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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation assay in bacteria (AMES test) is in progress and expected to be negative.

Gene mutation assays in mammalian cells (HPRT test): negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
NR deficient strains
Principles of method if other than guideline:
The traditional strains used for OECD 471 will be checked in parallel with the same strains deficient in the nitro-reductasi enzyme (present only in bacteria) as to avoid the NO2 group reduction present in the test item. Reduction of the nitro groups in fact produce in the substance aromatic amines that typically give false positive for the traditional tested strains.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
nitroreductase deficient
Remarks:
TA98NR, TA100 NR
Metabolic activation:
with and without
Metabolic activation system:
reductive (Prival) metabolic activation system
Test concentrations with justification for top dose:
Highest dose tested: 5000 μg/plate unless limited by cytotoxicity or solubility
Species / strain:
other: all strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: expert assessment
Adequacy of study:
supporting study
Reliability:
other: not applicable
Qualifier:
no guideline required
Principles of method if other than guideline:
epxert statement
GLP compliance:
no
Type of assay:
other: expert statement
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
in addition to traditional baceria strain, nitro-reductase deficinet strains for TA98 an TA100 are used, namely TA98NR and TA100NR
Metabolic activation:
with and without
Metabolic activation system:
uninduced rat hamster liver S9 mix
Test concentrations with justification for top dose:
0,5,16,50,150, 500, 1600 and 5000 micro/plate
Vehicle / solvent:
water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Species / strain:
other: refer to attached expert statament
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Based on the expert statament, the substance is not considerd as mutagenic in gene mutation studieis on bacteria
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From September 17 to November 10, 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
In Main Assay II, the mutant frequency was evaluated at day 9, instead of day 8. This deviation was not considered to have affected the integrity of the study.
Qualifier:
according to guideline
Guideline:
other: Test method B.17 ‘in vitro mammalian cell gene mutation test’ described in Council Regulation (EC) No. 440/2008.
Deviations:
yes
Remarks:
In Main Assay II, the mutant frequency was evaluated at day 9, instead of day 8. This deviation was not considered to have affected the integrity of the study.
GLP compliance:
yes
Type of assay:
bacterial forward mutation assay
Target gene:
enzyme hypoxanthine-guanin phosphoribosyl-transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (from Sprague-Dawley rat liver)
Test concentrations with justification for top dose:
600, 300, 150, 75.0, 37.5, 18.8, 9.38, 4.69 and 2.34 µg/ml (exposure of 3 h).
(By the end of treatment, precipitation of the test substance was noted starting from 75.0 µg/ml in the absence of S9 metabolism and at the three highest dose levels in its presence. Opacity of the treatment medium was observed starting from 18.8 µg/ml, in the absence of S9 metabolism. Selection of dose levels used in Main Assay I was performed taking into account precipitation and toxicity observed in the preliminary cytotoxicity assay. The dose range used in Main Assay II was modified to focus on the highest concentrations that could be tested. The highest concentrations were finally: 120 or 150 and 300 µg/ml, in the absence and presence of S9 metabolism, respectively).
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
- Cytotoxicity assay (preliminary test - large range of concentrations)
Treatments were performed both with or without S9 metabolism; a single culture was used at each test point and positive controls were not included.
- Mutation assay (determination of survival and determination of mutant frequency)
Two experiments were performed including negative and positive controls, with and without S9 metabolic activation. Duplicate cultures were prepared at each test point, with the exception of the positive controls, which were prepared in a single culture.
Evaluation criteria:
For a test substance to be considered mutagenic in this assay, it is required that:
- there is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test substance. If only the highest practicable dose level (or the highest dose level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
- there must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).
Statistics:
- Analysis of variance in which the effect of replicate culture and dose level in explaining the observed variation was examined. For each experiment, the individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = (x + a)b where a = 0 and b = 0.275. A two way analysis of variance was performed (without interaction) fitting to two factors:
- replicate culture: to identify differences between the replicate cultures treated.
- dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained with and without S9 metabolism.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 300 µg/ml (experiments I and II)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 37.5 µg/ml (experiment I) and at and above 60 µg/ml (experiment II)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Survival:
In Main Assay I, without S9 metabolism, moderate reduction in relative survival (RS = 18-19 %) was noted at the two highest dose levels (150.0 and 75.0 µg/ml), treatment at 37.5 µg/ml yielded a reduction of relative survival to 36 % of the negative control, while no relevant toxicity was noted over the remaining concentrations tested. With S9 metabolism, slight reduction in relative survival (RS = 70 %) was noted at the highest dose level (300 µg/ml), while no relevant toxicity was observed over the remaining concentrations tested. In Main Assay II, in the absence of S9 metabolism, treatment at the highest dose (120 µg/ml) level yielded a reduction of relative survival to 11 % of the negative control, moderate toxicity (RS = 55%) was noted at the next lower concentration of 60.0 µg/ml, while no relevant toxicity was observed over the remaining concentrations tested. With S9 metabolism, moderate toxicity (RS = 55 %) was noted at the highest dose level (300 µg/ml), while no relevant toxicity was observed over the remaining concentrations tested. Opacity of the treatment medium was noted starting from 30.0 µg/ml, in the absence of S9 metabolism. Opacity and slight precipitation were observed at the highest dose level, both with and without S9 metabolism.
- Mutation results:
no five-fold or greater increase in mutant frequency, compared with the negative control, was observed at the highest dose level or at two consecutive doses of the test substance, with or without S9 metabolic activation. No reproducible evidence of a dose effect relationship was noticed. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system.
- Osmolality and pH measurements: addition of test substance solution did not have any obvious effect on osmolality or pH of the treatment medium.

