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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the key study with the target substance and Category Member 2, 2-butyloctanoic acid, the test item was examined for mutagenic activity by assaying for reverse mutation in Salmonella typhimurium, strains TA1535, TA1537, TA98, TA100 and TA102. Experiments were performed both in the absence and presence of metabolic activation. 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 was concluded that 2-butyloctanic acid did not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.

In the supporting study the Source substance 1, Reaction mass of n-undecanoic acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2 -butyl-heptanoic-acid, was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. The study was designed to be compliant with OECD Guideline No. 471 and in accordance with the OECD Principles of Good Laboratory Practices.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. Toxic effects of the test item were noted in all tester strains used in experiment I and II. In experiment I toxic effects of the test item were observed at concentrations of 1.0 µL/plate and higher with and without metabolic activation depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 0.316 µL/plate and higher with and without metabolic activation, depending on the particular tester strain.

No biologically relevant increases in revertant colony number of any of the five tester strains were observed following treatment with the test substance.

Therefore, the test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.

The mouse lymphoma tests on both sides of the category, means for the smallest and the longest chain, ISOCARB 11 and ISOCARB 24, were negative with and without metabolic activation. Category member 1, ISOCARB 11, and the Source substance 2, docosanoic acid, did not induce chromosome aberration with and without metabolic activation. Altogether these results indicate due to intrapolation that the whole category has no genotoxic potential and classification according to EU classification criteria for genetic toxicity is not required.


Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
January - February 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, read-across
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
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 liver microsomal fraction, derived from male Wistar rats, phenobarbital (80 mg/kg bw) / ß-naphthoflavone (100 mg/kg bw) induced for three consecutive days by oral route
Test concentrations with justification for top dose:
Pre-experiment: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate
Experiment I: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate
Experiment II: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 µL/plate (additionally 5.0 µL/plate for E. coli WP2 uvrA only)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Remarks:
A. dest., treated in the same way as all dose groups
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, treated in the same way as all dose groups
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: S. typhimurium strains TA 100, TA 1535: sodium azide (10µg/plate); S. typhimurium strains TA 98, TA 1537: 4-nitro-o-phenylene-diamine (10 µg/plate for TA 98, 40 µg/plate for TA 1537); E.coli WP2 uvrA: methylmethanesulfonate (1 µg/plate)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
A. dest.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2- aminoanthracene (2.5 µg/plate for all S. typhimurium strains, 10 µg/plate for E. coli WP2 uvrA)
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in the pre-experiment and in experiment I, preincubation in experiment II

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approx. ≤ 0.5 in relation to the solvent control

OTHER:
The properties of the S. typhimurium and E. coli strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al.
The colonies were conted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH, Germany). Tester strains with a low spontaneous mutation frequency (TA 1535 and TA 1537) were counted manually.
Evaluation criteria:
Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA1537 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Criteria of validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100)
- the mean values of the spontaneous reversion frequencies of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on both negative control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate.
Statistics:
A statistical evaluation of the results was not regarded as necessary as the biological relevance of the results is the criterion for the interpretation of results (according to guideline).
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations 0.316 µl/plate and higher, depending on the particular tester strain
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was obseved an any tester strain used in experiment I and II with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determind with tester stains TA 98 and TA 100. As the experimental conditions and the tested concentrations were the same as for experiment I, the results of the pre-experiment were included as a part of experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: The control plates with and without S9 mix were within the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects were noted in all tester strains evaluated:
Experiment I: tester strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA at concentrations of 1.0 µL/plate and higher with and without metabolic activation
Experiment II: tester strains TA 98 and E coli WP2 uvrA at concentrations of 1.0 µL/plate and higher with and without metabolic activation;
tester strains TA 100 and TA 1537 at concentrations of 0.316 µL/plate and higher with metabolic activation and at concentrations of 1.0 µL/plate and higher without metabolic activation;
tester strain TA 1535 at concentrations of 0.316 µL/plate and higher with and without metabolic acitvation.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative

The test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item Reaction mass of n-undecanoic acid and 2-methyl-decanoic-acid and 2-ethyl-nonanoic-acid and 2-propyl-octanoic-acid and 2-butyl-heptanoic-acid was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. The study was designed to be compliant with OECD Guideline No. 471 and in accordance with the OECD Principles of Good Laboratory Practices.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I: 0.00316, 0.01, 0.0316, 0.1, 0.3165, 1.0, 2.5 and 5.0 µL/plate

Experiment II: 0.00316, 0.01, 0.0316, 0.1, 0.316, 1.0 and 2.5 µL/plate (additionally 5.0 µL/plate for E. coli WP2 uvrA only)

No precipitation of the test item was observed in any tester strain used in experiment I and II with and without metabolic activation.

