5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria:
key study, Ames test, S. typhimurium/ E.coli, with and without metabolic activation: negative (non-GLP, comparable to OECD 471; BASF SE, 1982) nanomaterial
supporting study, Ames test, S. typhimurium, with and without metabolic activation: negative (GLP, OECD 471; Clariant, 1996) nano not specified

Gene mutation in mammalian cells:
key study, HPRT test, CHO cells, with and without metabolic activation: negative (GLP, OECD 476; BASF SE, 2012) nanomaterial

Cytogenicity in mammalian cells:
Study ongoing with nanomaterial

Link to relevant study records

Reference

Reference 1

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:

(only 2-Aminoanthracene as positive control with metabolic activation)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: yellow powder
- Analytical purity: technically pure
- Storage condition of test material: +4°C

Target gene:

his, trp

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Remarks:

control for sterility

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene, MNNG, 4-nitro-o-phenylendiamine, 9-aminoacridine chloride monohydrate, N-ethyl-N'-nitro-N-nitrosoguanidin (see details below)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation)

DURATION
- Preincubation period:
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 4 in the first experiment, 2 in the second experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: Negative control: Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) is
carried out for each tester strain in order to determine the spontaneous mutation rate.

Positive controls
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix:
10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1538, TA 1537 and TA 1535
60 µg 2-aminoanthracene (dissolved in DMSO) for E. coli WP2 uvrA
without S-9 mix
5 µg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strains TA 98 and TA 1538
100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537
10 µg N-ethyl-N´-nitro-N-nitrosoguanidin (ENNG) (dissolved in DMSO) for the strain E. coli WP2 uvrA

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

Statistics:

The data were not statistically analyzed.

Species / strain:

other: S. typhimurium TA 1535, TA 100, TA 1537, TA 1538, TA 98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Standard plate test (20 - 5000 µg/plate): 1. Exp.
Strain	Metabolic activation system	Replicates	Mean revertants in Controls	Maximum revertant factor	Dose dependency	Assessment
TA 98	no	4	18	1.1	no	negative
	yes	4	42	1.0	no	negative
TA 1537	no	4	8	1.0	no	negative
	yes	4	12	1.0	no	negative
TA 1538	no	4	10	1.2	no	negative
	yes	4	28	0.9	no	negative
TA 1535	no	4	18	0.9	no	negative
	yes	4	13	1.1	no	negative
TA 100	no	4	110	1.0	no	negative
	yes	4	106	1.0	no	negative
WP2 uvr A	no	4	37	1.0	no	negative
	yes	4	37	1.1	no	negative

Standard plate test (20 - 5000 µg/plate): 2.Exp.
Strain	Metabolic activation system	Replicates	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	2	21	1.3	no	negative
	yes	2	35	1.1	no	negative
TA 1537	no	2	10	0.9	no	negative
	yes	2	8	1.1	no	negative
TA 1538	no	2	13	1.0	no	negative
	yes	2	24	1.1	no	negative
TA 1535	no	2	20	1.1	no	negative
	yes	2	24	1.1	no	negative
TA 100	no	2	120	1.0	no	negative
	yes	2	122	1.3	no	negative
WP2 uvr A	no	2	31	1.1	no	negative
	yes	2	35	1.3	no	negative

Incomplete solubility of the test substance in DMSO from about 500 µg/plate onward.

No bacteriotoxic effect was observed.

Mutagenicity: An increase in the number of his + or trp+ revertants was not observed either without S-9 mix or after the addition of a metabolizing system.

Conclusions:

Pigment Yellow 139 was not mutagenic in bacteria.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

liver S9 from rats treated with phenobarbital i.p. and β-naphthoflavone orally

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL

with S9 mix (4-hour exposure period)
0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL

2nd Experiment
without S9 mix (24-hour exposure period)
0; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00; 400.00 μg/mL

with S9 mix (4-hour exposure period)
0; 3.75; 7.50; 15.00; 30.00; 50.00 μg/mL

Vehicle / solvent:

culture medium (Ham's F12)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 300 μg/mL ethyl methanesulfonate (without S9) or 20 μg/mL methylcholanthrene (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24h
- Exposure duration: 4 hours both with and without metabolic activation and 24 hours without metabolic activation
- Expression time (cells in growth medium): 6 - 8 days
- Selection time (if incubation with a selection agent): 1 week
- Fixation time (start of exposure up to fixation or harvest of cells): 14 - 16 days

SELECTION AGENT (mutation assays): 6-thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
pH
Changes in the pH were recorded by a change in the indicator color in the culture medium
(phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two
top doses and for the negative controls with and without S9 mix.

