Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: Mutagenic

Gene Mutation: Not mutagenic

Chromosomal Aberration test: Non mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 13 to September 07, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted on a similar substance. Further details were reported in the attachment at section 13.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No E. Coli strain used; no statistical analysis
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
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:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver microsomal fraction
Test concentrations with justification for top dose:
Experiment I (TA 98 and TA 100): 3.00, 10.00, 33.00, 100.00, 333.00, and 1000.00 µg/plate
Experiment I (TA 1535, TA 1537, and TA 102): 1.00, 3.00,10.00, 33.00, 100.00, and 333.00 µg/plate
Experiment II
TA 1535 (without S9-mix): 3.00, 10.00, 33.00, 100.00, 150.00, and 200.00 µg/plate
TA 1535 (with S9-mix): 1.00, 3.00, 33.00, 100.00, and 333.00 µg/plate
TA 1537 (without S9-mix): 0.25, 0.50, 1.00, 5.00, 10.00, and 15.00 µg/plate
TA 1537 (with S9-mix): 1.00, 5.00, 10.00, 20.00, 30.00, and 40.00 µg/plate
TA 98 (without S9-mix): 1.25, 2.50, 5.00, 10.00, 20.00, and 30.00 µg/plate
TA 98 (with S9-mix): 0.30, 1.00, 3.00, 10.00, 33.00, and 100.00 µg/plate
TA 100, TA 102: 1.00, 3.00, 10.00, 33.00, 100.00, and 333.00 µg/plate
Untreated negative controls:
yes
Remarks:
Solvent
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -aminoanthracene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
Solvent
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Details on test system and experimental conditions:
The bacterial strains TA 1535, TA 1537, TA 100, and TA 102 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64292 Darmstadt)

The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes an inactivation of the excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB-mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.

Evaluation criteria:
A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice in strains TA 1535 and TA 1537 (3, 4).
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the criteria described above or not.
Statistics:
A statistical analysis of the data was not required
Species / strain:
other: TA 98 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA 100, TA 102, TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The plates incubated with the test item showed irregular background growth at 100 µg/plate and above with and without S9 mix in neariy all strains used.

In both experiments, a dose dependent and substantial increase in revertant colony numbers was observed following treatment with the test substance in strains TA 1537 and TA 98 with and without metabolic activation. In experiment (I), the threshold of thrice the number of the corresponding solvent control was exceeded in strain TA 1537 at 3 µg/plate without S9 mix and 10 µg/plate with S9 mix. In strain TA 98, the threshold of twice was exceeded at 10 µg/plate without S9 mix and at 33 µg/plate with S9 mix. In experiment (II), an increase in revertant colony numbers was observed in strain TA 1537 without metabolic activation, however, the required threshold of thrice was not reached. In the presence of metabolic activation the threshold was exceeded at 5 µg/plate. In strain TA 98 the number of revertant colonies exceeded the threshold of twice at 1.25 µg/plate without S9 mix and at 333 µg/plate with S9 mix. Due to overlapping toxic effects the number of colonies was reduced at higher concentrations in both strains (except strain TA 98 with S9 mix in experiment (II)).

In experiment (I), an isolated increase in revertant colony numbers occurred at 100 µg/plate in strain TA 1535 without S9 mix. This increase is jugded to be based upon a technical error and therefore, excluded from assessment. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Conclusions:
Positive
Executive summary:

Method

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum. The following specific strains were used: TA 98, TA 100, TA 1535, TA 1537, and TA 1538. The assay was performed in two independent experiments [(I) and (II)], both with and without liver microsomal activation. To verify the positive response in experiment (I) a second experiment was performed as plate incorporation. Each concentration, including the controls, was tested in triplicate.

 

Observation

In both experiments, a dose dependent and substantial increase in revertant colony numbers was observed following treatment with the test substance in strains TA 1537 and TA 98 with and without metabolic activation. In experiment (I), the threshold of thrice the number of the corresponding solvent control was exceeded in strain TA 1537 at 3 µg/plate without S9 mix and 10 µg/plate with S9 mix. In strain TA 98, the threshold of twice was exceeded at 10 µg/plate without S9 mix and at 33 µg/plate with S9 mix. In experiment II, an increase in revertant colony numbers was observed in strain TA 1537 without metabolic activation, however, the required threshold of thrice was not reached. In the presence of metabolic activation the threshold was exceeded at 5 µg/plate. In strain TA 98 the number of revertant colonies exceeded the threshold of twice at 1.25 µg/plate without S9 mix and at 333 µg/plate with S9 mix. Due to overlapping toxic effects the number of colonies was reduced at higher concentrations in both strains (except strain TA 98 with S9 mix in experiment (II)).

