negative control. However, given the fact that the occurrence frequency of the cells with structural aberrations was 1.7 % which was within the fluctuation range of the negative control which was calculated from the background data, the fact that the occurrence frequency of the cells of the negative control with structural aberrations was 0 % and the fact that an increase in dose dependency was recognized, it was determined that this increase was incidental and there was no meaningful biological increase. Meanwhile, other doses not metabolically activated using the short-term processing method and metabolically activated using the continuous processing method (24-hour processing), no statistically meaningful increase was confirmed. As a result, the occurrence frequency of the cells and the polyploids whose chromosomes had structural aberrations as evidenced in the processing of the test substance was determined not  to increase significantly biologically.

The occurrence frequency of the structural aberrations in the negative control was 0 to 1.0 % and the numerical aberration occurrence frequency was 0 to 0.3 %, in both cases indicating that these were within the fluctuation range of the negative control calculated from the background data under both processing conditions. The structural aberration occurrence frequency of the positive control was 12.7 to 33.7 % and was within the fluctuation range of the positive control in the background data under all of the processing conditions. There was also a significant statistical increase compared to the negative control. These results indicate that the tests were carried out appropriately.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

no guideline followed

Principles of method if other than guideline:

We carried out the test both with and without metabolic activation (without S9 mix) using the plain incubation method. In other words, a test tube (15.5 x 100 mm), cleansing test tube, “Rarbo”, Termo Ltd.) ① 0.05 mL of test substance prepared solution, medium control solution or negative control solution; and 0.1 mL of the positive control solution; ② 0.5 mL (without metabolic activation) of 0.1 mol/L Na-1) phosphoric acid buffer solution (pH 7.4) which had been steam sterilized at high pressure; ③ we added 0.1 mL of the bacterial solution, in that order; and incubated it for 20 minutes at 37°C using a repeat vibration type of incubator shaker. Then we added 2 mL of top agar which had been heated to 45°C and mixed it. Then, we spread a minimal amount on a glucose agar flat culture medium, turned the plate upside down and cultured it for approximately 48 hours in a low-temperature thermostat set to 37°C.

After the culturing was completed and we had observed any presence of a precipitate on the plate with the naked eye, we measured the number of reverse mutant colonies using a colony analyzer (CA-11D, System Science Ltd.). After we had measured these, we observed any presence of bacterial growth inhibition using a 100x stereoscopic microscope. Further, we observed the presence of a precipitate on the plate with the naked eye even when the culturing started.
We used 3 plates combined for the strain, the presence of metabolic activation and the concentration. In addition, the test tubes and the plate were identified by different colors using an oil-based ink for each strain.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test substance octanoic acid［alternate name: n-caproic acid: octanoic acid, CAS No. 124-07-2, Ministerial Circular Publication Adjustment No. (Chemical Evaluation Act): 2-608] chemical formula: CH3 (CH2) 6COOH (chemical structural formula: see Figure 1), molecular weight:
144.21, physical properties, characteristics: very faint yellow clear liquid, with distinctive odor, does not dissolve in water, readily dissolves in ethanol and acetone, ignition point: 109°C, density (20°C): 0.910 g/mL. In this test, we used［content (GC): 99.2%]. After we obtained it, we stored it at room temperature, away from the light, set temperature 23°C (actual measured value: 22.0 to 25.0°C) set humidity, 55 % (actual measured value: 40.8 to 67.0 %] in a test substance storage room at the test institution.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Vehicle / solvent:

anhydrous ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminoanthracene; 9—-aminoacridine hydrochloride; 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks on result:

other: it was determined that there was no gene mutation inducibility in the octanoic acid under the conditions for this test.

Executive summary:

We investigated the presence of the gene mutation inducibility using octanoic acid using salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2uvrA in reverse mutation tests using the PLA incubation method. We carried out the test with and without the S9 mix.

The test concentration in the octanoic acid treatment group involved setting bacterial stock for 5, 15, 50, 150, 500, 1500 and 5000 µg/plate both with and without the S9 mix in the dose setting tests.

