2-pentylcyclopentan-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 & E.coli WP2uvrA.
- in vitro Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): non mutagenic in the presence and absence of metabolic activation.
- Chromosome aberration test (read-across, OECD 473, GLP, K, rel. 2): non clastogenic to human lymphocytes in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across approach is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological, ecotoxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances (Table 1) are 1,2-branched pentyl cyclopentane. The source substance is an alcool, while the target substance is a ketone. The source substance is the secondary alcohol formed by metabolism of the Target substance.

3. ANALOGUE APPROACH JUSTIFICATION
In the CAT performed on the source substance according to OECD 473, none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations was induced. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities.

4. DATA MATRIX
See Iuclid section 13

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant increase observed. In Exp 1 pH solvent = 7.6 versus pH 1620 µg/mL = 7.3.
- Effects of osmolality: no relevant increased observed. In Exp 1 solvent = 365 mOsm versus 1620 µg/mL = 365 mOsm.
- Precipitation: In Experiment I, no visible precipitation of the test item in the culture medium was observed. However, phase separation occurred at 925.7 Lg/mL and above in the absence of S9 mix and at 529.0 Lg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II, in the presence of S9 mix, at 302.3 Lg/mL and above.

RANGE-FINDING/SCREENING STUDIES: since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA: the aberration rates of the cells after treatment with the test item (0.5-3.0 %, exl. gaps) were slightly above the range of solvent control values (0.5-1.0%, excl. gaps) but were within the range of laboratory's historical control data: 0.0-4.0%, excl. gaps.
In both experiments, either EMS or CPA showed distinct increases in cells with structural chromosome aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment I, in the absence and the presence of S9 mix, and in Experiment II in the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II, in the absence of S9 mix, cytotoxicity indicated by clearly reduced mitotic indices could be observed at the highest evaluated concentration (47.1 % of control).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.2 %) as compared to the rates of the solvent controls (0.0 – 0.2 %).

The proliferation index of the lymphocytes in solvent control cultures in Experiment I with and without S9 mix (4 hrs treatment; 1.25 and 1.52, respectively), in Experiment II with and without S9 mix (1.10 and 1.88, respectively) was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. This is also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the test conditions, the source substance was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human lymphocyte were exposed to the source substance diluted in DMSO. Three treatment conditions were used, i.e.

- 4 hours exposure with the addition of an induced rat liver homogenate metabolising system in standard co-factors (S9-mix) with cell harvest after 18 hours,

- 4 hours exposure with cell harvest after 18 hours in the absence of activation,

- 22 hours continuous exposures in the absence of activation.

The dose range for evaluation was selected from a series of 10 dose levels on the basis of toxicity.

 

In Experiment I, in the absence and the presence of S9 mix, and in Experiment II in the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II, in the absence of S9 mix, the mitotic indices were clearly reduced below 50 % of control at the highest evaluated concentration.

 

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures

 

All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

Under the test conditions, the source substance was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 07 to August 04, 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted according to OECD test Guideline No. 473 without any deviation. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection date: 2006-09-02

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were obtained from a healthy donor not receiving medication. For this study, blood was collected from a female donor (32 years old) for the 1st experiment and from 34 year-old female and 26 year-old male donor for experiment II. Blood samples were drawn by venous puncture and collected in heparinised tubes.
- Type and identity of media: Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks. The culture medium was DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; Life Technologies GmbH, 76339 Eggenstein, Germany) containing 10 % FCS (fetal calf serum). The antibiotic solution contains 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with Phytohemagglutinin (final concentration 3 µg/mL), the anticoagulant heparin (25,000 U.S.P.-U/mL), and HEPES (final concentration 10 mM).
- All incubations were done at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S-9

Test concentrations with justification for top dose:

Experiment I: 10.5, 18.4, 32.2, 56.4*, 98.7*, 172.7*, 302.3, 529.0, 925.7, 1620.0 µg/L (+/- S9 mix) - up to 10 mM.
Experiment II: 3.2, 5.7, 9.9, 17.4, 30.5*, 53.3*, 93.3*, 163.3, 285.7, 500 µg/L (- S9 mix).
Experiment II: 10.5, 18.4, 32.2, 56.4, 98.7*, 17207*, 302.3*, 529, 925.7, 1620 µg/L (+ S9 mix) - up to 10 mM.
* = evaluated.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration in the culture medium was 0.5 % v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

825 µg/mL (6.64 mM), Exp I. 660 µg/mL (5.32 mM), Exp II. Dissolved in nutrient medium.

