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Endpoint:
carcinogenicity
Data waiving:
other justification
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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:
2010-04-27 to 2010-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to guidelines EU B17 and OECD 476
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
according to German Chemikaliengesetz, §19 and Directive 2004/9/EEC
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of livers of Wistar rats induced with Phenobarbital/ß-naphthoflavone
Test concentrations with justification for top dose:
range finding experiment: 12.8 to 1640.0 µg/mL in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation
1st experiment: 102.5. 205.0, 410.0, 820.0, 1093.3, 1366.7 and 1640.0 µg/mL (without S9); 102.5, 205.0, 410.0, 820.0, 1230.0 and 1640.0 µg/mL (with S9), 4 h exposure
2nd experiment: 250.0, 500.0, 750.0, 1000.0, 1250.0, 1500.0 and 1640.0 µg/mL (24 h, without S9); 500.0, 750.0, 1000.0, 1250.0, 1500.0 and 1640.0 µg/mL (4 h, with S9)

Justification of top dose:
Relevant toxic effects measured as reduction of cloning efficiency to about 50 % of control
or less were observed at 820.0 Qg/mL and above following 4 hours treatment and at
1640.0 Qg/mL after 24 hours treatment in the absence of metabolic activation in a pre-test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the cells
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 24 h
- Exposure duration: experiment 1: 4 h (with and without metabolic activation); experiment 2: 24 h (without metabolic activation), 4 h (with metabolic activation)
- Expression time (cells in growth medium): 7 d
- Selection time (if incubation with a selection agent): 8 d

SELECTION AGENT (mutation assays): 6-TG

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT(R)11 (SYSTAT Software, Inc., Richmond, CA, USA) statistics software
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9: at 1093,3 µg/mL and above after 4 h exposure, at 1500 µg/mL and above after 24 h exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Under the conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

The study was performed to investigate the potential of 3,5,5-Trimethylhexanoic acid to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 hours in concentrations of 102.5; 205.0, 410.0, 820.0, 1093.3 and 1366.7 µg/mL without metabolic activation and 205.0, 410.0, 820.0, 1230.0 and 1640.0 µg/mL with metabolic activation.

The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test conentations were 500.0, 750.0, 1000.0, 1250.0, 1500.0 and 1640.0 µg/mL without metabolic activation and 750.0, 1000.0, 1250.0, 1500.0 and 1640.0 µg/mL with metabolic activation. The highest concentration in the main experiments (1640.0 µg/mL) was equal to a molar concentration of about 10 mM and was adjusted to purity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

Under the experimental conditions of this study the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, 3,5,5-Trimethylhexanoic acid is considered to be non-mutagenic in this HPRT assay (Carboxylic acids REACH Consortium, 2010). This study was performed according to guideline EU B17 and OECD 416 and is therefore considered to be of high reliability (RL1).

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-04-13 to 2018-06-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
In accordance with OECD TG 473, adopted on 2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
human lymphocytes.
Donor for preliminary toxicity test: male, aged 28 years
Donor for main experiment: male, aged 30 years
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Colcemid 0.1 µg/mL
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
4-hour exposure without S9-mix: 0*, 98.75, 197.5, 395, 790, 987.5*, 1185*, 1580* µg/mL. (1580 µg/mL corresponds to the maximum recommended dose level (10 mM).

4-hour exposure with S9-mix (2%): 0*,98.75, 197.5, 395, 790*, 987.5*, 1185*, 1580 µg/mL. 1185 µg/mL corresponds to the lowest precipitating dose level.

24-hour exposure without S9-mix: 0*, 24.69, 49.38, 98.75*, 148.13*, 197.5*, 296.25*, 395 µg/mL.

395 µg/mL was not used for metaphase analysis, as too few metaphases were present. Higher doses were not used for the 24-hour continuous exposure due to no metaphases present in the preliminary toxicity test.