Results of the dose setting test indicated that bacterial growth inhibition was confirmed at a concentration of 1500 µg/plate or more without the S9 mix for TA100, TA1535, TA98 and TA1537;

at a concentration of 5000 µg/plate at WP2uvrA; at a concentration of 1500 µg/plate or more with the S9 mix at TA100, TA98 and TA1537; and a concentration of 5000 µg/plate or more for TA1535 and WP2uvrA. As a result, the test concentration for this test was set at a concentration of 6 or 7 with a common ratio of 2 so that it contained a concentration of 4 or more wherein a bacterial growth inhibition is thought to be confirmed both with and without the S9 mix.

This means that it was 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/plate without the S9 mix for TA100, TA1535, TA98 and TA1537; 39.1 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/plate with the S9 mix for TA100, TA98 and TA1537; and 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate with TA1535 and WP2uvrA.

Results of the test did not indicate that the number of reverse mutation colonies increased 2 or more times over those of the medium control for any of the strains with or without the S9 mix. No reproducibility was noted in the results of the dose setting test and in this test.

In the positive control, the number of reverse mutation colonies increased more than twice that of the negative control and in the negative control, it was within a mean 2 S.D. range of the background data of the test institution. In addition, the number of reverse mutation colonies in the anhydrous ethanol (medium control) used as a medium was with a range of a mean 2. S.D. of the background data in the negative control for all of the strains and no effect of the medium on the test group was confirmed.

Based on the above results, it was determined that there was no gene mutation inducibility in the octanoic acid under the conditions for this test.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

no guideline followed

Principles of method if other than guideline:

We carried out a cell growth inhibition test to set the test concentration of the octanoic acid in the chromosomal aberration test. The test was carried out for 3 series both with the S9 mix added and without the S9 mix added using the short treatment method and the continuous treatment method (24-hour treatment method).

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

The test substance octanoic acid [alternate name: n-caproic acid, English name: octanoic acid, CAS No. 124-07-2 Ministerial Circular Announcement Adjustment No. (Chemical Evaluation Law): 2-608] has a chemical formula of C8H16O2 (see Figure 1 for chemical structural formula), 144.21, physical property. Characteristics: extremely faint yellow clear liquid with a peculiar odor. Insoluble in water and readily soluble in ethanol and acetone. Ignition point: 109°C, density (20°C): 0.910 g/mL. We obtained this for this test. [content: (GC): 99.2 %]. After we obtained it, we stored it under the following conditions: room temperature, away from the light, sealed tight, in a cabinet at room temperature in the test substance storage room of the test institution [set temperature: 23°C (actual measured value: 22.0 to 25.0°C), set humidity: 55 % (actual measured value: 40.8 to 67.0 %)

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

The S9 we used [Lot No.: 10081305 (cell growth inhibition test) and 10100807 (chromosomal aberration test)] was manufactured from the livers of 37 male rats (weight: 210.1 +/- 10.2 g) and 35 male rats (weight: 210.4 +/- 9.0 g) [Crl:CD(SD] which were one week old [manufacturing date: August 13, 2010 (usage limit: February 12, 2011) and October 8, 2010 (usage limit: April 7, 2011) which had been administered phenobarbital and 5,6-benzoflavon, the usage limit was 6 months after manufactured based pm the standards of the test institution. The S9 was purchased on September 2, 2010 and October 28, 2010 and was kept frozen in an ultra-low temperature freezer set at -80°C. We produced and mixed a variety of substances other than S9 as a solution and after we had filtered this using a membrane filter ⌀0.2μm NALGENE®, added S9 immediately before using it and produced it.