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

30 µg/mL (0.106 mM), Exp I. 15 µg/mL (0.053 mM), Exp II. Dissolved in saline (0.9 % NaCl w/v)

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 50-80 hours.
- Exposure duration: Exp I: 4 hours. Exp II: 4 hours with S9-mix, 22 hours without S9-mix.
- Expression time (cells in growth medium): 18 hours (except Exp II without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL) was added to the cultures 3 hours before harvesting.
STAIN (for cytogenetic assays): Giemsa or according to the fluorescent plus Giemsa technique, respectively.

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: At least 1000 cells were counted per culture for determination of the mitotic index. For structural chromosome aberrations at least 100 cells per culture were scored. The number of polyploid cells was scored in 250 metaphase cells.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

OTHER: Exp II with S9-mix was repeated 3 times due to missing cytotoxicity or technical reasons.

Evaluation criteria:

Acceptability of the assay:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range: 0.0 - 4.0 % aberrant cells, exclusive gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data:
EMS (330-880 µg/mL) : 4 - 47 %
CPA (15-45 µg/mL): 7.5 - 40 %

A test item is classified as non-mutagenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant increase observed. In Exp 1 pH solvent = 7.6 versus pH 1620 µg/mL = 7.3.
- Effects of osmolality: no relevant increased observed. In Exp 1 solvent = 365 mOsm versus 1620 µg/mL = 365 mOsm.
- Precipitation: In Experiment I, no visible precipitation of the test item in the culture medium was observed. However, phase separation occurred at 925.7 Lg/mL and above in the absence of S9 mix and at 529.0 Lg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II, in the presence of S9 mix, at 302.3 Lg/mL and above.

RANGE-FINDING/SCREENING STUDIES: since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA: the aberration rates of the cells after treatment with the test item (0.5-3.0 %, exl. gaps) were slightly above the range of solvent control values (0.5-1.0%, excl. gaps) but were within the range of laboratory's historical control data: 0.0-4.0%, excl. gaps.
In both experiments, either EMS or CPA showed distinct increases in cells with structural chromosome aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment I, in the absence and the presence of S9 mix, and in Experiment II in the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II, in the absence of S9 mix, cytotoxicity indicated by clearly reduced mitotic indices could be observed at the highest evaluated concentration (47.1 % of control).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.2 %) as compared to the rates of the solvent controls (0.0 – 0.2 %).

The proliferation index of the lymphocytes in solvent control cultures in Experiment I with and without S9 mix (4 hrs treatment; 1.25 and 1.52, respectively), in Experiment II with and without S9 mix (1.10 and 1.88, respectively) was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. This is also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.

Conclusions:

Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 and in compliance with GLP, human lymphocyte were exposed to the test material diluted in DMSO. Three treatment conditions were used, i.e.

- 4 hours exposure with the addition of an induced rat liver homogenate metabolising system in standard co-factors (S9-mix) with cell harvest after 18 hours,

- 4 hours exposure with cell harvest after 18 hours in the absence of activation,

- 22 hours continuous exposures in the absence of activation.

The dose range for evaluation was selected from a series of 10 dose levels on the basis of toxicity.

In Experiment I, in the absence and the presence of S9 mix, and in Experiment II in the presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In Experiment II, in the absence of S9 mix, the mitotic indices were clearly reduced below 50 % of control at the highest evaluated concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures

All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Under the test conditions, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).

