Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not considered to be mutagenic based on the results of the bacterial reverse mutation assays performed with two structural analogue substances.

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:
Apr 28 - Jul 07, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
HIS operon (S. thyphimurium)
TRP operon (E. coli)
Species / strain:
S. typhimurium TA 1535
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 1537
Details on mammalian cell lines (if applicable):
his C 3076, uvrB, rfa
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 98
Details on mammalian cell lines (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 100
Details on mammalian cell lines (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
S. typhimurium TA 102
Details on mammalian cell lines (if applicable):
his G 428, rfa + R-factor
Additional strain characteristics:
other: mutations in the histidine operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Species / strain:
E. coli WP2
Details on mammalian cell lines (if applicable):
uvrA pkM101
Additional strain characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 1.58, 5, 15.8, 50, and 158 µg/plate (S9 10 %)
1. Series: 1.58, 5, 15.8, 50, and 158 µg/plate (S9 30 %)
Vehicle:
acetone
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
cumene hydroperoxide
other: daunomycin
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and conditions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment: No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".

Interpretations:
A test material is defined as non-mutagenic in this assay if:

- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:

- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Evaluation criteria:
details see results
Statistics:
n.a.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
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:
Feb 05, 2003 - Jul 21, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Salmonella: histidine operon
E coli. tryptophane operon
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 pretreated rats
Species / strain:
S. typhimurium TA 102
Details on mammalian cell lines (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 pretreated rats
Test concentrations with justification for top dose:
1st series: 0.5, 1.58, 5.00, 15.8, 50.0, 158, and 500 µg/plate
2nd series: 5.00, 8.89, 15.8, 50.0, 88.9 µg/plate
Vehicle:
acetone
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: Sodium azide, 2-Aminoanthracene, Cumene hydroperoxide, 9-Aminoacridine, Daunomycin, 4-Nitroquinoline-N-oxide
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 3-5 hours
- Exposure duration: about 2 days


SELECTION AGENT (mutation assays): Histidine, Tryptophane

NUMBER OF REPLICATIONS:
Negative controls 6
Test material 3
Positive controls 3


NUMBER OF CELLS EVALUATED: about 1e9


DETERMINATION OF CYTOTOXICITY
- Method: other: background cytotox
Rationale for test conditions:
According to guideline
Evaluation criteria:
- valid assay (cf. laboratory historical data)
- no or weak increase in revertant colonies = negative
- clear, dose dependent (over at least two concentrations), and reproducible increase in revertant colonies= positve
Statistics:
not applied
Key result
Species / strain:
other: all strains/cell types tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: ok
- Precipitation:yes ( ≥ 50 µg/plate)
- Other confounding effects: no

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 471. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For this endpoint information from two structural similar compounds (1-Ethoxy-2,3-difluoro-4-[trans-4-(trans-4-propylcyclohexyl)-cyclohexyl]-benzene and 2,3-difluoro-1-methoxy-4-[(trans,trans)-4'-propyl[1,1'-bicyclohexyl]-4-yl]-benzene) are available. This study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 471. See chapter 13 report for a more detailed justification.
Reason / purpose:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Species / strain:
other: all strains tested for the source compounds
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Conclusions:
Read Across: negative, not genotoxic
Executive summary:

For this endpoint information from two structural similar compounds (1-Ethoxy-2,3-difluoro-4-[trans-4-(trans-4-propylcyclohexyl)-cyclohexyl]-benzene and 2,3-difluoro-1-methoxy-4-[(trans,trans)-4'-propyl[1,1'-bicyclohexyl]-4-yl]-benzene) are available. The studies for these similar compounds were performed according to GLP and the methods applied are fully compliant with OECD TG 471. See chapter 13 report for a more detailed justification.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For this endpoint reliable and adequate bacterial reverse mutations studies according to OECD Guideline and in compliant with GLP from two similar structural compounds are available. The results did not reveal any mutagenic potential of the structural analogue substances in any strains tested.

Justification for classification or non-classification

The test item is not considered to be mutagenic according to the Regulation (EC) No 1272/2008.