Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:

Ames test (according to GLP & OECD guideline 471): non-mutagenic with and without metabolic activation (BASF, 40M0059/954021, 1995)

HPRT test (according to GLP & OECD guideline 476): non-mutagenic with and without metabolic activation in CHO cells (BASF, 50M0059/954233, 2001)

Chromosome aberration test (GLP & OECD guideline 476): ambiguous results. with metabolic activation: not clastogenic; without metabolic activation: clastogenic effect at 2700 µg/mL (BASF, 32M0059/954023, 1995)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3043/V9Z
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM supplemented with 10 % FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for plating efficiency: yes
- Cell cycle duration: 13-14 h
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix extracted from Aroclor activated rat livers
Test concentrations with justification for top dose:
Pretest: 1; 5; 10; 50; 100; 500; 1000; 3000 µg/ml with and without S-9 mix
1. experiment: 900; 1800; 2700 µg/ml with and without S-9 mix
2. experiment: 1800; 2250; 2700 µg/ml without S-9 mix
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S-9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S-9 mix
Details on test system and experimental conditions:
DETAILS OF 1. and 2. EXPERIMENT:
METHOD OF APPLICATION: in medium

DURATION
- Attachment period: 24-30 h
- Exposure duration: 4 h with or without S-9 mix
- Expression time (cells in growth medium): 14 h
- Fixation time (start of exposure up to fixation or harvest of cells): ~ 18 h
The 2nd harvest time of 28 hours generally conducted was omitted due to the clear clastogenic effect of the pretest.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid dissolved in PBS
STAIN (for cytogenetic assays): Giemsa and Titrisol pH 7.2

NUMBER OF REPLICATIONS: 2 per dose

NUMBER OF CELLS EVALUATED: 50-200 metaphase cells depending on the incidence of chromosomally damaged cells

CHROMOSOMAL ANALYSIS
- Method: Structural and numerical chromosomal aberrations

DETERMINATION OF CYTOTOXICITY
-Method: cell counts, morphology, mitotic index

OTHER EXAMINATIONS:
- Treatment conditions: pH, osmolality, solubility of test substance (microscopically)

TEST SUBSTANCE ANALYSIS
The stability of the test substance throughout the study period will be proven by reanalysis at a later date. The homogeneity of the test substance was guaranteed by mixing before preparation of the test substance formulations. The stability of the test substance in water was determined analytically.
Statistics:
- statistical evaluation: MUCHAN program system (BASF AG).
- comparison of dose groups with control data: Fisher's exact test; correction using one-sided Bonferri-Holm test
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
chelating properties of test substance may be causative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S-9 mix at 2700 µg/mL
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: pH remained unchanged during the experiments (pH 7.4 - 7.8)
- Effects of osmolality: Osmolality remained unchanged.
- Water solubility: test substance completely soluble
- Precipitation: not observed.

RANGE-FINDING/SCREENING STUDIES:
Only the highest dose of 3,000 µg/mL exhibited chromosomal aberrations with a high proportion and a weakly toxic effect (weak suppression of the mitotic activity, decrease in the cell count, reduced cell attachment). Thus, according to the findings of the pretests and in agreement with current guidelines, 2,700 µg/mL (both with and without S-9 mix) were selected as top doses. Higher doses were not tested to avoid concentrations which may lead to artefactual chromosome breakage.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: OECD Guideline Nos. 471 and 472
Principles of method if other than guideline:
2-aminoanthracene is used as the only pos. ctrl. for S-9 metabolic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3043/V9Z
Target gene:
histidine (S.typhimurium); tryptophan (E.coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: strains TA98 and TA100 carry R-factor pkM101
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix extracted from Aroclor activated rat livers
Test concentrations with justification for top dose:
all experiments: 0, 100, 500, 2500, 5000 and 7500 µg/plate
Vehicle / solvent:
purified water
Untreated negative controls:
yes
Remarks:
sterility ctrl.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO
Remarks:
with S-9 mix for S. typhimurium (2.5 μg) and E.coli (60 μg)
Untreated negative controls:
yes
Remarks:
sterility ctrl.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine in DMSO
Remarks:
without S-9 mix for strains TA100 and TA1535 (5 μg) and Wp2 uvrA (10 μg)
Untreated negative controls:
yes
Remarks:
sterility ctrl.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine in DMSO
Remarks:
without S-9 mix for strain TA98 (10 μg)
Untreated negative controls:
yes
Remarks:
sterility ctrl.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine in DMSO
Remarks:
without S-9 mix for strain 1537 (100 μg)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (standard plate test)
DURATION
- Growth/exposure period: 48 h, in the dark; with addition of S-9 or phosphate buffer, respectively

METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min, with addition of S-9 or phosphate buffer, respectively
- Growth/exposure period: 48 h, in the dark

GROWTH MEDIA:
- S.typhimurium: Vogel-Bonner E medium + 20 g/L D-glucose
- E.coli: SAI selective agar

SELECTION AGENT (mutation assays): histidine or tryptophan proficiency

NUMBER OF REPLICATIONS: 3 per dose and control

NUMBER OF COLONIES EVALUATED: all revertant colonies

DETERMINATION OF CYTOTOXICITY
- Method: reduced his- background growth, decrease in the number of his+ revertants

ANALYSIS OF BACTERIA STRAINS:
- The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (A uvrB); ampicillin resistance (R factor plasmid). Histidine and tryptophan auxotrophy is automatically checked in each experiment via the spontaneous rate.



Evaluation criteria:
In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only for preincubation test; > 2500 µg/plate without S-9 mix and >7500 µg/plate with S-9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only for preincubation test; > 2500 µg/plate without S-9 mix and >7500 µg/plate with S-9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only for preincubation test; > 2500 µg/plate without S-9 mix and >7500 µg/plate with S-9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only for preincubation test; > 2500 µg/plate without S-9 mix and >7500 µg/plate with S-9 mix
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:
cytotoxicity
Remarks:
only for preincubation test; > 2500 µg/plate without S-9 mix and >7500 µg/plate with S-9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRECIPITATION
No test substance precipitation was found.

STABILITY IN WATER
The stability of the test substance throughout the study period will be praven by reanalysis. The stability of the test substance in water was assured analytically.
Endpoint:
in vitro gene mutation study in mammalian cells
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell gene mutation assay (HPRT)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 30/43V9Z
Target gene:
X-linked hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of pretreatment media: Ham's F12 medium with glutamine and hypoxanthine supplemented with 10% fetal caif serum (FCS), Hypoxanthine, Aminopterin, Thymidine, 1% (v/v) penicillin/streptomycin, 1 % amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes (during the week prior to treatment)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix extracted from Aroclor activated rat livers
Test concentrations with justification for top dose:
TEST CONCENTRATIONS
1. experiment:
without S-9 mix: 0; 109.38; 218.75; 437.50; 875.00; 1,750.00 µg/mL
with S-9 mix (cofactors 3:7): 0; 218.75; 437.50; 875.00; 1,750.00; 3,500.00 µg/mL

2. experiment:
without S-9 mix: 0; 109.38; 218.75; 437.50; 875.00; 1,750.00 µg/mL
with S-9 mix (cofactors 1:9): 0; 218.75; 437.50; 875.00; 1,750.00; 3,500.00 µg/mL
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S-9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

RATIONAL FOR DOSE SELECTION:
According to results of a pretest covering concentrations from 1 µg/mL - 3,500 µg/mL incubated for 4 hours with the cells. According to OECD guideline 476, maximal concentration should not exceed 10 mM. Without S-9 mix the cloning efficiency was effected from about 1,750 µg/mL onward. With S-9 mix the test substance did not exhibit any toxic effects after a treatment time of 4 hours up to the highest recommended concentration, i.e. 10 mM. On the basis of the findings from the pretests, the following doses (related to the test article) or concentrations (related to the active ingredient) were selected as top doses:
- Without S-9 mix, 4 hours exposure time 1,750 µg/mL (= 5.16 mM)
- With S-9 mix, 4 hours exposure time 3,500 µg/mL (= 10.33 mM)