Vehicle / solvent:

anhydrous ethanol

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other: dimethylnitrosamine

Details on test system and experimental conditions:

In the test, the cells divided early on and there were few chromosomes so that it was suitable for observing the chromosomes. In addition, we used these cells and a great deal of chromosome aberration data has been published, there was a great deal of background data and we used fibroblast cell stock (CHL/IU) originating in Chinese hamster lungs. The cells were kept and stored in liquid nitrogen and when used for the test we thawed the cells which had been frozen (number of passages: 15 times; number of chromosome modes: 25; cell doubling time: 16.8 hours; mycoplasma test: negative), grew then and then used then. The passage of the cells was carried out for 3-4 days each using a culturing vial (manufactured by Nunc). The number of cells from the second culturing was indicated in each test as indicated below.
Cell growth inhibition test: 3 times.
Chromosomal aberration test: 7 times
Further, we carried out the experiment operations in the Chromosomal Aberration Test Laboratory (wing G) which was provided with air conditioning.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

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

Remarks on result:

other: it was determined that there was no chromosomal aberration inducibility in the octanoic acid in the conditions in this test.

Executive summary:

We studied the presence of chromosomal aberration inducility of octanoic acid using mammalian cultured cells (CHL/IU cells) using the short treatment method (both with and without S9 mix added for 6-hour treatment) and using the continuous treatment method (24-hour treatment).  

Based on the results of the cell growth inhibition test, the test concentration of octanoic acid was set in 4 stages based on the common ratio of 2 using as indicators the 50 % cell growth inhibition concentration (IC50) and the cell survival rate. In other words, this was 93.8, 187.5, 375 and 750 µg/mL when S9 mix was added using the short treatment method, 187.5, 375, 750 and 1500 µg/mL when the S9 mix was not added and with 93.8, 187.5, 375 and 750 µg/mL using the continuous treatment method.

The results of the test indicated that the incidence of cells with numeric and structural aberrations was less than 5 % both for the short treatment method and the continuous treatment method.

The positive control used in each test series displayed clear positive results, the inducibility of the chromosomal aberrations satisfied the conditions for completing the test. In addition, the chromosomal aberration inducibility in anhydrous methanol used as a medium (medium control) was less than 5 % for each test series. No effect of the medium on the test group was confirmed. Based on the above results, it was determined that there was no chromosomal aberration inducibility in the octanoic acid in the conditions in this test.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation

Species / strain / cell type:

other: peripheral blood for lymphocytes cultures

Details on mammalian cell type (if applicable):

For each experiment, whole blood was collected from healthy volunteer donors. The volunteers were non-smoker and were not receiving any medication or radiation exposure prior to the time of sampling.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

First experiment: Dose levels 2.50, 1.67, 1.11, 0.741, 0.494, 0.329, 0.219, + 0.146 and 0.0975 µg/mL
Since for the continuous treatment, no concentration presented the adequate cytotoxicity for selecting the maximum dose level for scoring micronuclei, a Main Assay 2 was performed where treatment was as follows:
Second experiment: Dose levels 0.390, 0.339, 0.295, 0.256, 0.223, 0.194, 0.169, 0.147 and 0.127 µg/mL
Based on solubility results and pH values measured in the final treatment medium during a preliminary test, the maximum dose level of the test item selected for treatment was 2.50 µL/mL.

Vehicle / solvent:

DMSO. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: colchicine

Remarks:

Colchicine in absence of S9 metabolic activation and Cyclophosphamide in presence of S9 metabolic activation

Details on test system and experimental conditions:

Three treatment conditions were performed. A short termtreatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours.
Solutions of the test item were prepared in DMSO.
Based on solubility results and pH values measured in the final treatment medium during a preliminary test, the maximum dose level of the test item selected for treatment was 2.50 µL/mL. This concentration was considered adequate since the test item is not of defined composition (i.e. UVCB) and a higher dose level is needed to increase the concentration of each component as indicated in the Study Protocol.
Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.
The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity. Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum (55 ± 5%) to slight or no toxicity.

Evaluation criteria:

The criteria for identifying micronuclei are as follows: (i) the micronucleus diameter must be less than 1/3 of the diameter of the nucleus, (ii) the micronucleus diameter must be greater than 1/16 of the diameter of the nucleus, (iii) no overlapping with the nucleus must be osberved, (iv) the aspect must be the same as the chromatin. In this assay, the test item is considered as clearly positive if the following criteria are met: - Significant increases in the proportion of micronucleus cells over the concurrent controls occur at one or more concentrations. - The proportion of micronucleus cells at such data points exceeds the normal range. If the increases fall within the range of values normally observed in the negative control cultures, the test item can not be classified as positive. Any significant increases over the concurrent negative controls are therefore compared with historical control values derived from recent studies. - There is a significant dose-relationship

Statistics:

For the statistical analysis, a modified chi-squared test was used to compare the number of binucleated cells with micronuclei in control and treated cultures.