Osmolarity
Osmolarity was measured, at least for the top dose and for the negative controls with and
without S9 mix.

Solubility
Test substance precipitation was checked immediately after treatment of the test cultures and
at the end of treatment.

Cell morphology
The test cultures of all test groups were examined microscopically at the end of exposure
period with regard to cell morphology, which allows conclusions to be drawn about the
attachment of the cells.

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than
50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our
historical negative control data range of 0 – 15.95 mutants per 106 clonable cells (see
Appendix 5).
• The positive controls both with and without S9 mix must induce distinctly increased
mutant frequencies (historical positive control data; see Appendix 6).
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of
solubility under culture conditions should be tested. Freely soluble and apparently
non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent
negative control values and our historical negative control data range (see Appendix 5).
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse
relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e.
15 mutants per 106 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 substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant
increased above the concurrent negative control and is within our historical negative
control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

The test substance was poorly soluble in the most suitable vehicle culture medium.
Due to lacking distinct cytotoxicity in the pretests in the absence and presence of metabolic
activation after 4 hours exposure all experimental parts with short-term exposure were
performed using concentrations at the border of saturation of culture medium.
In the pretest with 24 hours exposure in the absence of metabolic activation cytotoxicity of
about 20% relative survival was observed at intermediate concentrations showing strong test
substance precipitation.

A table with the results is attached as a jpg file.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

micronucleus test, mouse, in vivo: negative (GLP, OECD 474; BASF SE, 2008) nanomaterial

No toxicity was observed, and no analytical determination of the test material was performed. As the test material is an insoluble pigment, it would not have been possible to detect it in plasma.

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

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories GmbH, Germany
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: mean: 30.0 g
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type M I; single housing
- Diet (ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 500 mg/kg bw (25 mg/mL), 1000 mg/kg bw (50 mg/mL), 2000 mg/kg bw (100 mg/mL)
- Amount of vehicle (if gavage or dermal): The usual application volume of 10 mL/kg body weight led to a non-applicable mass and therefore the volume was increased to 20 mL/kg body weight.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To achieve homogeneity of the test substance in the vehicle, the test substance preparation was stirred with an ultraturrax.
All test substance formulations were prepared immediately before administration.
The stability of the test substance at room temperature in the vehicle corn oil was determined analytically.
To keep the test substance homogeneously in the vehicle, the test substance preparation was constantly stirred.

Duration of treatment / exposure:

24 h (all test substance concentrations, vehicle control and both positive controls)
48 h (highest test substance concentration and vehicle control)

Frequency of treatment:

single oral administration

Post exposure period:

The animals were sacrificed 24 hours and 48 hours after the treatment, respectively.
The animals were examined for any clinically evident signs of toxicity several times.

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP) and Vincristine sulfate (VCR)
- Justification for choice of positive control(s): Both substances are well-established reference clastogens and aneugens, respectively.
- Route of administration: orally (CPP), intraperitoneal (VCR)
- Doses / concentrations: 20 mg/kg bw (CPP), 0.15 mg/kg bw (VCR)
The positive control substances were given with a volume of 10 mL/kg bw.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2 000 mg/kg body weight, recommended as the highest dose according to the OECD Guideline, was tolerated by all animals (male and female) without any clinical sign. Thus, only male animals were used for the cytogenetic investigations.
Therefore, a dose of 2 000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1 000 mg/kg and 500 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
Bone marrow/FCS (fetal calf serum) suspension (about 2 mL/femur), preheated up to 37°C and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS. One drop this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored.

The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
(The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.)
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
(The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.)
• Ratio of polychromatic to normochromatic erythrocytes
(An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.)
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
(The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4)).

