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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1) Ames Test (OECD 471, GLP) performed with the reference substance: non mutagenic up to the cytotoxic concentrations in S. typhimurium strains TA 1535, TA 1537, TA1538, TA 98 and TA 100.

2) Ames Test (performed following the Japan Guidelines for Screening Mutagenicity Testing Of Chemicals, equivalent to OECD 471, under GLP conditions) with a similar substance (2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol, CAS 28219-61-6): non mutagenic up to the cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA100 and in E. coli WP2 uvr A.

Read-Across:

- Chromosome aberration: negative

(Read across from substance 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol, CAS 28219-61-6)

- Mouse lymphoma assay: negative

(Read across from substance (2E)-2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-buten-1-ol, CAS 106185-75-5)

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:
experimental study
Adequacy of study:
key study
Study period:
from 13th of August to 28th of November, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test conducted in GLP according to the version of the OECD guideline 471 applicable then. However under the currently version of the guideline it is also required to include Salmonella typhimurium TA102 and/or Escherichia coli WP2 or E.coli WP2 (pKM101)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1st experiment: 8; 40; 200; 1000 and 5000 µg/plate
2nd experiment: 5, 10, 20, 50 and 100 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and the untreated fresh cell suspensions served as negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylendiamine
Remarks:
As positive controls, sodium azide was used in the tester strains TA 100 and TA 1535, 9-aminoacridine in the tester strain TA 1537, and 4-nitro-o-phenylendiamine in the tester strains TA 98 and TA 1538 without microsomal drug-metabolizing enzymes (S9-mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and the untreated fresh cell suspensions served as negative control
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: The enzyme activity of the Aroclor 1254 induced S9 fractions was controlled by testing with 2-aminoanthracene in all strains.
Remarks:
The enzyme activity of the S9 fractions was controlled by testing with 2-aminoanthracene in all strains.
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:
see table 1, 2 & 3
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
see table 1, 2 & 3
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item was tested in two independent test series at a concentration range of 5.0 - 5000 µg/plate (first and second test).
The test item was tested in the absence and presence of Aroclor 1254-induced S9-mix. In both test series (Tables 1 - 2) the plates were incubated for 48 hours at 37° C.
The findings show that the test item did not induce any reverse mutations in the absence of rat liver enzymes (without S9-mix), nor did occur any induced reverse mutations in the presence of Aroclor 1254-induced S9-mix (with S9-mix).

Toxic effects were noted, starting at a concentration of 40 µg/plate without S9-mix and above 200 µg/plate with S9-mix. Precipitations were not noted. The summarized results are presented in table 3.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In the described mutagenicity test and under the experimental conditions reported, the test item did not induce point mutations by base-pair changes or frameshifts in the genome of the strains used.
Therefore the test item is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

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

Therefore, the test item is not considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 20th June 2007 to 12th of July 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other:
Remarks:
no certificate of analysis of test substance; only duplicate cultures used /dose
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
test in accordance with "III Mutagenicity Test" of "Reverse-Mutation Assay in Bacteria" (notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No.2 (2003.11.13) of the METI & No. 031121002 of the MOE
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
minor deviations: only duplicate cultures used dose
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
10% S9 fraction of male SD rat liver induced with phenobarbital and 5, 6-benzoflavone and supplemented with Cofactor I® (Oriental Yeast Co., Ltd.)
Test concentrations with justification for top dose:
Range-finding test 1 (all 5 strains): 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 or 5000 µg/plate with or without S9 mix
Range-finding test 2 (all 4 Salmonella strains): 0.610, 1.22, 2.44, 4.88, 9.77 or 19.5 µg/plate without S9 mix
Main test:
- Without S9 (all 4 Salmonella strains): 0.610, 1.22, 2.44, 4.88, 9.77 or 19.5 µg/plate
- Without S9 (WP2 uvrA) or with S9 (all 5 strains): 2.44, 4.88, 9.77, 19.5, 39.1 or 78.1 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (for TA 100, TA 98 and WP2 uvrA); sodium azide (for TA 1535); 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCI (for TA 1537)
Remarks:
without metabolic activation
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:
see table 3 and figures 4 & 5
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes; in the range-finding test-1, precipitation was observed at 1250 µg/plate or more without S9 mix and at 5000 µg/plate or more with S9 mix.