.

Conclusions:
Under the study conditions, test substance did not induce gene mutation in Chinese hamster V79 cells (HPRT).
Executive summary:

Method

A study was conducted to determine the genetic toxicity in vitro of test substance according to OECD Guideline 476 and EU method B.17.

In an in vitro mammalian cell gene mutation test (forward gene mutation in HPRT), Chinese hamster V79 cells were exposed to the test substance at concentrations ranging from 2.34 to 300.0 µg/ml with or without metabolic activation (S9 mix).

Results

Positive and negative controls were valid. In Main Assay I, without S9 mix, moderate reduction in relative survival (RS = 18 - 19 %) was noted at the two highest dose levels (150.0 and 75.0 µg/ml), treatment at 37.5 µg/ml yielded a reduction of relative survival to 36 % of negative control, while no relevant toxicity was noted over the remaining concentrations tested. With S9 mix, slight reduction in relative survival (RS = 70 %) was noted at the highest dose level (300 µg/ml), while no relevant toxicity was observed over the remaining concentrations tested. In Main Assay II, without S9 mix, treatment at the highest dose (120 µg/ml) level yielded a reduction of relative survival to 11 % of negative control, moderate toxicity (RS = 55 %) was noted at the next lower concentration of 60.0 µg/ml, while no relevant toxicity was observed over the remaining concentrations tested. With S9 mix, moderate toxicity (RS = 55 %) was noted at the highest dose level (300 µg/ml), while no relevant toxicity was observed over the remaining concentrations tested. Opacity of the treatment medium was noted starting from 30.0 µg/ml, without S9 mix. Opacity and slight precipitation were observed at the highest dose level, both with and without S9 mix. No five-fold or greater increase in mutant frequency, compared with negative control, was observed at the highest dose level or at two consecutive doses of test substance, both with and without S9 mix. No reproducible evidence of a dose effect relationship was noticed. Under the study conditions, test substance did not induce gene mutation in Chinese hamster V79 cells both with and without S9 metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From December 16, 1997 to March 26, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
numbers of mutant colonies but no detrimental impact
Qualifier:
according to guideline
Guideline:
other: EPA; 40 CFR; Ch.I; Part 798; Detection of gene mutation in somatic cells in culture; pp. 717720 (7-1-86 Edition)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian cell gene mutation tests, forward gene mutation
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT enzyme)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
mammalian microsomal fraction S9 mix (liver) (50 µl/ml)
Test concentrations with justification for top dose:
- At least four concentration levels were tested. These concentration levels should yield a concentration related toxic effect. The highest concentration level should induce a reduced level of survival.
Two independent experiments were run:
- experiment I:
without S9 mix: 3.0; 5.0; 10.0; 25.0; and 50.0 µg/ml
with S9 mix: 3.0; 5.0; 10.0; 25.0; and 50.0 µg/ml
- experiment II:
without S9 mix: 0.5*; 1.0; 3.0; 5.0; and 10.0 µg/ml
with S9 mix: 3.0; 5.0; 10.0; and 50.0 µg/ml
The limit of solubility was 10 µg/ml in both experiments as indicated by a slight perturbation. Visible precipitation occurred above this concentration.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
cells were cultivated without interruption
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
cells cultivated the same without treatment
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
The assay was performed in two independent experiments with and without liver microsomal activation. Treatment duration was 4 h in the first experiment and in the second experiment with metabolic activation. In the second experiment without metabolic activation, treatment duration with test substance was extended to 24 h.
Rationale for test conditions:
V79 cells: high proliferation rate, good cloning efficiency of negative control cells, and stable karyotype with a modal chromosome number of 22.
Evaluation criteria:
A test substance is classified as positive if it induces either a concentration-related increase of mutant frequency or a reproducible and positive response at one of test points. A test substance producing neither a concentration-related increase of mutant frequency nor a reproducible positive response at any of test points is considered non-mutagenic in this system.
A significant response is described as follows: a test substance is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
Test substance is classified as mutagenic if there is a reproducible concentration-related increase of mutation frequency. Such evaluation may be considered also in the case that a threefold increase of mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5-33.1 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed.
Statistics:
Stained (with 10 % methylene blue in 0.01 % KOH solution) colonies with more than 50 cells were counted.
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only at maximal concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Cytotoxicity
Strong toxic effects occurred in the first experiment in both cultures at the maximal concentration without metabolic activation. Only minor toxic effects were observed up to the maximal concentration with metabolic activation. In the second experiment strong toxic effects occurred only in one out of two parallel cultures at the maximal concentration without metabolic activation. This deviation between parallel cultures is probably based upon erratic toxic effects caused by precipitation during long term exposure (24 h).