Evaluation criteria:

The mouse micronucleus test is considered valid if the following criteria are met:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: The administration of the test substance did not lead to any clinical signs of toxicity

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): summary table see below
- Ratio of PCE/NCE (for Micronucleus assay): test compound: 0.7, CPP: 0.97, VCS: 0.47

Induction of Micronuclei in bone marrow cells
Substance	Dose (mg/kg)	sex	post exposure period (h)	Micronuclei in PCE
				total^a [‰]	large^b [‰]	Number of NCE^c
vehicle	corn oil	male	24	0.80	0.0	3442
test substance	500	male	24	1.10	0.0	2753
test substance	1000	male	24	0.90	0.0	3094
test substance	2000	male	24	2.2*#	0.1	2495
vehicle	corn oil	male	48	0.90	0.0	2542
test substance	2000	male	48	1.30	0.0	3216
positive control cyclophosphamid	80	male	24	15.0**	0.0	2057
positive control vincristine sulfate	20	male	24	61.1**	22.6**	4253
PCE = polychromatic erythrocyts (2000 were scored for micronuclei)
NCE = normochromatic erythrocytes
^a= sum of small and large micronuclei
^b = large micronuclei (indication for spindle poison effect)
^c= number of NCEs observed when scoring 10 000 PCEs
^*=  p ≤ 0.05
^** = p ≤ 0.01

(# = historical control range 0.7 - 3.0)

Under the experimental conditions chosen here, the test substance Paliotol Gelb K 2142 does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation in bacteria:

In an available Ames test conducted comparable to OECD guideline 471, the 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 TA 1535, TA 100, TA 1537, TA 1538, TA 98 and Escherichia coli WP2 uvrA (BASF SE, 1982).

Based on the trade name given in the study report, the test material was a nanomaterial.

The investigations were performed with (Aroclor 1254-induced rat liver S-9 mix) and without microsomal activation at a dose range up to 5000 µg/plate. From 500 µg/plate onward precipitates of the test substance were visible in the agar.

In the experiments performed without and with microsomal activation, none of the tested concentrations of the test item led to an increase in the incidence of both histidine- or tryptophan-prototrophic mutants in comparison with the negative control. No evidence of the induction of point mutations by the test item or by the metabolites of the substance was found.

According to the results of the present study, the test substance is thus not mutagenic in the Ames test under the experimental conditions chosen.

 

In a GLP conform study conducted according to OECD guideline 471, the potential of the test substance to induce gene mutations was investigated using the Salmonella typhimurium strains TA 100, TA 1535, TA1537 and TA 98 (Clariant, 1996).

Two independent mutagenicity studies were conducted, each in the absence and in the presence of a metabolizing system derived from rat Iiver homogenate. For both studies the compound was suspended in DMSO, and each bacterial strain was exposed to 6 dose levels. Doses for both studies ranged from 4 to 5000 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All positive control compounds gave the expected increase in the number of revertant colonies.

No toxicity was found in the mutagenicity experiments with and without metabolic activation.

In the toxicity test using histidine enriched agar plates and a dilution of the tester strain TA 100 (designated TA 100 D), which was performed in parallel with the second experiment, the test compound proved to be not toxic to the bacterial strain.

In the absence and in the presence of the metabolic activation system did not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated that the test substance is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

 

Gene mutation in mammalian cells:

In a GLP conform study conducted according to the OECD guideline 476 the potential of the test item (purity: > 99 weight-%) to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro was assessed (BASF SE, 2012). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the trade name given in the study report, the test material was a nanomaterial.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, doses in the range from 0 – 400 µg/ml were tested in this study.

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

The test substance was poorly soluble in the most suitable vehicle culture medium. Due to lacking distinct cytotoxicity in the pretests in the absence and presence of metabolic activation after 4 hours exposure all experimental parts with short-term exposure were performed using concentrations at the border of saturation of culture medium. In the pretest with 24 hours exposure in the absence of metabolic activation cytotoxicity of about 20% relative survival was observed at intermediate concentrations showing strong test substance precipitation.

The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

Cytogenicity in mammalian cells:

Study ongoing

 

Cytogenicity in vivo

A micronucleus assay conducted according to OECD guideline 474 and GLP requirements was performed with the test item (purity: > 99%) (BASF SE, 2008). This test is performed to detect both chromosome breaking substances (clastogens) and aneuploidy inducing substances (aneugens) in NMRI mice. Based on the trade name given in the study report, the test material was a nanomaterial.

For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 500, 1000 and 2000 mg/kg body weight in a volume of 20 mL/kg body weight. A control group received the vehicle by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. Only the 24 hour sacrifice interval was investigated in the test groups of 1000 and 500 mg/kg body weight and in the positive control groups. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

The rate of micronuclei was always close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

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 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and a study for the in vitro chromosome aberration assay (OECD 473, GLP) is ongoing. Based on the available information, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.