RANGE-FINDING/SCREENING STUDIES:

Range-finding test-1:
- Number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control
- Precipitation was observed at 1250 µg/plate or more without S9 mix and at 5000 µg/plate or more with S9 mix.
- Bacterial growth inhibition was observed at 19.5 µg/plate or more in TA100, TA1535, TA98 and TA1537 without S9 mix, at 78.1 µg/plate or more in WP2uvrA without S9 mix and at 78.1 µg/plate or more in all test strains with S9 mix.

Range-finding test-2:
- Number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in TA100, TA1535, TA98 and TA1537 without S9 mix
- Number of revertant colonies was increased nearly twice in TA1537
- Bacterial growth inhibition was observed at 19.5 µg/plate in all test strains


COMPARISON WITH HISTORICAL CONTROL DATA: Yes; numbers of revertant colonies in the negative control and the positive controls were within the range of the historical data (December, 2006 - May, 2007)

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding test-1, complete cytotoxicity was observed at 78.1 µg/plate or more in TA100, TA1535 and TA1537 without S9 mix, at 313 µg/plate or more in TA98 without S9 mix and at 313 µg/plate or more in TA100, TA1535 and TA1537 with S9 mix
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

see attached documents

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

Therefore it was concluded that, under the test conditions, the test item is not considered mutagenic according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In this reverse gene mutation assay in bacteria, performed following a guideline similar to the OECD guideline 471 and in compliance with GLP conditions, four strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and one Escherichia coli strain (WP2 uvrA) were exposed to the test item in both the absence and presence of S9 metabolic activation.

The test substance was judged to be negative because the number of the revertant colonies in the test substance treatment groups in all test strains was less than twice that in the negative control regardeless of the presence or absence of S9 mix.

The number of the revertant colonies in the positive controls were above two times that in the negative control. The test results showed that the numbers of revertant colonies in the vehicle control and in the positive controls were within the range of the historical data at the testing facility. It was also confirmed that the test system was free from bacterial contamination, which indicates the test results to be valid.

Therefore it was concluded that, under the test conditions, the test item is not considered mutagenic according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
0. INTRODUCTION:
The analogue approach is based on it's structural similarity, with the only difference being in the terminal ethyl group of the source chemical instead of the methyl of the target chemical (as defined in this dossier section number 1). The read-across is based in the hypothesis that this ethyl group will have the same behaviour as the methyl GROUP in regard of the gene mutation towards bacteria.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read read-across is from a source substance with the same functional groups than the target but only with a different alkyl chain (ethyl instead of methyl), which is believed not to have an effect on the mutagenic properties of the substance.
The breakdown products are predicted to be the same except for the methyl group, which is not expected to have a mutagenic potential also.
The mechanisms of action both products are predicted to be the same. The physical properties are also very similar, including it’s characteristic odour which is very similar in both cases.
About the trans/cis isomerism: in the source substance such ratio is unfortunately not specified in the report (the CAS number refers to the mixture of cis & trans) and in the target substance is typically 95% trans and 1% cis. However, considering all the safety data available on similar substances, and the lack of mutagenic effect related, not for the racemic nor for its trans o cis isomers, this possible difference is also not believed to have an effect on the mutagenic potential of the substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both substances have a similar purity. In the source substance report, the purity is reported as 93.2%. In the target substance the typical purity content is 95%.

3. ANALOGUE APPROACH JUSTIFICATION
As can be seen in the data matrix detailed below, the physical properties of the target and source substances are very similar. The differences in the water solubility, vapour pressure and skin irritation potential are believed to be not significant related to the mutagenic potential towards bacteria.

