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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance underwent testing for mutagenicity potential in the Ames test employing Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia coli strain WP2uvrA, both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. In a genotoxicity study cultured human lymphocytes exposed to the substance at 52μg/ml was not cytotoxic or clastogenic. The substance was also found to be negative in the gene mutation/clastogenicity test using mouse lymphoma cells (L5178Y), both in the presence and absence of S9 -metabolic activation.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28th April 2016 to 26th May 2016
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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) from Aroclor 1254 treated rats.
Test concentrations with justification for top dose:
Dose range finding test was conducted with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix employing strains TA100 and WP2uvrA.

The highest concentration of the test item used in the subsequent mutation assays was the level at which the test item exhibited limited solubility.

Doses used for the final direct plate assay were 17, 52, 164, 512 and 1600 μg/plate employing TA1535, TA1537 and TA98 in the absence and presence of S9-mix.

Doses used for the final pre-incubation assay were 17, 52, 164, 512 and 1600 μg/plate employing TA1535, TA1537, TA98, TA100 and WP2uvrA in the absence and presence of S9-mix.
Vehicle:
Hexane.
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR - 191; 2-Aminoanthracene
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Pre-incubation assay only
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Pre-incubation assay only
Conclusions:
Based on the results of this study it is concluded that 4,4’-(1-phenylethane-1,1-diyl)bis(heptyloxybenzene) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 May 2016 - 06 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
In the second cytogenetic assay in one of the cultures of the positive control (24 h exposure) only 98 metaphases were examined for chromosome aberrations. The study integrity was not adversely affected by the deviation.
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells
Test material information:
Composition 1
Species / strain:
lymphocytes:
Details on mammalian cell lines (if applicable):
Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 homogenate was obtained from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight; dosing on 4 consecutive days) and ß-naphthoflavone (100 mg/kg; single dosing).
Test concentrations with justification for top dose:
In the first cytogenetic assay, the substance was tested up to 52 μg/ml for a 3 h exposure time.
In the second cytogenetic assay, the substance was again tested up to 52 μg/ml for a 24 and 48h continuous exposure time.
The highest tested concentration was determined by the solubility and >52 μg/ml resulted in precipitation.
Vehicle:
Hexane
Negative controls:
yes
Remarks:
Culture medium
Solvent controls:
yes
Remarks:
Hexane
True negative controls:
not specified
Positive controls:
yes
Remarks:
Mitomycin C was used as a direct acting mutagen. Cyclophosphamide was used as an indirect acting mutagen, requiring metabolic activation. The solvent for positive control was Hanks' Balanced Salt Solution.
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) All results are inside the 95% control limits of the negative historical control data range.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: The substance is not clastogenic in human lymphocytes.
Conclusions:
The substance is not clastogenic in human lymphocytes.
Executive summary:

In a genotoxicity study cultured human lymphocytes were exposed to the substance at 52 μg/ml. This was the concentration at which the substance was found to be soluble in the solvent. The results indicates that the substance was not cytotoxic or clastogenic at the concentration used.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 September 2016 - 28 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Test material information:
Composition 1
Species / strain:
mouse lymphoma L5178Y cells
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
First experiment - up to 300 µg/ml
Second experiment - up to 150 µg/ml (precipitation observed at 150-300 µg/ml in the first experiment.
Vehicle:
Hexane.
Negative controls:
no
Solvent controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10-6).
Statistics:
A test item 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 item 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 item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Conclusions:
It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.
Executive summary:

The substance was evaluated for its mutagenic potential ) in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period (rat liver S9-mix was induced by a combination of phenobarbital and ß-naphthoflavone).  The study procedures followed the most recent OECD and EC guidelines. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that the substance is not considered mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the findings of the Ames test (bacterial reverse mutation study), the mouse lymphoma assay and the in vitro chromosome aberration study conducted on the substance, classification of the substance is not justified.