Toxic effects of the test item were noted in all tester strains used in experiment I and II. In experiment I toxic effects of the test item were observed at concentrations of 1.0 µL/plate and higher with and without metabolic activation depending on the particular tester strain. In experiment II toxic effects of the test item were noted at concentrations of 0.316 µL/plate and higher with and without metabolic activation, depending on the particular tester strain.

No biologically relevant increases in revertant colony number of any of the five tester strains were observed following treatment with the test substance in experiment I and II.

The reference mutagens induced a distinct increase or revertant colonies indication the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the test strains used.

Therefore, the test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.

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:
2002-03-29 to 2002-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Deficiencies/Proficiencies: histidine auxotroph
- Metabolic activation system: rat liver S9 of Phenobarbital/beta-Naphthoflavone induced male Sprague-Dawley rats
ADMINISTRATION:
- Dosing:
experiment I:
313, 625, 1250, 2500 and 5000 µg/plate (TA 1535, TA 98 and TA 1537 only with metabolic activation)78.1, 156, 313, 625, 1250, 2500 µg/plate (TA 102, TA 100 and TA1537 only without metabolic activation)
experiment II:
156, 313, 625, 1250, 2500 and 5000 µg/plate (TA 1535 and TA 1537 only with metabolic activation)
78.1, 156, 313, 625, 1250, 2500 µg/plate (TA 100 and TA1537 only without metabolic activation)
313, 625, 1250, 2500 and 5000 µg/plate (TA 98)
78.1, 156, 313, 625, 1250 µg/plate (TA 102)
experiment III:
9.77, 19.5, 39.1, 78.1, 156 µg/plate (TA 1535, TA 100 and TA 1537 only with metabolic activation)
4.88, 9.77, 19.5, 39.1, 78.1 µg/plate (Ta 102 and TA 1537 only without metabolic activation)
19.5, 39.1, 78.1, 156, 313 µg/plate (TA 98)
- Number of replicates: 3
- Application:
Experiment I (plate-incorporation method): Bacteria cultures, test item, S9 mix or phosphate buffer were added to the molten overlay agar and vortexed. The mixture was poured onto the surface of a medium agar plate and allowed to solidify prior to incubation. Plates were incubated upside down for approx. 72 hours at 37° C.
Experiment II and III (pre-incubation method): Bacteria cultures, test item, S9 mix or phosphate buffer were mixed and placed at 37° C for 30 minutes. Afterwards overlay agar was added and the mixture was vortexed and poured onto the surface of a medium agar plate and allowed to solidify. Plates were incubated upside down for approx. 72 hours at 37° C.
- Positive control: without metabolic activation: Sodium azide (1 µg/plate with TA 1535 and TA 100), 9-aminoacridine (50 µg/plate with TA 1537), 2-nitrofluorene (2 µl/plate with TA 98), cumene hydroperoxide (100 µg/plate with TA 102); with metabolic activation: with metabolic activation: 2-aminoanthacene (1 µg/plate with TA 1535, Ta 1537, TA 98 and TA 100; 10 µg/plate with TA 102)
- Negative control: solvent control (DMSO) and untreated control
- Pre-incubation time: 30 minutes (only in experiment II and III)
DESCRIPTION OF FOLLOW UP REPEAT STUDY: see above
Evaluation criteria:
The test item is considered as a mutagen if at least a two-fold increases in mean revertant numbers are 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.
Key result
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:
cytotoxicity
Remarks:
toxicity was observed at higher dose-levels with all tester strains, both in the absence and presence of S9 metabolic activation, with the exception of TA98
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity was observed at higher dose-levels, both in the absence and presence of S9 metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results:
negative