4. DATA MATRIX
Appearance. Source substance: clear liquid at 20°C. Target substance: clear liquid at 20°C
Melting Point. Source substance: < - 20 °C. Target substance: < -20 °C
Boiling Point. Source substance: 279 °C. Target substance: 272 °C.
Relative density at 20°C. Source substance: 0.916 ± 0.001 Target substance: 0.921 ± 0.001
Vapour Pressure.
Source Substance: 1.71 * 10-2 Pa at 20°C; 4.24 * 10-2 Pa at 25°C.
Target Substance: 1.35* 10-01Pa at 20 °C; 2.08* 10-01Pa at °C.
Water Solubility. Source Substance: 25.83 mg/L at 20 °C. Target Substance: 59.36 at 20 °C
Flash Point: Source Substance: 140 °C. Target Substance: 126 °C
Skin Irritation Potential: Source Substance: Not Irritant Target Substance: Irritant
Eye Irritation Potential: Source Substance: Irritant Target Substance: Irritant
Acute Oral Toxicity: Source Substance: LD50 > 5000 mg/kgbw . Target Substance: LD50 > 5000 mg/kgbw
Skin Sensitization Potential: Source Substance: Not sensitizer. Target Substance: Not sensitizer
Reason / purpose:
read-across source
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The test item is not considered to induce mutations to E. coli WP2 uvr A in both the absence and presence of S9 metabolic activation.
Therefore it was concluded that, the test item is not considered mutagenic according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.5.87 - 22.12.87
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: In -vitro chromosome aberration assays
Version / remarks:
Scott,D., Danford,N., Dean,B., Kirkland,D., and Richardson,C. In: Report of the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Part 1: Basic Test Battery; Minimal Criteria; Professional Standards; Interpretation; Selection of Supplementary Assays. The United Kingdom Environmental Mutagen Society, 1983, pp 41-64.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
without metabolism - 20, 6.3, 2.0 and 0.63 µg/ml
with S9 - 46, 14.6, 4.6, and 1.46 µg/ml
When solutions of the tset item at 500mg/ml and 100mg/ml in DMSO were diluted at 1% v/v with culture medium the chandanol was seen to separate out to give cloudy suspensions. When a 10mg/ml solution of chandanol in DMSO was diluted the solution formed (100pg/ml) was only slightly cloudy and this was selected as the top dose for treatment in the initial toxicity assays.
Vehicle / solvent:
Dimethylsulphoxide
Untreated negative controls:
yes
Remarks:
Medium only; 1% v/v dimethylsulphoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Rationale for test conditions:
Provide information on the mutagenic potential of the test item in in vitro cytogenetics assays both with and without exogenous metabolism.
Evaluation criteria:
Metaphases from each culture were scored for the presence of chromosome aberrations.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed in the two top doses.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity assay:
Without S9 Mix: Observation of the cultures at the end of treatment showed toxicity in the top two concentrations (100 and 46 µg/ml) with cells rounded up and detached and these flasks were discarded. 21µg/ml showed some evidence of toxicity with some cells starting to round up. The mitotic index scores showed no reduction in mitotic index but it was noted that the number of cells present was greatly reduced.
With S9 Mix: Observation of the cultures at the end of treatment showed toxicity in the top concentration (100µg/ml) with cells rounded up and detached and this flask was discarded. Other cultures appeared normal. The mitotic index scores indicate a reduction in mitotic index to about 50% at 46µg/ml.

Cytogenic assay:
Without S9 Mix:Taking into account the results of the toxicity assays described above, the top dose for the cytogenetics assay without exogenous metabolism was set at 20µg/ml. There was no evidence to suggest that the test substance induced chromosome aberrations above the control levels.
With S9 Mix: There was no evidence of clastogenicity at any dose.