In experiment (I), an isolated increase in revertant colony numbers occurred at 100 µg/plate in strain TA 1535 without 89 mix. This increase is jugded to be based upon a technical error and therefore, excluded from assessment. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

 

Conclusion

Under the experimental conditions reported, the test item with and without metabolic activation induced gene mutations by frameshifts in the genome of the strains TA 1537 and TA 98. Therefore the test substance is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

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:
key study
Study period:
2015-12-16 to 2016-05-03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
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:
other: mammalian cell gene mutation assay
Target gene:
The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single step forward mutation to the TK-/- genotype in which little or no TK activity remains. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium supplemented with 10% Foetal Calf Serum (RPMI complete)
- Properly maintained: yes; permanent stock of mouse lymphoma L5178Y cells are stored in liquid nitrogen and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue fraction: Species: Rat, Strain: Sprague Dawley, Tissue: Liver, Inducing Agents: P henobarbital – 5,6-Benzoflavone, Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number 3512
Test concentrations with justification for top dose:
A preliminary cytotoxicity assay was performed at the following dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 μg/mL. Based on the results obtained in the preliminary cytotoxicity assay, two independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (-S9; treatment time 3 hours): 60.2, 43.0, 30.7, 22.0, 15.7 and 11.2 μg/mL
Main Assay I (+S9; treatment time 3 hours): 100, 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL
Main Assay II (-S9; treatment time 24 hours): 35.0, 25.0, 17.9, 12.8, 9.11 and 6.51 μg/mL
Vehicle / solvent:
Test item solutions were prepared using dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. The mutation assays were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained without metabolic activation, the second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and incubated to allow expression of the mutant phenotype. At the end of the expression period cells were plated for the evaluation of 5-trifluorothymidine resistance and for viability.
Rationale for test conditions:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126x10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Survival after treatment:
In the first experiment, in the absence of S9 metabolic activation, no cell survived after treatment at the highest dose level, moderate toxicity reducing relative total growth (RTG) to 14% of the concurrent negative control was noted at 43.0 μg/mL, slight to mild toxicity was observed between 15.7 and 30.7 μg/mL, while no relevant toxicity was noted at the lowest concentration tested. In the presence of S9 metabolism, treatment with the test item at 100 μg/mL yielded moderate toxicity reducing RTG to 24% of the concurrent negative control value, slight toxicity was noted at the next lower concentration, while no relevant toxicity was observed over the remaining concentrations tested. In the second experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (35 μg/mL) yielded marked toxicity reducing RTG to 8% of the concurrent negative control value. The next two lower dose levels of 25.0 and 17.9 μg/mL yielded moderate toxicity reducing RTG to 15 and 26%, respectively. Dose-related toxicity was seen over the remaining dose levels tested.

Mutation results:
In Main Assay I, statistically significant increases in mutant frequency were observed at the lowest and at two intermediate concentrations, in the absence of S9 metabolism, and at the highest dose level in its presence. A linear trend was indicated, both in the absence and presence of S9 metabolism. However, the observed increases were lower than the Global Evaluation Factor both in the absence and presence of S9 metabolism, thus they were considered of no biological relevance. In Main Assay II, no increases in mutant frequency were observed at any concentration tested.
Conclusions:
Under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay using mouse lymphoma L5178Y cells.
Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD 490, mouse lymphoma L5178Y cells cultured in vitro were exposed to the test item (87.9% purity) in dimethylsulfoxide at concentrations of 11.2, 15.7, 22.0, 30.7, 43.0 and 60.3 μg/mL (main assay I, without S9 mix) and at 3.13, 6.25, 12.5, 25.0, 50.0 and 100 μg/mL (main assay I, with S9 mix) and in main assay II, without S9 mix at concentrations of 6.51, 9.11, 12.8, 17.9, 25.0 and 35.0 μg/mL. The test item was tested in the main assay I up to 60.3 μg/mL (without metabolic activation) and up to 100 μg/mL (with metabolic activation) and in main assay II up to 35.0 μg/mL (without metabolic activation) based on data obtained from the preliminary cytotoxicity test. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490

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

Genetic toxicity in vivo

Description of key information

Chromosomal Aberration: Negative

Non-mutagenic with respect to clastogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1992-03-17 to 1993-01-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 12 weeks of age
- Weight at study initiation: 28 - 43 g
- Housing: females: in groups of a maximum of three mice; males: individually in Macrolon type I cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 40 -70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: physiological saline
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in physiological saline solution, stirred with a magnetic mixer during administration and injected intraperitoneally.