- Genotoxicity
No relevant and reproducible increase in mutant colony numbers was observed up to the highest investigated concentration, neither in the presence nor in the absence of metabolic activation.
Taking into account mutation rates found in groups treated with test substance compared to negative and solvent controls, it can be concluded that no relevant increase of gene mutations was observed. Test substance did not induce a reproducible concentration-related increase in mutant colony numbers. Mutant values of groups treated with test substance remained in the range of historical negative controls. The highest value of mutant colonies (30.0 colonies per 10^6 cells) occurred at the lowest concentration in culture II in the second experiment with metabolic activation. This increase was considered as biologically irrelevant since it was not reproduced in the parallel culture in experiment II nor in both cultures of experiment I under identical conditions. In this study in both experiments (with and without S9 mix) the range of the negative controls was from 2.5 up to 22.0 mutants per 10^6 cells; the range of groups treated with test article was from 3.0 up to 30.0 mutants per 10^6 cells. EMS (0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.
Conclusions:
Not mutagenic.
Executive summary:

Method

An in vitro mammalian cell gene mutation test (forward gene mutation HPRT) was run according to OECD guideline 476. Chinese hamster V79 cells were exposed to test substance at concentrations ranging from 0.5 to 50.0 µg/plate with or without metabolic activation (S9 mix). The assay was performed in two independent experiments with and without liver microsomal activation. Treatment duration was 4 h in the first experiment and in the second experiment with metabolic activation. In the second experiment without metabolic activation, treatment duration was extended to 24 h. Positive and negative controls were valid.

Results

Strong cytotoxic effects occurred in the first experiment in both cultures at the maximal concentration without metabolic activation. Only minor toxic effects were observed up to the maximal concentration with metabolic activation.

In the second experiment strong cytotoxic effects occurred only in one out of two parallel cultures at the maximal concentration without metabolic activation. No relevant and reproducible increase in mutant colony numbers was observed up to the highest investigated concentration, neither in the presence nor in the absence of metabolic activation. Under study conditions, test substance did not induce gene mutations at the HPRT locus in V79 cells and thus was not considered as genotoxic.

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

Genetic toxicity in vivo

Description of key information

Micronucleus assay in bone marrow cells of mouse: negative.

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

Additional information

Evaluation of available information on genotoxicity was done as reported in ECHA Guidance Chapter R.7a: Endpoint specific guidance, Version 5.0 – December 2016.

A preliminary assessment should normally include data from a gene mutation test in bacteria unless existing data for analogous substances indicates this would be inappropriate.

When the result of the bacterial test is positive, it is important to consider the possibility of the substance being genotoxic in mammalian cells.

In order to ensure the necessary minimum level of information is provided, at least a further test is required in addition to the gene mutation test in bacteria. This should be an in vitro mammalian cell test capable of detecting both structural and numerical chromosome aberrations. Suitable options are in vitro chromosome aberration test (OECD guideline 473), i.e. a cytogenetic assay for structural chromosome aberrations, or in vitro micronucleus test (OECD guideline 487), i.e. a cytogenetic assay to detect not only structural chromosomal aberrations but also aneuploidy. It is possible to present data from an in vivo cytogenetic test as an alternative to the first in vitro mammalian cell test. For instance, if an adequately performed in vivo micronucleus test is available, it may be presented as an alternative.