The test substance failed to induce chromosome aberrations either in the presence or the absence of a rat liver S9 microsome mix. The positive control mutagens induced cytogenetic damage as expected.
The test substance showed no evidence of clastogenic activity in this in vitro cytogenetics study. The data from this study indicate that the test substance does not present a genotoxic hazard.
Conclusions:
Cytogenetic damage is normally only detectable at concentrations which cause toxicity. Assays of mitotic index were carried out to determine the toxicity of the test item so that the top dose for treatment each cytogenetic assay could be set at about 50%.
The results of the cytogenetic assays both with and without exogenous metabolism gave no indication of clastogenic effect by the test substance at any concentration above the background level observed in the negative controls.
Use of the two positive control mutagens cyclophosphamide and methyl methanesulphonate, with and without exogenous metabolism respectively, ensured that the cytogenetics assays performed were capable of detecting mutagens.
It is concluded that under the conditions of these experiments the test substance did not exhibit mutagenic activity.
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 April 19 to June 15, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline 476 with minor deviations: 3 test concentrations were based on one replicate only as flask contents were lost during expression period and in experiment 1, no concentration gave 10-20% relative survival.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
3 test concentrations were based on one replicate only as flask contents were lost during expression period and in experiment 1, no concentration gave 10-20% relative survival.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/- locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Type and identity of media: RPMI 1640 medium
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes; each batch was purged of TK- mutants
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder experiment: 0, 12.5, 25, 50, 100, 200 and 400 μg/mL (with and without S-9)
Main study:
- Experiment 1: without S-9: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL; with S-9: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL
- Experiment 2: without S-9: 0, 10, 20, 25, 30, 35, 37.5, 40, 42.5, 45 and 50 μg/mL; with S-9: 0, 10, 20, 40, 45, 50, 55, 60, 65, 70 and 75 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was soluble in anhydrous analytical grade DMSO at concentrations up to at least 269.29 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation Migrated to IUCLID6: 2 or 3 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.10 or 0.15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 12 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Duplicates (single cultures only used for positive control treatments and range-finding experiment)

NUMBER OF CELLS EVALUATED: 20000 cells/well plated for survival, viability and 6TG resistance

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

OTHER: Cell viability was identified by eye using background illumination and counted; cell densities were determined using a coulter counter
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05)
3. The effects described above were reproducible.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
The experimental data was analysed using UKEMS recommended statistical guidelines
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The highest concentrations to give >10% Relative Survival were 25 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 66% and 61% relative survival, respectively.
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
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: No data; solubility limit in the culture medium: 84.1 to 168.3 μg/mL
- Precipitation: Yes; upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (50 to 400 µg/mL).

RANGE-FINDING/SCREENING STUDIES: Six concentrations were tested in the absence and presence of S-9 ranging from 12.5 to 400 µg/mL (limited by solubility in culture medium).
- Precipitation was observed precipitate was observed at the highest four concentrations tested in the absence and presence of S 9 (50 to 400 µg/mL).
- The highest concentrations to give >10% Relative Survival were 25 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 66% and 61% Relative Survival, respectively.
- See table 1 for more details

COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: strain/cell type: TK+/- (3.7.2C) cells
Remarks:
Migrated from field 'Test system'.

Table 1: RS values - range-finder experiment

Treatment (µg/mL)

-S-9 %RS

+S-9 %RS

0

100

100

12.5

100

112

25

66

92

50 P

0

61

100 P

NP

0

% RS: Percentage Relative Survival

P: Precipitation observed at time of treatment

NP: Not plated for viability due to excessive toxicity

Table 2: Summary of mutation data

Experiment 1: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

6.51

 

0

 

100

4.96

 

10

 

97

4.91

NS

10

 

92

3.37

NS

15

 

90

3.61

NS

20

 

78

3.89

NS

20

 

91

5.68

NS

30

 

72

5.39

NS

30

 

64

7.11

NS

40

 

52

5.15

NS

35

 

55

4.84

NS

50

P

27

2.51

NS

40

 

32

2.73

NS

60

P

32!

4.55

NS

45

 

7

4.44

NS

70

P

4

5.67

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

70

17.73

 

2

 

85

17.87

 

0.15

 

64

62.76

 

3

 

26

47.67

 

Experiment 2: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

0.74

 

0

 

100

6.83!