Reference 3

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:

From June 10 to August 26, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD test Guideline No. 476 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on March 4 to 8, 2013/ signed on May 6, 2013

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK+/- gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation).
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically "cleansed" against high spontaneous background: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (4 % v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats orally induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Dose range finding test: 17, 52, 164, 512 and 1545 μg/mL, in the absence of S9-mix with a 3 and 24 h treatment period and in the presence of S9-mix with a 3 h treatment period
First mutagenicity test:
- Without S9-mix (3 h exposure): 0.17, 0.54, 1.7, 5.4, 17, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium
- With S9-mix (3 h exposure): 17, 52, 70, 90, 110, 130, 150, 170, 190 and 200 μg/mL exposure medium
Second mutagenicity test:
- Without S9-mix (24 h exposure): 0.54, 1.7, 5.4, 17, 30, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test substance preparation: No correction was made for the purity/composition of the test substance. The test item was immiscible in RPMI 1640 medium at a concentration of 155 mg/mL, but was fully miscible in DMSO at the required concentration, as determined in solubility checks. The test substance was therefore dissolved in DMSO (SeccoSolv, Merck Darmstadt, Germany). Test item concentrations were used within 1.5 h after preparation. The final concentration of the solvent in the exposure medium was 1.0% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix: 15 and 5 μg/mL for a 3 and 24 h treatment period, respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix: 7.5 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
Exposure medium: For 3 h exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 h exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

DURATION
- Exposure duration: Dose range finding test: 3 h (±-S9-mix); 24 h (- S9-mix); First mutagenicity test: 3 h (±-S9-mix); Second mutagenicity test: 24 h (- S9-mix)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 or 12 days
- All incubations were carried out in a humid atmosphere (80 - 100%, actual range 50 – 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.6–37.7 °C).

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Dose range finding test: single cultures/dose for test item and vehicle control; mutagenicity test: single cultures/dose for test item and positive control; duplicate cultures for vehicle control

NUMBER OF CELLS EVALUATED:
- For determination of the CE on day 2 the cell suspensions were diluted and seeded in wells of a 96-well dish. 1 cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the MF a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). In the treatment group of 0.17 μg/mL in the first experiment (absence of S9-mix) a total number of 464 wells was used for determination of the mutation frequency.
- Stain: The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 h, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency (CE), Relative survival (RS), Relative Total Growth (RTG), Suspension Growth (SG), Relative Suspension Growth (RSG)

Evaluation criteria:

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF (controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF (mean solvent control) + 126.
In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Statistics:

No data

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Since the test substance precipitated in the exposure medium at concentrations of 512 μg/mL and above, the pH and osmolality were not determined.
- Precipitation: Although test item precipitated in the exposure medium at concentrations of 512 μg/mL and above, the test item could be tested up to and including the concentration of 1545 μg/mL (= 0.01 M, recommended maximum dose level in the guidelines).

DOSE RANGE FINDING TEST:
- In the absence of S9-mix (3 h treatment), no toxicity in the relative suspension growth was observed up to test substance concentrations of 52 μg/mL compared to the solvent control. Hardly any cell survival was observed at test substance concentrations of 164 μg/mL and above.
- In the presence of S9-mix (3 h treatment), the relative suspension growth was 25% at the test substance concentration of 164 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 512 μg/mL and above.
- In the absence of S9-mix (24 h treatment), the relative suspension growth was 67% at the test substance concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at test substance concentrations of 164 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The spontaneous mutation frequencies in the solvent-treated control cultures were within the minimum and maximum value of the historical control data range. Historical control data generated from experiments performed between May 2011 to May 2014.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test:
- In the absence of S9-mix, the dose levels of 52 to 90 μg/mL showed similar cytotoxicity. Therefore, the dose level of 90 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 130 and 150 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 0.17, 0.54, 1.7, 5.4, 17, 52, 70 and 110 μg/mL exposure medium.
- In the presence of S9-mix, too many dose levels showed severe cytotoxicity, this part of the experiment was repeated (experiment 1A): the following dose range was selected: 5.4, 17, 52, 70, 100, 110, 120, 130, 140 and 150 μg/mL. The dose levels of 140 and 150 μg/mL were too toxic for further testing. Therefore, the dose level of 140 and 150 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels selected to measure mutation frequencies at the TK-locus were: 5.4, 17, 52, 70, 100, 110, 120 and 130 μg/mL exposure medium.
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 83% compared to the total growth of the solvent controls.