Duration of treatment / exposure:
test item dose groups: 16, 24 and 48 hours,
negative/positive control: 24 hours
Frequency of treatment:
once
Post exposure period:
no
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
three test item related experimental dose groups: 16, 24 and 48 hours
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control used: cyclophosphamide
- Route of administration: intraperitoneal
- Dosis: 20 mg/kg bw
Tissues and cell types examined:
bone marrow derived erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. For the results please box "Additional information on results".

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
see Table 1 in box "Any other information on materials and methods incl. tables".

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were automatically stained with an Ames HemaTek Slide Stainer and then destained with methanol, rinsed with deionized water and dried. After drying, the slides were covered with xylene and a cover glass.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. Moreover, the ratio of polychromatic to normochromatic erythrocytes was determined (number of normochromatic erythrocytes per 1000 polychromatic ones). Additionally, the number of normochromatic erythrocytes showing micronuclei was also established.
Evaluation criteria:
A test was considered positive if at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory’s experience, was within the range of negative controls. A test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.
Statistics:
The test item group(s) with the highest mean and the positive control were checked by Wilcoxon’s non parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant, if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (1s ranges) were calculated for all the means.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. The following symptoms were recorded for up to 48 hours, starting at 10 mg/kg bw: apathy, roughened fur, red discoloration of hairless parts of skin, staggering gait, spasm, leaping coloured urine. In addition, 3 of 5 animals died in the 75 mg/kg group and all animals died in the 100 mg/kg bw group. Based on the results, 50 mg/kg bw was considered as 1 MTD for the main test.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. No symptoms were recorded for the control groups.
- Mortality: One of forty treated animals died during the test period due to acute toxicity. No animals died in the control group.
- Induction of micronuclei (for Micronucleus assay): no clastogenic effect, see Table 2
- Ratio of PCE/NCE (for Micronucleus assay): slightly changed, see Table 2
- Statistical evaluation: see Table 2

Table 2: Summary of results of micronucleus test with the test item
experimental groups Number of evaluated poly-chromatic erythrocytes (PCE) Number of normo-chromatic erythrocytes per 1000 PCE micronucleated cells per 1000
normo-chromatic erythrocytes poly-chromatic erythrocytes
Negative control 10000 811 +/- 208 1.2 +/- 1.6 1.5 +/- 1.1
Test item_16 hours 10000 1321* +/- 319 1.3 +/- 1.0 1.6 +/- 1.0
Test item_24 hours 10000 1088 +/- 549 1.3 +/- 1.6 1.5 +/- 1.0
Test item_48 hours  10000 775 +/- 199 0.4 +/- 0.7 1.3 +/- 1.2
Positive control 10000 557 +/- 231 0.4 +/- 1.3 12.6* +/- 6.9

Concentration: test item: 50 mg/kg bw; positive control: 20 mg/kg bw

* p< 0.01 (tested by non-parametric Wilcoxon ranking test)

Conclusions:
Non-mutagenic with respect to clastogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.
Executive summary:

Method

In a NMRI mouse bone marrow micronucleus test conducted in accordance with OECD Guideline 474, 5 mice/sex/treatment group were treated intraperitoneally once with the test item (95.6% purity) at doses of 0 and 50 mg/kg bw. The vehicle used was physiological saline. The animals were sacrificed and bone marrow cells were harvested after 16 hours (test item), 24 hours (negative/positive control and test item) and after 48 hours (test item).

After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. The test item was tested at an adequate dose based on a preliminary dose range finding test. The positive control induced the appropriate response.

Results

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time (16, 24 and 48 hours).

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— 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.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as:

- In vivo somatic cell mutagenicicty tests such as these indicated in paragraph 3.5.2.3.5:

— mammalian bone marrow chromosome aberration test;

— mouse spot test;

— mammalian erythrocyte micronucleus test.

- In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

In the "Bacterial Reverse Mutation Assay" the test item shows mutagenic effects. As indicated in the "Flow Chart of the Mutagenicity testing strategy" R.7.7 -1 of ECHA Guidance chapter r.7a (v. 5.0, December 2016), in case of positive response in the gene mutation test in bacteria, it is necessary to fulfil REACH Annex VIII requirements.

An in vitro cytogenicity test is available and the test substance does not show any mutagenic effect.

As per REACH Regulation requirements, a further in vivo study is considered in which the test substance didn’t induce increase the number of polychromatic cells with micronuclei.

Therefore, as reported in the Flow chart R.7.7 -1, no further test are required and it is possible to conclude that the test substance is Not Classified for Mutagenic Toxicity.