Species / strain:

lymphocytes: human lymphocytes

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

Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively.

The harvest time of 32 hours corresponding to approximately 2.0 cells cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 31 hours was used. Solutions of the test item were prepared in DMSO. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity. In addition, there

are many historical control data demonstrating that no cytogenetic effects are induced by this solvent. Since the test item is not of defined composition (i.e. UVCB), this concentration, corresponding to the final dose level of 5 µL/mL, was considered the upper limit to testing as indicated in the Study Protocol.

The addition of an aliquot of this solution to culture medium in the ratio 1:100 did not give any precipitation or opacity of the medium; however a remarkable reduction of the pH of the medium over the physiological value was observed (pH 5.45). On the basis of these obervations a final concentration of 2.50 µL/mL which presented a pH of 6.23 was selected as the maximum dose level for treatment.

For the first Main Assay, following the 3 hour treatment in the absence and presence of S9 metabolism, severe cytotoxicity was observed at the highest dose level of 2.50 µL/mL where mainly mononucleated cells or few cells were recovered. Moderate cytotoxicity was seen at the next lower dose level of 1.67 (61% or 55%), while slight or no cytotoxicity was noticed over the remaining dose range.

Following the continuous treatment in the absence of S9, marked or severe cytotoxicity was observed from 0.329 µL/mL onwards. Mild cytotoxicity was seen at the lower dose level of 0.219 µL/mL (37%). No remarkable cytotoxicity was noticed over the remaining dose range. A mild cytotoxicity (33%) was observed at the highest dose level of 2.50 µL/mL which was probably due to the observed precipitation of test item and therefore to a low bioavailability of the test item.

For the second Main Assay, moderate cytotoxicity was seen at the two highest dose levels of 0.390 and 0.339 µL/mL (53% and 46% respectively). Mild to no remarkable cytotoxicity was seen over over the remaining dose range.On the basis of the above results, the dose levels selected for scoring of micronuclei were as follows:

Main Assay	S9	Treatment time (hours)	Harvest time (hours)	Dose level (µL/mL)	Cytotoxicity (%)
1	-	3	32	1.67 1.11 0.741	61 27 7
	+	3	3	1.67 0.741 0.329	55 25 15
2	-	3	3	0.390 0.295 0.223	53 28 21

For the positive control, the following dose levels were selected for scoring:

Main Assay	S9	Treatment time (hours)	Harvest time (hours)		Dose level (µL/mL)	Cytotoxicity (%)
1	- +	3		Cyclophosphamide	15.0	13
2	-	31		Colchicine	0.0400	92

Results are presented below:

Main Assay	S9	Treatment time (hours)	Harvest time (hours)		Dose level (µL/mL)	Incidence of micronucleated cells (%)	Statistical significance
1	- +	3	32	Cyclophosphamide	15.0	1.85	***
2	-	31	31	Colchicine	0.0400	3.20	***

*** = p < 0.001

The study was accepted as valid. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment

series and no concentration related increase was seen. All incidenceswere within the normal distribution of historical control data (95% confidence limits).

Conclusions:

Interpretation of results: negative

On the basis of this results, it is concluded that Carboxylic Acids, C5-9 do not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test item Carboxylic Acids, C5 -9 was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. This study was performed in accordance with OECD guideline 487 and GLP without any deviation.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Secondary source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9; 9000g supernatant from homogenates of livers from Aroclor 1254 induced male SD rats

Test concentrations with justification for top dose:

without metabolic activation: 150, 200, 300, 600, 800, 1000, 1200, and 1600 µg/mL
with metabolic activation: 37.5, 50, 75, 100, 150, 200, 300, 500, and 600 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: low solubility of pelargonic caid

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: without S9: methylmethanesulfonate; with S9: 3-methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-14 days


SELECTION AGENT (mutation assays): trifluorothymidine


NUMBER OF REPLICATIONS: 2 separate trials


NUMBER OF CELLS EVALUATED: all colonies counted


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A positive result was considered if the mutation frequency was increased at least 2-fold over the solvent control, is at least double the background, and relation to dose for at least 3 doses. Moreover, cloning efficiency was required to be at least 10%, and the number of clones in the selection plating plates exceeds about 60.