Second mutagenicity test:
- The dose levels of 0.54 to 17 μg/mL showed similar cytotoxicity. Therefore, the dose level of 0.54 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 130 and 150 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 1.7, 5.4, 17, 30, 52, 70, 90 and 110 μg/mL exposure medium.
- In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 84% compared to the total growth of the solvent controls.

Remarks on result:

other: strain/cell type: L5178Y/TK+/--3.7.2C mouse lymphoma cells

Remarks:

Migrated from field 'Test system'.

Evaluation of the mutagenicity:

First mutagenicity test: No significant increase in the mutation frequency (MF < MF_{mean solvent control}+ GEF) at the TK locus was observed after treatment with test item either in the absence (MF < 236 x 10^-6) or in the presence of S9-mix (MF < 205 x 10^-6). The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test: No significant increase in the mutation frequency at the TK locus was observed after treatment with test item (MF < 239 x 10^-6). The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Table 7.6.1/1: Cytotoxic and mutagenic response of test item in the mouse lymphoma L5178Y test system

 

Dose (μg/mL)	RSG (%)	CEday2 (%)	RSday2	RTG (%)	mutation frequency per 10^6survivors
					Total	small	large
Experiment 1: Without metabolic activation - 3 h treatment
DMSO	100	80	100	100	104	37	63
DMSO		94			115 (110^1)	40	68
0.17	88	95	109	97	101	39	57
0.54	64	77	88	57	99	46	49
1.7	108	76	87	94	104	54	46
5.4	182	66	76	138	92	40	49
17	126	76	87	110	90	53	34
52	50	81	93	47	92	42	46
70	37	88	100	37	103	45	54
110	13	76	87	11	83	35	46
MMS	53	47	54	29	1050	586	343
Experiment 1: With metabolic activation - 3 h treatment
DMSO	100	88	100	100	79	24	52
DMSO		93			79 (79^1)	29	47
5.4	103	98	109	112	66	17	47
17	91	99	110	101	80	26	52
52	86	99	110	95	70	32	35
70	73	91	101	74	90	32	55
100	74	111	124	92	64	24	38
110	54	101	112	61	55	25	28
120	46	108	120	55	88	40	43
130	17	90	100	17	92	25	64
CP	73	71	79	58	633	291	271
Experiment 2: Without metabolic activation - 24 h treatment
DMSO	100	81	100	100	112	76	31
DMSO		86			114 (113^1)	82	28
1.7	91	81	97	89	121	79	37
5.4	91	77	92	84	149	98	43
17	81	80	96	77	137	92	39
30	64	83	99	63	124	84	35
52	56	95	114	63	108	83	21
70	34	80	96	33	100	74	22
90	23	75	89	20	94	61	30
110	15	86	103	16	104	66	34
MMS	96	64	77	74	806	560	168

 

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth;

MMS = Methylmethanesulfonate; CP = Cyclophosphamide

1) Mean no of mutants in the solvent control groups {SC1 + SC2}/2

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/-(3.7.2C) mouse lymphoma cells were exposed to test item at the following concentrations:

Dose range finding test: 17, 52, 164, 512 and 1545 μg/mL, in the absence of S9-mix with a 3 and 24 h treatment period and in the presence of S9-mix with a 3 h treatment period

First mutagenicity test:

- Without S9-mix (3 h exposure): 0.17, 0.54, 1.7, 5.4, 17, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium

- With S9-mix (3 h exposure): 17, 52, 70, 90, 110, 130, 150, 170, 190 and 200 μg/mL exposure medium

Second mutagenicity test:

- Without S9-mix (24 h exposure): 0.54, 1.7, 5.4, 17, 30, 52, 70, 90, 110, 130 and 150 μg/mL exposure medium 

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 4 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone.

 

In dose range finding test, hardly any cell survival was observed at test substance concentrations of 164 μg/mL and above in the absence of S9-mix and at test substance concentrations of 512 μg/mL and above in the presence of S9-mix. In the first experiment, test item was tested up to concentrations of 110 and 130 μg/mL in the absence and presence of S9-mix, respectively. Relative total growth (RTG) was reduced to 11 and 17% in the absence and presence of S9-mix, respectively. In the second experiment, test item was tested up to concentrations of 110 μg/mL in the absence S9-mix. The RTG was reduced to 16%.