An in vitro gene mutation study in mammalian cells (OECD guideline 476) is taken into account to complete the assessement.

In general, substances that are:

- positive in the gene mutation test in bacteria,

- negative in in vitro or in vivo tests to measure chromosomal damage, e.g. chromosome aberration or micronucleus assays

- negative in in vitro or in vivo gene mutation tests, e.g. HPRT assay

may be considered as non-genotoxic.

A modified AMES test with nitroreductase deficient strains is in progress on the target substance. The genotoxic potential of target substance was assessed also based on data on Similar Substance 01. Available studies, namely in vitro HPRT assay and in vivo micronucleus assay, examined the potential of test substance to induce in vitro gene mutations, both in bacteria and in mammalian cells, as well as in vivo chromosomal aberrations. Available data was considered as sufficient to draw a conclusion on genotoxicity of target substance.

HPRT assays were conducted on Similar Substance 01 following OECD guideline 476.

In the first assay (1998), a pre-test on cytotoxicity was run to select the concentrations. Two experiments were run in the main assay using V79 cells of Chinese hamster. In particular:

- exp. I with and without metabolic activation, 4 h exposure to concentrations of 3, 5, 10, 25 and 50 µg/ml;

- exp. II with metabolic activation, 4 h exposure to concentrations of 3, 5, 10 and 50 µg/ml; without metabolic activation, 24 h exposure at concentrations of 1, 3, 5 and 10 µg/ml.

Toxicity was evaluated in terms of cloning efficiency. Strong toxic effects were noted in exp. I without metabolic activation at the highest concentration adopted. Only minor toxic effects were seen up to the maximal concentration in the presence of metabolic activation. In exp. II, toxic effects were seen in one out of two parallell cultures at the highest concentration without metabolic activation. Mutagenicity was evaluated by considering the number of mutant colonies.

In the second assay (2016), dose levels were selected based on precipitation and toxic effects recorded in a preliminary cytotoxicity assay. Two assays were run in duplicate using chinese hamster lung fibroblasts (V79):

- main assay I, with and without metabolic activation, 3 h exposure to dose levels of 2.34, 4.69, 9.38, 18.8, 37.5, 75, 150, 300 and 600 µg/ml. Precipitation and opacity were noted, depending on the concentration.

- main assay II, 3 h exposure to concentrations of 120 or 150 and 300 µg/ml without and with metabolic activation.

Positive and negative (solvent DMSO) controls were used.

Cytotoxic effects were noted at 300 µg/ml with metabolic activation and starting at 37.5 µg/ml without metabolic activation. Mutagenic effects, in terms of increase in mutant frequency, were not noted.

A micronucleus assay in bone marrow cells of mouse was conducted using Similar Substance 01 according to OECD guideline 474.

Test substance was administered orally by gavage. Males were dosed for 2 consecutive weeks prior to pairing, through the mating period up to the day before necropsy (day 38 - 39); females were doses 2 consecutive weeks prior to pairing, during pairing, up to day 3 post partum. Doses of 62.5, 250, 500 and 1000/500 mg/kgt bw/d were used. Vehicle control and positive controls were used.

Bone marrow cells of the mouse were collected after 24 h and 48 h for micronucleus analysis. Ten animals (5/sex) per test group were evaluated. For each animal, 4 slides were prepared. 4000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei; number of normal and normochromatic erythrocytes (NCEs) was also recorded. Cytotoxicity was estimated by the ratio of polychromatic (PCE) and normochromatic erythrocytes (NCE) in the same sample; a reduction of the ratio is indicative of inhibition of cell division. The incidence of micronucleated PCEs provides an index of induced genetic damage.

Under the study conditions, no relevant inhibitory effect of erythropoietic cell division was seen at any dose level. Therefore, test substance is considered to be non-genotoxic in rats.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), Annex I, Part 3, substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans are classified in Category 2. This classification is based on positive evidence obtained in:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

In vitro mutagenicity tests are the following:

in vitro mammalian chromosome aberration test;

in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

The overall assessement on the genotoxic potential was based on: expected negative outcome in bacterial reverse mutation assay (AMES test), negative outcomes in in vitro HPRT assays and negative result in in vivo micronucleus test in bone marrow cells of mouse.

All studies were conducted according to OECD guidelines and have a high reliability.

Based on these results, target susbstance was considered as non genotoxic and it was not classified within the CLP Regulation (EC 1272/2008).