DURATION
Attachment period: 24 h
- Exposure duration: 4 h (with and without S-9 mix)
- Expression time (cells in growth medium): 1 week (in HAT medium)
- Selection time (if incubation with a selection agent): 1 week (in TG medium)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 2 weeks


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): Fixation using methanol and staining with Giemsa

NUMBER OF REPLICATIONS: 2 per dose

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (survival of treatment and cell viability without mutant selection); mutant frequency; cell morphology after test substance exposure

OTHER:
During exposure period:
- pH
- osmolality
- solubility of test substance



Evaluation criteria:
EVALUATION CRITERIA:
The criteria for a positive response are:
- Increases of the corrected mutation frequencies above the concurrent negative control values and above 15 mutants per 10^6 clonable cells and/or the evidence of a dose-response relationship
- Reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above 15 mutants per 10^6 clonable cells or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test chemical is considered non-mutagenic according to the following criteria.
-The corrected mutation frequency in all dose groups is within the historical control range and is not significantly above the concurrent negative control.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>875 µg/mL after 4 h exposure
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: During the 2. experiment, the S-9 mix (cofactors 1:9) precipitated during the exposure time.
- Effects of pH: pH remained unaffected during the experiments
- Effects of osmolality: Osmolality remained unaffected during the experiments.

CELL MORPHOLOGY:
- >875 µg/ml: reduced attachment
- Other test concentrations: cells were completely attached after the 4 h exposure period
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

MNT (GLP & OECD guideline 474): non-clastogenic when administered orally to NMRI mice (BASF, 26M0059/954226, 1997).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF
Type of assay:
other: mammalian micronucleus assay in vivo
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3043/V9Z
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, Sulzfeld, FRG
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: small groups for acclimatization, individually after administration
- Identifications of animals: cage cards
- Diet: (Kliba Haltungsdiät, Klingentalmühle AG, Kaiseraugst, Switzerland; ad libitum
- Water: from bottles, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 – 24°C
- Humidity: 30-70 %
- Air changes (per hr): fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: immediately before administration; all concentrations were administered at 10 mL volume.

Frequency of treatment:
single treatment
Post exposure period:
24 h (all concentrations and controls); 48 h (negative control and 2000 mg/kg)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5/sex (vehicle ctrl.; all dose groups) 5 animals (2 males and 3 females or 3 males and 2 females) for pos. ctrl.
Control animals:
yes, concurrent vehicle
Positive control(s):
As a positive control, 20 mg of cyclophosphamide (CPP)/kg body weight or 0.15 mg of vincristine sulphate (VCR)/kg body weight, both, dissolved in purified water, were administered to male and female animals once orally or intraperitoneally each in a volume of 10 mL/kg body weight.
Tissues and cell types examined:
erythrocytes of the femural bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The respective OECD guideline suggests 2000 mg/kg bw as the highest dose. In a pretest, all animals survived this dosage.

DETAILS OF SLIDE PREPARATION: Bone marrow was prepared from the femura and transferred onto microscopic slides using a standard protocol. The resulting slides were stained with eosin-methylene blue and Giemsa and finally embedded in Corbit-Balsam.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes were counted for each animal. The number of nor-chromatic erythrocytes was scored, too.

Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met: A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals. The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if: There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time. The frequencies of cells containing micronuclei were within the historical control range.
Statistics:
Statistical evaluation of data: MUKERN (BASF AG).
Comparison of dose groups: One-sided Wilcoxon test for the hypothesis of equal medians.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No inhibition of erythropoiesis was observed.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro, the mutagenicity of the test item was studied in an Ames test, a HPRT test and a chromosome aberration assay.

