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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance showed no genotoxic effects in the Ames test (OECD TG 471), cytogenetic assay with CHO cells (OECD TG 473, GLP) and HGPRT assay (OECD TG 476, GLP) when tested up to the cyto/bacteriotoxic range.

Link to relevant study records

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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)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1 BH4
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The Chinese hamster ovary (CHO-K1 BH4) cell line, isolated by Kao and Puck (1967) and cloned by O'Neill et al (1977) was used.

MEDIA USED
Ham's F-12 medium, supplemented with 10% foetal bovine serum and antibiotics, at 37°C with 5% CO2 in air
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9
It was prepared from the livers of male Sprague-Dawley rats weighing ca. 200 g. These had received a single i .p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation . The S9 was stored at -196°C.
- concentration or volume of S9 mix and S9 in the final culture medium
10% S9- mix was freshly prepared by mixing 2 mL of S9 with 1 mL of 0.1M NADP, 1 mL of 0 .1M G6P, 2 mL of 330mM KC1/80mM MgCl2 and 14 mL of 0 .1M Phosphate buffer (pH 7 .4) . The S9 mix was stored at 4°C for a maximum of 20 minutes before use .
Test concentrations with justification for top dose:
0, 78.13, 156.25, 312.5 μg/mL (- S-9, 12-hour culture)
0, 156.25, 312.5, 625 μg/mL (+ S-9, 12-hour culture)
0, 156.25, 312.5, 625 μg/mL (+ S-9, 20-hour culture)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.5 x 10E6 cells per flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Without S9 mix
i) 12 hours exposure to the test material
With S9 mix
ii) 4 hours exposure to the test material and S9 mix (0.5 mL per 4 .5 mL culture medium, of 10% S9 in standard cofactors). A phosphate buffered saline wash and then a further 8 hours in treatment-free media prior to cell harvest .
iii) 4 hours exposure to the test material and S9 mix (0.5 mL per 4 .5 mL culture medium, of 10% S9 in standard cofactors). A phosphate buffered saline wash and then a further 16 hours in treatment-free media prior to cell harvest .

- Spindle inhibitor: Mitosis was arrested by addition of demecolcine (0.1 µg/mL) two hours before the required harvest time .

- Methods of slide preparation and staining technique used including the stain used:
The cells were resuspended in 3.0 mL of fresh fixative if necessary before centrifugation and resuspension in 0.5 mL of fixative. Three or four drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was
permanently labelled with the appropriate identification data. When the slides were dry they were stained in 2% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and mounted in Depex mounting medium.

- Number of cells spread and analysed per concentration:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 18 to 22 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976)
If the cell had more than 22 chromosomes then it was recorded on a separate sheet as an aneuploid or polyploid cell, chromosome aberrations in such cells were not recorded. Endoreduplicated cells are included as polyploid cells but can also be evaluated separately if necessary. All chromosome aberrations were checked by a senior cytogeneticist prior to decoding the slides .

Mitotoc index: A total of 2000 cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value .

METHODS FOR MEASUREMENT OF CYTOTOXICITY
A cytotoxicity test was performed on cell cultures using a 4-hour exposure time with metabolic activation followed by an 8 and 16-hour culture period in treatment free media. Treatment without metabolic activation was continuous with cell harvest at 12 hours. Growth inhibition was estimated by couating the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent negative control value .
Evaluation criteria:
A positive response was recorded for a particular treatment if the % diploid cells with aberrations (gaps excluded) exceeded the maximum historical value and gave a statistically significant increase over the concurrent control value. If only the % diploid cells with aberrations (gaps included) exceed
historical values then a ± response was recorded.

Positive responses were also recorded if the % cells with aberrations (gaps excluded) was statistically significantly greater than the concurrent control level, even if it was below historical levels, but only if there was an indication of a dose response. However, consideration is given to a number of factors, such as the frequency of chromosome exchange events which are comparatively rare in control cultures, and the ultimate designation must rely upon experience and sound scientific judgement (UKEMS Guidelines for Mutagenicity Testing, 1983).
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity was observed at doses of 313 μg/mL without and 625 μg/mL with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
The pH of culture medium containing S9 mix and test item at 0, 312.5 and 625 µg/mL was measured and values of 7.05, 7.45 and 7.55 were obtained.

RANGE-FINDING/SCREENING STUDIES:
It was observed that the test item showed evidence of a dose-related increase in cell toxicity at dose levels up to 312.5 µg/mL without S9 and up to 625 µg/mL with S9 .