 

10

 

117

1.21

NS

20

 

106

1.59

NS

20

 

100

1.74

NS

40

 

89

2.56

NS

30

 

67

4.70

*

50

 

71

3.32

NS

35

 

54

0.68

NS

55

 

77

2.23

NS

37.5

 

36

1.58

NS

60

 

60

1.07!

NS

40

 

23

1.01

NS

65

P

65

0.70

NS

42.5

 

11

4.17

NS

70

P

25

2.03

NS

 

 

 

 

 

75

P

10

1.32

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

70

21.29

 

2

 

45

24.82

 

0.15

 

55

22.80

 

3

 

47

27.60

 

§: 6‑TG resistant mutants/106 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

*: Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

!: Based on one replicate only as flask contents lost during expression period

P: Precipitation observed at time of treatment

Conclusions:
Interpretation of results: negative

The test item is not considered as mutagenic in L5178Y cells and should not be classified as mutagen according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD guideline 476, in compliance with GLP, mouse lymphoma L5178Y TK+/- (3.7.2C) cells were exposed to the test item in DMSO at concentrations of 0, 12.5, 25, 50, 100, 200 and 400 μg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test.

In the main test, two experiments were performed at the following concentrations:

- Experiment 1: without S-9: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL; with S-9: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL

- Experiment 2: without S-9: 0, 10, 20, 25, 30, 35, 37.5, 40, 42.5, 45 and 50 μg/mL; with S-9: 0, 10, 20, 40, 45, 50, 55, 60, 65, 70 and 75 μg/mL.

Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). In the absence and presence of S-9 in Experiment 1 and in the presence of S-9 in Experiment 2, there were no significant increases in mutant frequency at any concentration analysed. For Experiment 2 in the absence of S-9, significant increase in mutant frequency was noted at a single intermediate concentration.

As the response is not concentration related, there is no linear trend and the response is not well reproduced, this increase was not considered as biologically relevant. Therefore, the test item is not considered as mutagenic in L5178Y cells and should not be classified as mutagen according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 19 to June 15, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline 476 with minor deviations: 3 test concentrations were based on one replicate only as flask contents were lost during expression period and in experiment 1, no concentration gave 10-20% relative survival.
Justification for type of information:
See attached justification in section 13.2
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
3 test concentrations were based on one replicate only as flask contents were lost during expression period and in experiment 1, no concentration gave 10-20% relative survival.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/- locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co. Cells
- Type and identity of media: RPMI 1640 medium
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic acid (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20% v/v), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: Yes; each batch was purged of TK- mutants
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder experiment: 0, 12.5, 25, 50, 100, 200 and 400 μg/mL (with and without S-9)
Main study:
- Experiment 1: without S-9: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL; with S-9: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL
- Experiment 2: without S-9: 0, 10, 20, 25, 30, 35, 37.5, 40, 42.5, 45 and 50 μg/mL; with S-9: 0, 10, 20, 40, 45, 50, 55, 60, 65, 70 and 75 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was soluble in anhydrous analytical grade DMSO at concentrations up to at least 269.29 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation Migrated to IUCLID6: 2 or 3 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation Migrated to IUCLID6: 0.10 or 0.15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 12 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Duplicates (single cultures only used for positive control treatments and range-finding experiment)

NUMBER OF CELLS EVALUATED: 20000 cells/well plated for survival, viability and 6TG resistance

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

OTHER: Cell viability was identified by eye using background illumination and counted; cell densities were determined using a coulter counter
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05)
3. The effects described above were reproducible.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
The experimental data was analysed using UKEMS recommended statistical guidelines
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The highest concentrations to give >10% Relative Survival were 25 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 66% and 61% relative survival, respectively.
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
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: No data; solubility limit in the culture medium: 84.1 to 168.3 μg/mL
- Precipitation: Yes; upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (50 to 400 µg/mL).

RANGE-FINDING/SCREENING STUDIES: Six concentrations were tested in the absence and presence of S-9 ranging from 12.5 to 400 µg/mL (limited by solubility in culture medium).
- Precipitation was observed precipitate was observed at the highest four concentrations tested in the absence and presence of S 9 (50 to 400 µg/mL).
- The highest concentrations to give >10% Relative Survival were 25 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 66% and 61% Relative Survival, respectively.
- See table 1 for more details

COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: strain/cell type: TK+/- (3.7.2C) cells
Remarks:
Migrated from field 'Test system'.