The test item ISOCARB 12 does not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January to March, 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus/TK+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Complete culture medium: RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Treatment medium: RPMI medium supplemented with 5% heat-inactivated horse serum (in case of short term exposure) resp. 7.5% heat-inactivated horse serum (in case of long term exposure), 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Selective medium: RPMI 1640 medium supplemented with 20% heat-inactivated horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL trifluorothymidine (TFT)
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
liver S9 microsomal fraction of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced male Wistar rats
Test concentrations with justification for top dose:
Pre-experiment for toxicity: 0.2/1.0/2.6/5.3/8.0 and 10.6 mM for experiment I with and without S9 mix, 0.05/0.1/0.2/0.5/1.0 and 1.5 mM for experiment II, long term exposure, without S9 mix
Experiment I: 0.05/0.1/0.2/0.5/0.7/0.9/1.1, and 1.3 mM without S9 mix, 0.02/0.05/0.1/0.2/0.5/1.0/1.2 and 1.4 mM with S9 mix
Experiment II: 0.002/0.005/0.01/0.02/0.05/0.1/0.2 and 0.5 mM without S9 mix, 0.15/0.3/0.7/0.9/1.1/1.2/1.3 and 1.4 mM with S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: in the absence of metabolic activation: ethyl methanesulphonate (EMS, 200/300 µg/mL) and methyl methanesulphonate (MMS, 8/10µg/mL); in the presence of metabolic activation: benzo[a]pyrene (B[a]P, 2.5 µg/ml)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h
Experiment 2: 24 h (without metabolic activation) and 4 h (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (incubation with the selection agent): 14 days (mutation selection assay)

SELECTION AGENT: trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1 (test item and positive controls, 2 (negative control)

NUMBER OF CELLS EVALUATED: mutation selection assay: cells from each experimental group were seeded in four 96-well plates at a density of 200 cells/well in 200 µL selective medium; cloning efficiency assay: seeding a statistical number of 1.6 cells per well in two 96-well plates

DETERMINATION OF CYTOTOXICITY
- Method: measuring the colony-forming ability and the growth rate of cultures (relative suspension growth, recorded over 2 days following treatment)

OTHER EXAMINTATIONS
- Colony sizing: ratio of small to large type mutants
Evaluation criteria:
The assay is considered acceptable if it meets the following criteria:
- at least three out of four of the negative and/or solvent controls is in the range 65% - 120%
- the spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E6 cells
- the cell number of the negative/solvents controls should undergo 8-32 fold increase during a 2 day growth period (short-term treatment) or 32-180 fold increase during a 3 day growth period (long-term treatment)
- the clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E6 cells. The relative total growth (RTG) must be greater than 10%.

Criteria for a positive result:
- the induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E6 cells
- a dose-dependent increase in mutant frequency is detected.
Additionally, combined with a positive effect in the mutant frequency, an increased occurence of small colonies (>= 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. The biological relevance is considered first for the interpretation of results. Statistical methods might be used an aid in evaluation of the test result.