 

In the absence of S9-mix, test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, test item did not induce a significant increase in the mutation frequency in the first experiment. The mutation frequencies in the vehicle and positive control cultures were within the acceptable range indicating the validity of the study.

 

Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

Reference 4

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:

From May 31 to July 17, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 471 Guideline without any deviation.

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on August 30, 2005/ signed on November 21, 2005)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 from liver of male Sprague-Dawley rats orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day prior to S9 preparation on Day 4

Test concentrations with justification for top dose:

Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 100 and WP2 uvr A strains, with or without S9-mix using the direct plate incorporation method.

Mutation Test (direct plate incorporation method):

Experiment 1 (Range-finding Test)
Salmonella strains and E.coli strain WP2uvrA- (with and without S9): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with or without S9-mix.

Experiment 2 (Main Test)
All Salmonella strains (with and without S9): 5, 15, 50, 150, 500 and 1500 μg/plate.
E.coli strain WP2uvrA- (with and without S9): 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material formed an emulsion in sterile distilled water at 50 mg/mL , but was fully miscible in DMSO at 50 mg/mL. Therefore, DMSO was selected as vehicle.
- Preparation of test materials: The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

Evaluation criteria:

- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis recommended by UKEMS (Kirkland, 1989)

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: Emulsion in water
- Precipitation: None
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY: The test material was toxic at and above 1500 μg/plate to TA 100 and WP2uvrA-.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains initially at 1500 μg/plate.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate and found to be satisfactory.
- Test material formulation, amino acid supplemented top agar and the S9 mix used in the experiments were shown to be sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/2: Preliminary Toxicity Test

S9 mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
- S9	TA 100	87	73	64	90	70	75	78	66	69	0*	0*
+ S9	TA 100	77	69	82	75	78	63	85	71	57	0*	0*
- S9	WP2 uvr A	26	18	20	19	18	27	22	20	26	14*	0*
+ S9	WP2 uvr A	25	29	25	28	24	21	28	30	23	20*	0*

 

*: Partial or total absence of bacterial background lawn

See the attached document for information on tables of results – mutagenicity test

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A according to the criteria of the Annex VI of the of the Regulation (EC) No 1272/2008 (CLP).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 μg/plate. The experiment was repeated on a separate day using an amended dose range (5 to 5000 µg/plate), fresh cultures of the bacterial strains and fresh test material formulations. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.   The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the tester strains initially at 1500 μg/plate. The test material was tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.   Under the test condition, the test material is not mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP). This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Substance tested	Test / Guideline Reliability	Focus	Strains tested	Test concentration	Statement
1   Safepharm, 2006	Registered substance	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100 E.coli WP2 uvrA	Up to cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic
2   WIL Research Europe BV, 2014	Registered substance	L5178YTK+/-/MLA test (OECD 476) K, rel. 1	Gene mutation	L5178Y tk+/-(3.7.2C) mouse lymphoma cells	Up to cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic
3   Bohnenberger, 2008	Read-across 2-pentylcyclopentan-1-ol	HL/CAT (OECD 473) K, rel.2	Chromosomal aberration	Human lymphocyte	Up to cytotoxic concentration	-S9 : non clastogenic +S9 : non clastogenic

Gene mutation Assays (Tests n° 1 -2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria under the test condition whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammalian cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells (L5178Y TK+/- /MLA test) (Test n°2). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore the substance was considered as negative for inducing gene mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined with a supporting substance (see IUCLID section 13 for read-across justification) using an in vitro chromosome aberration test in Human lymphocytes, which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured Human lymphocytes. None of the dose levels up to the cytotoxicity limit with the supporting substance, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The supporting substance does not induce structural aberrations in the chromosomes of Human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Therefore both the registered substance and the supporting substance are considered as negative for inducing chromosomal mutations in human lymphocytes in vitro under activation and non-activation conditions used in this assay.

Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro and in vivo studies performed on the substance itself or on supporting substances were negative and of high quality.

Short description of key information:
- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- L5178Y/MLA Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): non mutagenic.
- Human lymphocytes chromosome aberration test (OECD 473, GLP, Read-across, K, rel. 2): non clastogenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).