Table 1: RS values - range-finder experiment

Treatment (µg/mL)

-S-9 %RS

+S-9 %RS

0

100

100

12.5

100

112

25

66

92

50 P

0

61

100 P

NP

0

% RS: Percentage Relative Survival

P: Precipitation observed at time of treatment

NP: Not plated for viability due to excessive toxicity

Table 2: Summary of mutation data

Experiment 1: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

6.51

 

0

 

100

4.96

 

10

 

97

4.91

NS

10

 

92

3.37

NS

15

 

90

3.61

NS

20

 

78

3.89

NS

20

 

91

5.68

NS

30

 

72

5.39

NS

30

 

64

7.11

NS

40

 

52

5.15

NS

35

 

55

4.84

NS

50

P

27

2.51

NS

40

 

32

2.73

NS

60

P

32!

4.55

NS

45

 

7

4.44

NS

70

P

4

5.67

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

70

17.73

 

2

 

85

17.87

 

0.15

 

64

62.76

 

3

 

26

47.67

 

Experiment 2: (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RS

MF§

 

%RS

MF§

0

 

100

0.74

 

0

 

100

6.83!

 

10

 

117

1.21

NS

20

 

106

1.59

NS

20

 

100

1.74

NS

40

 

89

2.56

NS

30

 

67

4.70

*

50

 

71

3.32

NS

35

 

54

0.68

NS

55

 

77

2.23

NS

37.5

 

36

1.58

NS

60

 

60

1.07!

NS

40

 

23

1.01

NS

65

P

65

0.70

NS

42.5

 

11

4.17

NS

70

P

25

2.03

NS

 

 

 

 

 

75

P

10

1.32

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

70

21.29

 

2

 

45

24.82

 

0.15

 

55

22.80

 

3

 

47

27.60

 

§: 6‑TG resistant mutants/106 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

*: Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

!: Based on one replicate only as flask contents lost during expression period

P: Precipitation observed at time of treatment

Conclusions:
Interpretation of results: negative

The test item is not considered as mutagenic in L5178Y cells and should not be classified as mutagen according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD guideline 476, in compliance with GLP, mouse lymphoma L5178Y TK+/- (3.7.2C) cells were exposed to the test item in DMSO at concentrations of 0, 12.5, 25, 50, 100, 200 and 400 μg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test.

In the main test, two experiments were performed at the following concentrations:

- Experiment 1: without S-9: 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μg/mL; with S-9: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL

- Experiment 2: without S-9: 0, 10, 20, 25, 30, 35, 37.5, 40, 42.5, 45 and 50 μg/mL; with S-9: 0, 10, 20, 40, 45, 50, 55, 60, 65, 70 and 75 μg/mL.

Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). In the absence and presence of S-9 in Experiment 1 and in the presence of S-9 in Experiment 2, there were no significant increases in mutant frequency at any concentration analysed. For Experiment 2 in the absence of S-9, significant increase in mutant frequency was noted at a single intermediate concentration.