Criteria for a negative result:
a test item is considered negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
Statistical significance of mean mutant frequency was evaluated at the 5% level (p<0.05) by means of the non-parametric Mann-Whitney test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for details see tables 1 and 2 below
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Water solubility: no data, the test substance was soluble in cell culture medium after being treated with ultrasound for 3 minutes at 37°C
- Precipitation: no precipitation was noticed
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA: All values found in the test were within the range of the historical laboratory control data
Table 1a: Pre-experiment for Toxicity, without metabolic activation
Test group Concentration
mM
Suspension Growth Relative Suspension Growth* [%]
Negative control 0 16.5 100.0
0 16.4
1 0.2 15.5 94.2
2 1.1 7.7 46.8
3 2.6 0.3 1.9
4 5.3 no viable cells -
5 8.0 no viable cells -
6 10.6 no viable cells -
Table 1b: Pre-experiment for Toxicity, with metabolic activation
Test group Concentration
mM
Suspension Growth Relative Suspension Growth* [%]
Negative control 0 13.4 100.0
0 13.6
1 0.2 12.2 90.1
2 1.1 9.1 67.7
3 2.6 0.3 1.9
4 5.3 no viable cells -
5 8.0 no viable cells -
6 10.6 no viable cells -
Table 1c: Pre-experiment II for Toxicity, long-term exposure, without metabolic activation
Test group Concentration
mM
Suspension Growth Relative Suspension Growth* [%]
Negative control 0 35.9 100.0
0 33.9
1 0.05 32.9 94.3
2 0.1 31.5 90.2
3 0.2 23.0 65.7
4 0.5 6.0 17.2
5 1.0 0.3 0.8
6 1.5 0.1 0.4
* = (Suspension Growth/Suspension Growth of corresponding controls) x100
Table 2a: Experiment I, without metabolic activation
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 101.2 100.0 100.0 80.1 / / 15.8
108.2 / / 14.8
3 0.05 104.6 99.9 97.9 71.1 -8.8 - n.d.
4 0.1 112.0 106.9 109.1 66.5 -13.6 - n.d.
5 0.2 112.0 106.9 97.6 63.8 -16.3 - n.d.
6 0.5 84.1 80.3 75.2 94.0 13.9 - n.d.
7 0.7 98.0 93.6 81.1 50.3 -29.8 - n.d.
8 0.9 95.0 90.7 61.9 89.7 9.6 - 18.3
9 1.1 114.0 108.8 58.2 79.2 -0.9 - 22.2
10 1.3 130.0 124.1 21.8 63.3 -16.8 - 24.1
EMS 300 µg/mL 93.5 89.3 68.9 690.8* 610.7* + n.d.
MMS 10 µg/mL 80.5 76.8 59.4 511.6* 431.5* + 41.9
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2b: Experiment II, without metabolic activation (24 h treatment)
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 114.0 100.0 100.0 56.7 / / 6.1
102.9 / / 22.5
1 0.002 96.5 89.0 87.1 60.3 3.5 - n.d.
2 0.005 90.7 83.6 127.2 75.8 10.9 - n.d.
3 0.01 85.4 78.7 107.5 101.4* 44.7* - n.d.
4 0.02 106.4 98.1 92.7 67.2 10.5 - n.d.
5 0.05 112.0 103.3 112.6 69.6 12.9 - n.d.
6 0.1 102.9 94.9 74.3 52.1 -4.6 - 15.4
7 0.2 102.9 94.9 72.2 62.0 5.3 - 13.0
8 0.5 101.2 93.4 20.3 67.6 10.9 - 16.3
EMS 200 µg/mL 32.0 29.5 19.4 3673.2* 3616.5* + n.d.
MMS 8 µg/mL 38.9 35.9 25.0 1191.8* 1135.1* + 51.1
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2c: Experiment I, with metabolic activation
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 90.7 100.0 100.0 81.0 / / 10.9
110.7 / / 20.8
2 0.02 80.5 80.2 90.2 109.8 28.9 - n.d.
3 0.05 101.2 100.9 110.3 89.1 8.1 - n.d.
4 0.1 86.6 86.3 99.7 96.4 15.5 - n.d.
5 0.2 75.9 75.6 77.3 100.1 19.1 - n.d.
6 0.5 90.7 90.3 92.7 92.1 11.2 - n.d.
7 1.0 93.5 93.2 73.4 95.9 15.0 - 22.2
8 1.2 93.5 93.2 46.4 83.0 2.0 - 21.8
9 1.4 93.5 93.2 10.1 121.4* 40.5* - 20.5
B[a]P 2.5 µg/mL 92.1 91.7 57.7 593.8* 512.8* + 42.9
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Table 2d: Experiment II, with metabolic activation
Test group Concentration CE RCE RTG MF IMF GEF exceeded colony sizing
mM % % % mutants/106cells mutants/106cells % small colonies
Negative control 0 110.1 100.0 100.0 72.2 / / 14.3
95.0 / / 18.4
5 0.15 90.7 88.4 79.9 75.6 3.5 - n.d.
6 0.3 95.0 92.6 100.1 78.5 6.3 - n.d.
7 0.7 125.0 121.9 103.3 68.1 -4.1 - n.d.
8 0.9 98.0 95.6 81.9 66.7 -5.5 - n.d.
9 1.1 82.1 89.8 56.2 79.2 7.1 - n.d.
10 1.2 120.3 117.4 57.4 63.0 -9.2 - 11.1
11 1.3 120.3 117.4 31.8 64.7 -7.5 - 12.7
12 1.4 104.6 102.0 8.7 77.2 5.1 - 17.5
B{a]P 2.5 µg/mL 85.4 83.3 59.2 477.9* 405.7* + 42.1
CE: Cloning efficiency, CE = (-ln ((96 - (mean value of plates)) / 1.6) x 100
RCE: Relative Cloning Efficiency, RCE = (CE of dose group/CE of corresponding controls)x100
RTG: Relative Total Growth, RTG = (RSG x RCE)/100
MF: Mutant Frequency
GEF: Global Evaluation Factor (126): + = GEF exceeded, - = GEF not exceeded
* statistical significant increase in mutant frequency compared to negative controls
Conclusions:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