Statistics:

Statistical significance of results was not calculated

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

RANGE-FINDING/SCREENING STUDIES: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Range finding study:
The test material was less toxic without metabolic activation than with S9 in the cytotoxicity assay. Without activation, total cell
killing was observed at 4000 µg/mL , and the 2000 µg/mL was highly toxic, with a cell density of only 0.9% of the vehicle control.
With S9 activation, total cell killing was observed at 2000 µg/mL, and the 1000 and 500 µg/mL doses were highly toxic, with cell
densities < 5% of vehicle controls. The pH was adjusted to 7.0-7.2.

Definitive study:
Without S9: Relative suspension growth generally decreased with increasing dose, from 80% at 150 µg/mL to 9.6% at 1200 µg/mL in
trial 1, and 121% at 200 µg/mL to 11% at 1600 µg/mL in trial 2. There was no cytotoxicity < 600 µg/mL.

With S9: Relative suspension growth was reduced in a dose-dependent manner at doses, ranging from 94.6% at 37 µg/mL (in
trial 1) to 11% at 600 µg/mL (trial 2).

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Without metabolic activation: criteria for positive test result were not met.

With metabolic activation: increases in the numbers of mutants per plate were seen at all test material concentrations, and doubled at ≥ 300 µg/mL in trial 1, and at doses ≥ 100 µg/mL in trial 2. Mutation frequencies exceeded the minimum for trial 1 of 136.7 x 10^-6at doses ≥ 75 µg/mL, and exceeded the minimum of 118.1 x 10^-6for trial 2 at all test doses. Colony sizing revealed a bimodal distribution in small and large mutant colonies. An increase in small colonies is consistent with the induction of chromosome deletions.

 

Conclusions:

Interpretation of results (migrated information):
ambiguous with metabolic activation

No effect in the absence of metabolic activation. A weak positive effect was seen in the presence of metabolic activation, but marked cytotoxicity may have contributed.

Executive summary:

The genotoxic potential of pelargonic acid was examined in mammalian cells (mouse lymphoma L5178Y TK +/- cells, equivalent to OECD test guideline No. 476) under GLP conditions. The test concentrations were 150, 200, 300, 600, 800, 1000, 1200, and 1600 µg/mL without metabolic activation, and 37.5, 50, 75, 100, 150, 200, 300, 500, and 600 µg/mL with metabolic activation (S9 mix, from livers of Aroclor 1254 induced male Sprague Dawley rats).

Cytotoxicity was more pronounced in the presence of S9 mix. In the absence of S9 the relative suspension growth generally decreased with increased doses to 9.6% at 1200 µg/mL; no cytotoxicity was seen < 600 µg/mL. With S9, the relative suspension growth generally decreased with increased doses to approx. 11% at 600 µg/mL. No increased mutation frequencies were not without metabolic activation. With metabolic activation, mutation frequencies doubled at >300 µg/mL. Colony sizing revealed a bimodal distribution in small and large colonies, which could indicate the induction of chromosome deletions.

Overall, pelargonic acid, in the presence of S9 metabolic activation, induced a weak mutagenic response in mouse TK+/- lymphoma cells. This occurred in the presence of increasing cytotoxicity, however, and may indicate damage to the chromosome carrying the TK locus, rather than actual mutagenicity at that locus. The observed weak positive result in the presence of metabolic activation is therefore considered to be ambiguous. (EPA OPPT, 1995).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

As no effects on Ames bacteria and on micronuclei in human lymphocytes after in vitro treatment, together the read across infomation from octanoic acid, decanoic acid and nonanoic acid

the substance Carboxylic acids, C5 -9 is not to be classified according to the criteria described in EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).