Chromosome aberration test (CA) in mammalian cells:
The negative control cultures gave values of chromosome aberrations within the expected range. The frequency of aberrations was consistent between the four negative control groups, the highest frequency (4 .0% cells with aberrations without gaps) being seen in the 20-hour culture group with S9.
The positive control cultures gave significant increases in the frequency of aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. Cyclophosphamide (10 µg/mL) gave a stronger response at the 20-hour harvest time point than that seen after 12 hours .

The test item was seen to induce no significant, dose-related increases in the frequency of aberrations in any of the treatment groups .

The test item induced a significant increase in the numbers of polyploid cells at the 625 µg/mL dose-level in the 20-hour treatment with S9. The duplicate slides from cultures A and B were rechecked for the incidence of polyploid cells and values of 26.5 and 14.5% respectively were obtained, thus confirming the original observations. An Increase in the frequency of polyploid cells was observed at one dose level only, but this was considered to be possibly due to the pH change induced by the test item when added to the culture medium .
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5th August 1991 - 17 September 1991
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:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HGPRT locus in V79 cells of the Chinese hamster
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without-S9 mix: 0.10 ; 0.30 ; 0.80 ; 1.20 ; 2.00* and 3.00* mg/mL
with S9 mix: 0.30 ; 1.00 ; 2.00 ; 3.00* ; 4.00* and 5.00* mg/mL
* toxic, culture not continued

Experiment II:
without S9 mix: 0.03 ; 0.10 ; 0.30 ; 0.60° ; 0.80° and 1.00 mg/ml
with S9 mix: 0.10 ; 0.30° ; 0.60 ; 1.00 ; 1.50° and 2.00 mg/ml
*culture not continued
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 7,12-dimethylbenzanthracene, ethylmethanesulphonate
Details on test system and experimental conditions:
The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation.
Evaluation criteria:
A test article is classified as positive if it induces either a significant concentration-related increase in the mutant frequency for a reproducible and significant positive response for at least one of the test points.
Statistics:
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=2.0 mg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2016 - 4 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
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 E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from male Wistar rats livers (received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days)
Test concentrations with justification for top dose:
Experiment 1: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate; (Standard plate test with and without S9 mix) all strains
Experiment 2: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate; (Standard plate test without S9 mix) TA 1537
Experimant 3: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate; (Preincubation test with and without S9 mix) all strains
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix, TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix, E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S9 mix, TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
without S9 mix, TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S9 mix, TA 1535, TA 100, TA1537, TA 98, E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Both tests: In each experiment 3 Test plates per dose control used.

Standard plate test
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. (7) and Matsushima et al. (8).
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Positive controls
The following positive controls were used to check the mutability of the bacteria and the activity of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA) ; 2.5 μg/plate, TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO, Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 5 μg/plate TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD); 10 μg/plate TA 98
• 9-aminoacridine (AAC); 100 μg/plate TA 1537
• 4-nitroquinoline-N-oxide; 5 μg/plate E. coli WP2 uvrA


Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Key result
Species / strain:
S. typhimurium 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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.
TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward.
MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

For detailed result tables see attached document.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Additional information

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (OECD 471). The doses ranged form 10 μg - 5000 μg/plate (SPT) and 10 μg - 2500 μg/plate (PIT). No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. The test substance not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF SE, 2016).

In further tests, the substance was also not capable to induce point mutations with or without metabolic activation (OECD TG 471, but only four strains tested). The doses ranged from 4 to 5000 μg/plate and bacteriotoxicity was noted at doses of 2500 μg/plate and above (BASF AG, 1986). In another Ames test the test substance was also negative up to 2832 μg/plate with and without using S9-mix (BASF AG, 1980).

The test compound was also negative in gene mutation test in mammalian cells in a HGPRT assay with Chinese hamster V79 cells (OECD TG 476). The cells were exposed to concentrations ranging from 0.03 to 1.2 mg/mL without metabolic activation and 0.1 to 2 mg/mL with metabolic activation. Higher concentrations could not be tested due to severe cytotoxic effects (BASF AG, 1992b).

Negative results were also obtained in the cytogenetic chromosome aberration assay with CHO (Chinese hamster ovary) cells according to OECD TG 473. The doses ranged from 78 to 313 μg/mL without and 156 to 625 μg/mL with metabolic activation. Cytotoxicity was observed at doses of 313 μg/mL without and 625 μg/mL with S9-mix (BASF AG, 1992c).

In conclusion, the substance showed no mutagenic and no cytogenetic effect in three different test systems in vitro. No in vivo data were available and in accordance with column 2 of REACH Annex VIII, these data are not needed as all in vitro studies are negative.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.