In the described mutagencity test under the experimental conditions reported, the test item Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The test item Reaction mass of 2 -butylheptanoic acid and 2 -ethylnonanoic acid and 2 -methyldecanoic acid and 2 -proyloctanoic acid was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiment. In experiment I 1.3 mM (without metabolic activation) and 1.4 mM (with metybolic activation) were selected as the highest concentrations. In experiment II 0.5 mM (without metabolic activation) and 1.4 mM (with metabolic activation ) were selected as the highest concentrations. Experiment I without and with metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I without metabolic activation: 0.05, 0.1, 0.2, 0.5, 0.7, 0.9, 1.1 and 1.3 mM and with metabolic activation: 0.02, 0.05, 0.1 ,0.2, 0.5, 1.0, 1.2 and 1.4 mM

Experiment II without metabolic activation: 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mM and with metabolic activation: 0.15, 0.3, 0.7, 0.9, 1.1, 1.2, 1.3 and 1.4 mM.

No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiment I and II without and with metabolic activation.

In experiment I without metabolic activation the relative total growth (RTG) was 21.8% for the highest concentration (1.3 mM) evaluated. The highest concentration evaluated with metabolic activation was 1.4 mM with a RTG of 10.1%. In experiment II without metabolic activation the relative total growth was 20.3% for the highest concentration (0.5 mM) evaluated. The two highest concentrations evaluated with metabolic activation were 1.3 and 1.4 mM with a RTG of 31.8% and 8.7% respectively.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF, defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration. No dose-response relationship was observed. Aditionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental condition (with and without metabolic activation).

Ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS) and benzo[a]pyrene (B[a]P) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a)P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagencity test under the experimental conditions reported, the test item Reaction mass of 2-butylheptanoic acid and 2-ethylnonanoic acid and 2-methyldecanoic acid and 2-propyloctanoic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
On the basis of all evaluated data, the similarity of all category members of the ISOCARB is justified on basis of the physico-chemical properties, toxicological and ecotoxicological profiles. There is convincing evidence that these chemicals possess an overall common category profile (Please see Category approach).
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
On the basis of all evaluated data, the similarity of all category members of the ISOCARB is justified on basis of the physico-chemical properties, toxicological and ecotoxicological profiles. There is convincing evidence that these chemicals possess an overall common category profile (Please see Category approach).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see details below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Conclusions:
Interpretation of results:
negative

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Justification for type of information:
The carbon chain length of this acid and that of the Category member 2-decyltetradecanoic acid, is very similar. Both substances are long chain acids which have no other funcitional group. Based on the similar structure and therefore similar expected toxicokinetic it is likely that the test result of this substance is also valid for the Category member (Please see attached Category Approach.)
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung (CHL) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle-MEM liquid medium
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
+S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
-S9 mix (24 h continuous exposure): 0, 350, 700, 1400, 2800 µg/mL
-S9 mix (48-hour continuous exposure): 0, 288, 575, 1150, 2300 µg/mL
Vehicle / solvent:
1.0 % carboxymethylcellulose sodium
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: continuous exposure: mitomycin C (0.05 µg/mL for 24 hours and 0.025 µg/mL for 48 hours); short-term exposure: cyclophosphamide (12.5 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: 6 (short-term exposure), 24, 48 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
The frequency of polyploid cells or cells with abnormal structure of each test group were determined according to the criteria of Ishidate.
Key result
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble in water, soluble in alcohol, ether, chloroform and acetone
- Precipitation: observed on the slide of continuous exposure high dose group

RANGE-FINDING/SCREENING STUDIES: see 'Any other information on material and method incl. tables'

COMPARISON WITH HISTORICAL CONTROL DATA: this test was valid, since the frequency of chromosomal aberration in positive control was within background data.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The Ames-test of the Source substance 1, ISOCARB L11, Category Member 2, ISOCARB 12 and Category Member 3, ISOCARB 24, were negative, also the mouse lymphoma tests on both sides of the category, means for the smallest and the longest chain, ISOCARB 11 and ISOCARB 24, were negative with and without metabolic activation. Category member 1, ISOCARB 11, and the Source substance 2, docosanoic acid, did not induce chromosome aberration with and without metabolic activation. Altogether these results indicate due to intrapolation that the whole category has no genotoxic potential and classification according to EU classification criteria for genetic toxicity is not required.