As the response is not concentration related, there is no linear trend and the response is not well reproduced, this increase was not considered as biologically relevant. Therefore, the test item is not considered as mutagenic in L5178Y cells and should not be classified as mutagen according to Directive 67/548/EEC and CLP Regulation (EC) n° 1272/2008.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11.5.87 - 22.12.87
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See attached justification in section 13.2
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: In -vitro chromosome aberration assays
Version / remarks:
Scott,D., Danford,N., Dean,B., Kirkland,D., and Richardson,C. In: Report of the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Part 1: Basic Test Battery; Minimal Criteria; Professional Standards; Interpretation; Selection of Supplementary Assays. The United Kingdom Environmental Mutagen Society, 1983, pp 41-64.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
without metabolism - 20, 6.3, 2.0 and 0.63 µg/ml
with S9 - 46, 14.6, 4.6, and 1.46 µg/ml
When solutions of the tset item at 500mg/ml and 100mg/ml in DMSO were diluted at 1% v/v with culture medium the chandanol was seen to separate out to give cloudy suspensions. When a 10mg/ml solution of chandanol in DMSO was diluted the solution formed (100pg/ml) was only slightly cloudy and this was selected as the top dose for treatment in the initial toxicity assays.
Vehicle / solvent:
Dimethylsulphoxide
Untreated negative controls:
yes
Remarks:
Medium only; 1% v/v dimethylsulphoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Rationale for test conditions:
Provide information on the mutagenic potential of the test item in in vitro cytogenetics assays both with and without exogenous metabolism.
Evaluation criteria:
Metaphases from each culture were scored for the presence of chromosome aberrations.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed in the two top doses.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity assay:
Without S9 Mix: Observation of the cultures at the end of treatment showed toxicity in the top two concentrations (100 and 46 µg/ml) with cells rounded up and detached and these flasks were discarded. 21µg/ml showed some evidence of toxicity with some cells starting to round up. The mitotic index scores showed no reduction in mitotic index but it was noted that the number of cells present was greatly reduced.
With S9 Mix: Observation of the cultures at the end of treatment showed toxicity in the top concentration (100µg/ml) with cells rounded up and detached and this flask was discarded. Other cultures appeared normal. The mitotic index scores indicate a reduction in mitotic index to about 50% at 46µg/ml.

Cytogenic assay:
Without S9 Mix:Taking into account the results of the toxicity assays described above, the top dose for the cytogenetics assay without exogenous metabolism was set at 20µg/ml. There was no evidence to suggest that the test substance induced chromosome aberrations above the control levels.
With S9 Mix: There was no evidence of clastogenicity at any dose.

The test substance failed to induce chromosome aberrations either in the presence or the absence of a rat liver S9 microsome mix. The positive control mutagens induced cytogenetic damage as expected.
The test substance showed no evidence of clastogenic activity in this in vitro cytogenetics study. The data from this study indicate that the test substance does not present a genotoxic hazard.
Conclusions:
Cytogenetic damage is normally only detectable at concentrations which cause toxicity. Assays of mitotic index were carried out to determine the toxicity of the test item so that the top dose for treatment each cytogenetic assay could be set at about 50%.
The results of the cytogenetic assays both with and without exogenous metabolism gave no indication of clastogenic effect by the test substance at any concentration above the background level observed in the negative controls.
Use of the two positive control mutagens cyclophosphamide and methyl methanesulphonate, with and without exogenous metabolism respectively, ensured that the cytogenetics assays performed were capable of detecting mutagens.
It is concluded that under the conditions of these experiments the test substance did not exhibit mutagenic activity.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an Ames test in the same substance as defined under section 1 and performed under GLP conditions, the test item did not show in evidence of mutagenic activity in the strains S. typhimurium TA1535, TA1537, TA1538 TA 98 and TA 100 with or without metabolic activation.

A second Ames has been included to assess the effects on all the required strains in the current guideline. In this case the test was performed in a similar substance, in the S. typhimurium strains TA 1535, TA 1537, TA 98, TA100 and in E. coli WP2 uvr A. In this fully valid test the substance tested did not show either mutagenic activity.

The source substance has the same functional groups than the target but only with a different alkyl chain, which is believed not to have an effect on the mutagenic properties of the substance.

In an in vitro mammalian cell gene mutation test performed on a similar substance according to OECD guideline 476, the substance did not show any mutagenic activity in mouse lymphoma L5178Y TK+/- (3.7.2C) cells.

In an in vitro chromosome aberration study, conducted on a similar substance in Chinese hamster ovary cells, no cytogenic activity was observed.

Therefore it was concluded that the substance under registration as defined in section 1 is not considered mutagenic according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

 

Justification for classification or non-classification

Based on negative results in the in-vitro studies conducted, the substance does not need to be classified for germ cell mutagenicity.