The GLP and OECD guideline conform Ames test included both a standard plate test and a preincubation test (BASF, 40M0059/954021, 1995). The S. typhimurium strains TA98, TA100, TA1535 and TA1537 and the E.coli strain Wp2 uvrA were exposed to six concentration levels ranging from 0 -7500 μg/plate with and without metabolic activation by S9 -mix. Negative and positive controls (2 -aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4 -nitro-o-phenylendiamine, 9 -aminoacridine) were included. The number of revertants was comparable between negative control and the different concentration groups. According to the Ames test, the test item is not mutagenic which is supported by another Ames test (BASF, 40M0107/944098, 1995). Here, the S. typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to 0-20000 μg/plate in the presence and absence of metabolic activation by S9. Both, a standard plate test and a preincubation test were applied.

For the GLP and OECD guideline conform HPRT, chinese hamster ovary cells (CHO) were exposed to six dose levels from 0 -1750 μg/mL without S9 -mix and from 0 -3500 μg/mL with metabolic activation (BASF, 50M0059/954233, 2001). Negative and positive controls (ethyl methane sulfonate, methylcholanthrene) were included. In the presence of metabolic activation, the test item was cytotoxic at dose levels equal or higher than 875 μg/mL. On the basis of the present study, the test substance did not cause an increase of the mutant frequencies in two experiments performed independently of each other. According to the conditions chosen in the HPRT test, the test item is non-mutagenic.

For the GLP and OECD guideline conform chromosome aberration assay, V79 chines hamster lung fibroblasts were exposed to four dose levels from 900-2700 μg/mL with or without S9 -mix (BASF, 32M0059/954023, 1995). Negative and positive controls (ethyl methane sulfonate, cyclophosphamide) were included. Cytotoxicity was observed in the absence of S9 metabolic activation at the highest concentration level. According to the results of the present study, the test substance caused a significant increase in the number of structurally aberrant metaphases without S-9 mix in two experiments independent of each other. After adding a metabolizing system the test substance exhibited only a weak clastogenic activity. Thus, under the experimental conditions chosen here the test item is considered to be chromosome damaging (clastogenic) agent in V79 cells. However, it can not be ruled out that these findings are the result of an indirect mechanism due to the chelating properties of the test substance which might interfere with cellular cationic pools.

In vivo, the genotoxicity of the test item was tested by a GLP and OECD guideline conform micronucleus assay (BASF, 26M0059/954226, 1997). 500, 1000 and 2000 μg/kg bw test substance (vehicle: water) were singularly administered to NMRI mice by gavage. After 24 or 48 h, animals were sacrificed and erythrocytes of the femural bone marrow were analyzed for micronucleus formation. Positive (cyclophosphamide, vincristine sulphate) and negative controls were included. The number of micronuclei was not increased after the administration of the test item when compared to the negative controls. Similar, the duration of the sacrifice intervals did not have an influence on the number of micronuclei. No inhibition of erythropoiesis induced by the treatment of mice with the test item was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups. Under the chosen test conditions, the test item can thus be considered as non-mutagenic in vivo.

In conclusion, the test item turned out to be non-mutagenic in an Ames test and a mammalian HPRT test according to the OECD guidelines even at the highest dose levels tested and irrespective of metabolic activation. In vivo, this results are confirmed by a Micronucleus Test in mice. However, a chromosome aberration assay gave an ambiguous result. This study is considered to be of minor relevance as a concentration relationship could not be clearly demonstrated and the ambivalent outcome of the experiment might be influenced by the chelating properties of the test substance. Considering the in vivo results of the MNT, the test item does not exert mutagenic effects in the bone marrow upon single oral administration to mice under the chosen test conditions. Similar, the test item is non-mutagenic upon repeated exposure, too. A 24-month GLP guideline feeding study in Wistar rats did not show an increased incidence of neoplastic transformations (see chapter 7.7; BASF AG 82S0059/95121, 1998).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.