*: dose levels selected for CA analysis
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
- Short-term exposure: 4 h exposure with and without metabolic activation followed by 20-hour culture in treatment-free media
- Long-term exposure: 24 h exposure without metabolic activation


SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 lymphocyte cultures from the same donor for each of the exposure conditions. 2 flasks per concentration for the 4 h(20 h) experiments, 1 flask per concentration for the 24 h experiment.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: 2000 cells for mitotic index

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300 (150 from each culture).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: If a cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, hematolysis

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Rationale for test conditions:
OECD TG 473
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the
vehicle control cultures was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p≤0.01) and
demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top
concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed

Providing that all of the acceptability criteria are fulfilled, the test item was considered to be
clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be
within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with
structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level



When all of the following criteria are met, the test item was considered able to induce
chromosomal aberrations in human lymphocytes.
1) The number of cells with structural chromosome aberrations is outside the range of
the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of
cells with structural chromosome aberrations compared to the concurrent negative
control.
3) The observed increase in the frequency of cells with structural aberrations is
considered to be dose-related
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes: human (male donor)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Unambiguously negative results in all three testing conditions (4h(+20h) with/without S9, 24h without S9). No polyploidies or endoreplicates were observed.

Metaphase cells were present up to the maximum dose level of 1580 µg/mL in the 4(20)-hour exposures in the presence and absence
of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 395 µg/mL.

Precipitate observations were made at the end of exposure in the blood cultures and was noted at 1580 μg/mL in the 4(20)-hour exposure group in the absence of S9 and at and above 1185 μg/mL in the 4(20)-hour exposure group in the presence of S9. No precipitate was observed in the blood cultures in the 24-hour continuous exposure group. Hemolysis was observed at and above 395 μg/mL in the 4(20)-hour exposure groups and at and above 197.5 μg/mL in the 24-hour exposure group. As hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes this was not regarded as relevant.

In the 4(20)-hour exposure group in the absence of S9, 37% mitotic inhibition was achieved at 1580 µg/mL.
However, this dose level was also the lowest precipitating dose level and, therefore, the maximum dose level
selected for metaphase analysis.

In the presence of S9, 26% mitotic inhibition was observed at 1185 µg/mL. However, this dose level was also
the lowest precipitating dose level and, therefore, the maximum dose level selected for metaphase analysis.
In the 24-hour continuous exposure group, an inhibition of mitotic index of 30% and 44% was observed at 197.5
and 296.25 µg/mL, respectively.
Remarks on result:
other: clearly negative result

For details see attachment

Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

An in vitro mammalian chromosomal aberration test according to OECD TG 473 and GLP has been performed with human lymphocytes.

Duplicate cultures of human lymphocytes, treated with the test item (vehicle DMSO), were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels selected for the main experiment were as follows (up to the recommenden highest dose level of 10 mM (1580 µg/mL or the dose level with a tolerable cytotoxicity):

4(20)-hour without S9: 0*, 98.75, 197.5, 395, 790, 987.5*, 1185*, 1580* µg/mL.

4(20)-hour with S9 (2%): 0*, 98.75, 197.5, 395, 790*, 987.5*, 1185*, 1580 µg/mL.

24-hour without S9: 0*, 24.69, 49.38, 98.75*, 148.13*, 197.5*, 296.25*, 395 µg/mL.

Dose levels marked with * were selected for analysis of chromosomal aberrations.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that approached 55±5% mitotic inhibition or was the maximum recommended dose level, depending on exposure group. Positive and vehicle controls were valid.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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:
1988-07-05 to 1988-07-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to guideline OECD 471
Reason / purpose for cross-reference:
other: Purity statement
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay
Specific details on test material used for the study:
For details on test material see attached report in section 13
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of livers from Sprague-Dawley rats, induced with Aroclor 1254
Test concentrations with justification for top dose:
first experiment: 4, 20, 100, 500, 2500, 10000 µg/plate
second experiment: 4, 20, 100, 500, 2500, 5000 µg/plate

top dose selected on basis of highest concentration requested in guideline
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide, 9-aminoacridine, 2-nitrofluorene; with S9: 2-aminoanthracene, benzo[a]pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: 2 ml of molten top agar (plate incorporation) with 100 µl of overnight nutrient broth culture of bacteria tester strain and 100 µl of test compound solution with or without 500 µl of S9 mix are poured into a petri dish with a layer of minimal agar. His+ revertants were counted after 48-72 h incubation at 37 °C in the dark.
Evaluation criteria:
A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn (controlled microscopically) were used as indicator for toxicity.
Statistics:
Statistical analysis was not stated.
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:
at 2500 µg/plate and above in most strains
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: WP2 uvr A
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the conditions of this study, the test substance was not mutagenic with or without metabolic activation.
Executive summary:

The test substance was not mutagenic in an Ames-test with the Salmonella typh. strains TA98, TA100, TA1535, TA1537 and TA1538 and E.coli WP2uvrA in concentrations up to 10000 µg/plate, both with or without metabolic activation (Hoechst AG, 1988).

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion