Zirconium dinitrate oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria

An Ames test was performed with zirconium dinitrate oxide in accordance with OECD guideline 471 to assess the potential for genotoxicity of the test item (Hargitai, 2015). In this reliable (Klimisch 1) GLP study, zirconium dinitrate oxide was not observed to be mutagenic in the absence and presence of metabolic activation, under the conditions of the test.

In vitro cytogenicity in mammalian cells

An in vitro Mammalian Cell (human lymphocytes) Micronucleus Test was performed with zirconium oxynitrate hydrate, the hydrated form of the reference substance zirconium dinitrate oxide, in accordance with OECD guideline 487 to assess the potential of the genotoxicity of the test item (Envigo, 2018). In this reliable (Klimish 1) GLP study the test substance did not induce micronuclei in human lymphocytes in the absence and presence of metabolic activation, when tested up to precipitating or to the highest evaluable concentrations.

In vitro gene mutation in mammalian cells

An in vitro Mammalian Cell Gene Mutation Test was performed with zirconium oxynitrate hydrate, the hydrated form of the reference substance zirconium dinitrate oxide, in accordance with OECD guideline 476 to assess the potential for genotoxicity of the test item (BASF SE, 2018). In this reliable (Klimish 1) GLP study the test substance was not found to be mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and presence of metabolic activation.

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

Study period:

10/9/2014-22/10/2014

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus (Salmonella typhimurium)
Triptophan locus (Escherichia coli WP2 uvrA)

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:

phenobarbital and beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary concentration range finding test: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate
Based on the results of the preliminary experiment, the examined test concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate with and without metabolic activation.

Vehicle / solvent:

The following chemicals were used for vehicle (solvent) control groups:

Distilled water:
Supplier: TEVA Hungary Ltd.
Batch No.: 9321113
Expiry date: 30 November 2016

Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich Co.
Batch No.: SZBE0220V
Expiry date: 06 January 2017

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine (NPD)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preliminary concentration range finding test, two independently performed main tests: in agar (plate incorporation); confirmatory mutation test: preincubation method.

DURATION
- Procedure for growing cultures: The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 µL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37°C in a Gyratory Water Bath Shaker.
- Exposure duration: 48 ± 1 hours
- Selection time (if incubation with a selection agent): 48 ± 1 hours

SELECTION AGENT (mutation assays): histidine for the Salmonella typhimurium strains and tryptophan for the Escherichia coli strain

NUMBER OF REPLICATIONS: In the test, each sample (including the controls) was tested in triplicate.

NUMBER OF CELLS EVALUATED:
The viability of each testing culture was determined by plating 0.1 mL of the 1E05, 1E06, 1E07 and 1E08 dilutions prepared by sterile physiological saline on Nutrient Agar plates. For each culture, the number of viable cells of the cultures was determined by manual counting after approximately 24-h incubation at 37°C.

DETERMINATION OF CYTOTOXICITY
- Inhibition of the background lawn of auxotrophic cells.

Evaluation criteria:

Criteria for a positive response:
A test item was considered mutagenic if a dose-related increase in the number of revertants occurred and/or a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if, in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA strains, the number of reversions was more than twice higher than the spontaneous reversion rate of the negative (vehicle/solvent) control plates; the number of reversions was more than three times higher than the reversion rate of the negative (vehicle/solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Criteria for a negative response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant response in any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft Excel software.

Key result

Species / strain:

S. typhimurium TA 1535

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:

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:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The solubility of the test item was examined in a short solubility test using distilled water, dimethyl sulfoxide and N,N-dimethylformamide as vehicles. The test item was insoluble at 100 mg/mL in any of these vehicles (partial dissolution seen). However, the formulation at 50 mg/mL concentration using distilled water resulted in a homogeneous suspension, suitable for the test (while still partial dissolution was seen at the same concentration using the other two vehicles). Therefore, distilled water was selected as vehicle for the study.
- Precipitation: Precipitate/slight precipitate was detected on the plates in the main tests (initial mutation test and confirmatory mutation test) in all examined bacterial strains at 5000 and 1581 µg/plate concentrations with and without metabolic activation, and in the confirmatory mutation test at 500 µg/plate concentration with metabolic activation. However, the precipitate did not interfere with the scoring in those cases.

RANGE-FINDING/SCREENING STUDIES
In the preliminary concentration range finding test (informatory toxicity test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (± S9) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in this test. The number of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No signs of inhibitory, cytotoxic effects were observed in the preliminary experiment in any examined bacterial strain at any concentration with or without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range in all strains.

Validity of the tests:

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. Three replicates were used for each control and test item concentration. The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range in all strains. The examined concentration range was considered to be adequate as the test item was examined up to the recommended maximum concentration. At least five analyzable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Conclusions:

In conclusion, the test item zirconium dinitrate oxide had no mutagenic activity in the examined bacterial strains under the test conditions of this study with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2017 - Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Material tested is a substance related to the reference substance which is considered sufficiently similar to be covered as well by the dossier of the reference substance. This merging is also agreed by the SIEF and is reflected in the substance identity section of the dossier (Section 1.1). Therefore, even though the CAS numbers might differ, the tested material sufficiently represents the reference substance. Therefore, provision of a read across justification is not required.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: This study was performed in an aqueous test system. Due to the use of deionised water as vehicle, the verification of the stability of the test substance in the vehicle is not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock formulations of the test item and serial dilutions were made in deionised water. The final concentration of deionised water in the culture medium was 10 %. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. All formulations were prepared freshly before treatment and used within two hours of preparation.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human blood samples
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors if applicable: one female donor, 35 years old, for the pre-experiment; one male donor, 34 years old, for experiment I; one female donor, 33 years old, for experiment II
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 - 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly. Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium / Ham's F12 (DEMEM / F12, mixture 1 : 1) already supplemented with penicillin / streptomycin (100 U/mL / 100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM / F12, mixture 1 : 1

Cytokinesis block (if used):

Cytochalasin B
Cytotoxicity was measured by a reduced cytokinesis-block proliferation index (CBPI) in 500 cells per culture and expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

2000 µg/mL was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 5.3 to 2000 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item in the absence of S9 mix was observed at the end of treatment at 267 µg/mL and above. Due to a technical error, precipitation at the end of treatment could not be determined in the experimental part in the presence of S9 mix. Therefore, the solubility limit was determined by performing an independent serial dilution under identical conditions. At the end of treatment, precipitation of the test item was observed in the absence of S9 mix at 267 µg/mL and above and in the presence of S9 mix at 49.8 µg/mL and above.
Since the solvent controls of both experimental parts were invalid, this preliminary test was repeated with a top dose of 400 µg/mL (with and without S9 mix) and designated Experiment I. In the absence of S9 mix relevant dose groups were not evaluable, due to the properties of the test item (cells were overlain by precipitation on the slides). Therefore, this part was not evaluated for cytogenetic damage at first and was repeated on request of the Sponsor with additional washing steps during preparation of the cells and with a top dose of 400 µg/mL. Again, cells were overlain by precipitation on the slides, which led to invalidity of this experiment, because only two dose groups were evaluable for cytogenetic damage. For this reason, the experimental part without S9 mix of Experiment I was evaluated for micronuclei after all.
No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 300 µg/mL was chosen as top treatment concentration for Experiment II (without S9 mix), using the standard protocol. This experiment was repeated with the same concentrations due to invalid solvent controls.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of deionised water in the culture medium was 10 %.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

PRE-EXPERIMENT
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hours (with and without S9 mix). The preparation interval was 40 hours after start of the exposure.

CYTOGENETIC EXPERIMENT - PULSE EXPOSURE
About 48 hours after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration and each control. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation, the culture medium was supplemented with approx. 2.5 % S9 fraction (50 µL S9 mix/mL culture medium). After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose * H2O, 192 mg/L Na2HPO4 * 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once (three times for the experimental part with additional washing steps) as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

CYTOGENETIC EXPERIMENT - CONTINUOUS EXPOSURE (without S9 mix)
About 48 hours after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration and each control. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were resuspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

PREPARATION OF CELLS
The cultures were harvested by centrifugation 40 hours after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in approximately 5 mL "saline G" and spun down once again by centrifugation for 5 minutes. The washing procedure was repeated once as described for the experimental part with additional washing steps. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.

DETERMINATION OF CYTOTOXICITY
- Method: percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
- None of the test item concentration exhibits a statistically significant increase compared with the concurrent solvent control.
- There is no concentration-related increase.
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control range (95 % control limit realised as 95 % confidence interval).
A test item can be classified as clastogenic and aneugenic if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control.
- The increase is concentration-related in at least one experimental condition.
- The results are outside the range of the laboratory historical solvent control data (95 % control limit realised as 95 % confidence interval).

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of "R", a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: human

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: test item was soluble in water
- Precipitation: In Experiment I, precipitation of the test item in the culture medium was observed at 400 µg/mL in the absence of S9 mix and at 185 µg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 115 µg/mL and above at the end of treatment.
- Definition of acceptable cells for analysis: Cells showing a clearly visible cytoplasm area. Micronuclei should only be evaluated in cells that have completed mitosis during exposure to the test item or during post-exposure period.

RANGE-FINDING/SCREENING STUDIES: Precipitation of the test item was observed in the absence of S9 mix at 267 µg/mL and above and in the presence of S9 mix at 49.8 µg/mL and above

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
Experiment I without S9 mix: 0.4 %
Experiment I with S9 mix: 0.35 %
Experiment I solvent control: 0.55 %
Experiment II without S9 mix: 0.55 %
Experiment II solvent control: 0.5 %
- Indication whether binucleate or mononucleate where appropriate: binucleated cells

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%))
- Positive historical control data:
without S9 mix, pulse treatment: 14.63 %
without S9 mix, continuous treatment: 3.68 %
with S9 mix, pulse treatment: 5.45 %
- Negative (solvent/vehicle) historical control data:
without S9 mix, pulse treatment: 0.62 %
without S9 mix, continuous treatment: 0.56 %
with S9 mix, pulse treatment: 0.73 %

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI (Cytochalasin B)

Table 1: Summary of results

Exp.	Prepearation interval	Test item concentration [µg/mL]	Proliferation index CBPI	Cytostasis [%]*	Micronucleated cells [%]**	95 % Control limit
Exposure period 4 hours without S9 mix
I	40 hours	Solvent control^1 Positive control² 185 222 267	1.88 1.45 1.99 1.98 1.96	48.7 n.c. n.c. n.c.	0.60 7.95^S 0.30 0.55 0.30	0.06 – 1.19 3.92 – 25.34
Exposure period 20 hours without S9 mix
II	40 hours	Solvent control^1 Positive control³ 28.7 57.4 115^P	1.57 1.30 1.68 1.70 1.72	47.1 n.c. n.c. n.c.	0.50 4.15^S 0.45 0.70 0.50	0.00 – 1.11 1.47 – 5.89
Exposure period 4 hours with S9 mix
I	40 hours	Solvent control^1 Positive control^4 129 154 185^P	2.17 1.56 2.02 2.02 2.05	52.4 13.1 12.7 10.1	0.55 5.75^S 0.30 0.15 0.60	0.08 – 1.38 0.70 – 10.20

*For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^PPrecipitation occured at the end of treatment

^SThe number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

¹Deionized water 10.0 % (v/v)

² MMC 0.8 µg/mL

³ Demecolcine 100 ng/mL

⁴CPA 15.0 µg/mL

Table 2: Toxicity - Pretest

Concentration [µg/mL]	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis [%]
Without S9 mix
Solvent control 5.3 9.3 16.2 28.4 49.8 87.1 152 267^P 667^P 2000^P	4 hours	40 hours	1.95 1.96 1.92 1.92 1.89 1.92 1.94 1.96 n.e. n.e. n.e.	- n.c. 3.1 2.5 5.6 3.0 0.5 n.c. n.e. n.e. n.e.
With S9 mix
Solvent control 5.3 9.3 16.2 28.4 49.8^P 87.1^P 152^P 267^P 667^P 2000^P	4 hours	40 hours	1.87 1.85 1.84 1.82 1.83 1.83 1.95 1.92 1.86 n.e. n.e.	- 2.1 3.6 6.2 4.4 4.4 n.c. n.c. 1.5 n.e. n.e.

*Mean value of two cultures

^PPrecipitation was observed at the end of treatment by the unaided eye in an additional experiment for solubility testing

n.e. Not evaluable due to strong precipitation

n.c. Not calculated as the CBPI was equal or higher than solvent control value

Table 3: Historical solvent control data

Micronucleated cells in [%]
	Without S9 mix		With S9 mix
	Pulse treatment (4/40)	Continuous treatment (20/40)	Pulse treatment (4/40)
No. of experiments	61*	65**	80***
Mean	0.62	0.56	0.73
95 % Control limit	0.06 – 1.19	0.00 – 1.11	0.08 – 1.38
1 x SD	0.28	0.28	0.33
Min – Max	0.00 – 1.18	0.05 – 1.10	0.10 – 1.85

*Aqueous solvents – 21 Experiments; Organic solvents – 40 Experiments

** Aqueous solvents – 24 Experiments; Organic solvents – 41 Experiments

*** Aqueous solvents – 30 Experiments; Organic solvents – 50 Experiments

Table 4: Historical positive control data

Micronucleated cells in [%]
	Without S9 mix		With S9 mix
	Pulse treatment (4/40)	Continuous treatment (20/40)	Pulse treatment (4/40)
	MMC	Demecolcin	CPA
No. of experiments	62	65	86
Mean	14.63	3.68	5.45
95 % Control limit	3.92 – 25.34	1.47 – 5.89	0.70 – 10.20
1 x SD	5.35	1.11	2.37
Min – Max	2.60 – 28.50	2.10 – 8.80	2.25 – 13.30

 

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating or to the highest evaluable concentrations.

Executive summary:

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments.

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in the pre-experiment (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. The highest concentration, which showed precipitation on the slides by the unaided eye, however, was not evaluable for cytogenetic damage. In Experiment I in the presence of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In both experiments in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating or to the highest evaluable concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Material tested is a substance related to the reference substance which is considered sufficiently similar to be covered as well by the dossier of the reference substance. This merging is also agreed by the SIEF and is reflected in the substance identity section of the dossier (Section 1.1). Therefore, even though the CAS numbers might differ, the tested material sufficiently represents the reference substance. Therefore, provision of a read across justification is not required.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: This study was performed in an aqueous test system. Due to the use of culture medium (Ham's F12) as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in culture medium (Ham's F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance solutions were prepared immediately before administration. To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before removal.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Suspension

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length, doubling time or proliferation index: about 12 - 16 hours
- Number of passages if applicable: at least 2 passages were performed before cells were taken for the experiment; a further passage was also necessary in order to prepare test cultures
- Methods for maintenance in cell culture if applicable: For cell cultivation, deep-frozen cell suspensions were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL Ham's F12 medium including 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: about 12 - 16 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) fetal calf serum (FCS), supplemented with 1 % (v/v) penicillin / streptomycin (stock solution: 10000 IU / 10000 µg/mL) and 1 % (v/v) amphotericin B (stock solution: 250 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Based on the solubility properties of the test substance and the data and the observations from the pre-test and taking into account the current guidelines, the following doses were selected in this study:
- 1st Experiment: 1.09; 2.19; 4.38; 8.75; 17.50; 35.00 and 70.00 µg/mL
- 2nd Experiment: 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 and 70.00 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (Ham's F12)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 20 * 10^6 cells in 40 mL

DURATION
- Attachment period: 20 - 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 6 - 8 days
- Selection time (if incubation with a selection agent): one week
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 15 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: pH, osmolality, solubility

Evaluation criteria:

A test substance is considered to be clearly positive if all following critera are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MFcorr.) exceed both the concurrent negative / vehicle control value and the range of the laboratory's historical negative control data (95 % control limit).
Isolated iincreases of mutant frequencies above the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups are close to the concurrent vehicle control value and within the range of the laboratory's historical negative control data (95 % control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels are printed in the tables. However, both biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Water solubility: The test substance was poorly soluble in many vehicles.
- Precipitation: In the absence and presence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at 35.00 µg/mL and above and at 50.00 µg/mL and above in the 1st and the 2nd Experiment, respectively.

RANGE-FINDING/SCREENING STUDIES:
No cytotoxicity was observed in the pre-test when tested up to the highest tested concentration of 500.0 µg/mL, however, test substance precipitation was observed from 31.3 µg/mL onwards. Therefore, in the main experiments of this HPRT study concentrations at the border of solubility in culture medium were tested for gene mutations.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: without S9 mix: MF(corr.) = 42.47 - 419.90 per 10^6 cells; with S9 mix: MF(corr.) = 21.52 - 270.48 per 10^6 cells
- Negative (solvent/vehicle) historical control data: without S9 mix: MF(corr.) = 0.00 - 5.97 per 10^6 cells; with S9 mix: MF(corr.) = 0.00 - 7.91 per 10^6 cells

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cloning efficiency

The dose selection of this study was based on the solubility properties of the test substance in culture medium in accordance with the recommendations of the current guidelines.

According to the results of the present in vitro study, in two experiments performed independently of each other, the test substance did not lead to a biologically relevant or dose-dependent increase the number of mutant colonies, either without S9 mix or after the addition of a metabolising system. The mutant frequencies at any concentration were within the range of the concurrent negative control values and within the 95 % control limit of the historical negative control data.

Additionally, no statistically significant dose-dependent increase in mutant colonies was observed in any experimental part of this study after 4 hours treatment either in the absence or presence of metabolic activation.

The mutation frequencies of the vehicle control groups were within the historical negative control data range (95 % control limit) and thus fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory's historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.

The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and thus fulfilled the acceptance criteria of this study.

Table 1: Pre-test (4-hour exposure)

Test groups Doses	S9 mix	pH value	Solubility				Relative survival [%]
			Vehicle Ma	Culture medium
				0 h	3 – 4 h
				Ma	Ma	Mi
Negative control 2.0 µg/mL 3.9 µg/mL 7.8 µg/mL 15.6 µg/mL 31.3 µg/mL 62.5 µg/mL 125.0 µg/mL 250.0 µg/mL 500.0 µg/mL	- - - - - - - - - -	7.3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 7.3	n.d. S S S S Su Su Su Su Su	n.d. S S S S P P P P P	n.d. S S S S P P P P P	n.d. S S P P P P P P P	100.0 124.4 133.5 130.6 118.2 119.5 113.8 113.2 106.1 109.9
Negative control 2.0 µg/mL 3.9 µg/mL 7.8 µg/mL 15.6 µg/mL 31.3 µg/mL 62.5 µg/mL 125.0 µg/mL 250.0 µg/mL 500.0 µg/mL	+ + + + + + + + + +	7.3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 7.3	n.d. S S S S Su Su Su Su Su	n.d. S S S S P P P P P	n.d. S S S S P P P P P	n.d. S S S S P P P P P	100.0 122.8 153.5 129.3 128.4 126.3 135.0 105.9 113.6 123.2

Ma macroscopically

Mi microscopically

n.d. not determined

S solution

Su suspension

P precipitation

Table 2: Mutant frequency and viability (4-hour exposure)

Exp.	S9 mix	Test groups [µg/mL]	Cloning efficiency 2 (viability)				Mutant frequency
			Number of colonies		CE2		Number of colonies^a		MF (per 10^6cells)
			Dish 1	Dish 2	Abs. [%]	Rel. [%]	Flask 1	Flask 2	Uncorrected	Corrected^b
1	- - - - - - - - -	Negative control 1.09 2.19 4.38 8.75 17.50 35.00 70.00 EMS 400.0	157 n.c.^1 n.c.^1 175 188 149 131 n.c.^2 152	171 n.c.^1 n.c.^1 158 133 172 163 n.c.^2 163	82.0 n.c.^1 n.c.^1 83.3 80.3 80.3 73.5 n.c.^2 78.8	100.0 n.c.^1 n.c.^1 101.5 97.9 97.9 89.6 n.c.^2 96.0	6 n.c.^1 n.c.^1 5 1 5 3 n.c.^2 242	4 n.c.^1 n.c.^1 4 0 1 5 n.c.^2 215	2.50 n.c.^1 n.c.^1 2.25 0.25 1.50 2.00 n.c.^2 114.25	3.05 n.c.^1 n.c.^1 2.70 0.31 1.87 2.72 n.c.^2 145.08^s
1	+ + + + + + + + +	Negative control 1.09 2.19 4.38 8.75 17.50 35.00 70.00 DMBA 1.25	142 n.c.^1 n.c.^1 125 126 153 108 n.c.^2 107	155 n.c.^1 n.c.^1 152 131 130 116 n.c.^2 112	74.3 n.c.^1 n.c.^1 69.3 64.3 70.8 56.0 n.c.^2 54.8	100.0 n.c.^1 n.c.^1 93.3 86.5 95.3 75.4 n.c.^2 73.7	8 n.c.^1 n.c.^1 7 1 4 1 n.c.^2 154	4 n.c.^1 n.c.^1 5 1 1 0 n.c.^2 144	3.00 n.c.^1 n.c.^1 3.00 0.50 1.25 0.25 n.c.^2 74.50	4.04 n.c.^1 n.c.^1 4.33 0.78 1.77 0.45 n.c.^2 136.07^s
2	- - - - - - - - -	Negative control 1.56 3.13 6.25 12.50 25.00 50.00 70.00 EMS 400.0	158 n.c.^1 n.c.^1 150 179 140 137 n.c.^2 148	145 n.c.^1 n.c.^1 157 171 161 141 n.c.^2 134	75.8 n.c.^1 n.c.^1 76.8 87.5 75.3 69.5 n.c.^2 70.5	100.0 n.c.^1 n.c.^1 101.3 115.5 99.3 91.7 n.c.^2 93.1	4 n.c.^1 n.c.^1 3 3 3 0 n.c.^2 230	0 n.c.^1 n.c.^1 4 2 6 6 n.c.^2 255	1.00 n.c.^1 n.c.^1 1.75 1.25 2.25 1.50 n.c.^2 121.25	1.32 n.c.^1 n.c.^1 2.28 1.43 2.99 2.16 n.c.^2 171.99^s
2	+ + + + + + + + +	Negative control 1.56 3.13 6.25 12.50 25.00 50.00 70.00 DMBA 1.25	168 n.c.^1 n.c.^1 143 126 131 136 n.c.^2 138	183 n.c.^1 n.c.^1 149 136 149 137 n.c.^2 122	87.8 n.c.^1 n.c.^1 73.0 65.5 70.0 68.3 n.c.^2 65.0	100.0 n.c.^1 n.c.^1 83.2 74.6 79.8 77.8 n.c.^2 74.1	3 n.c.^1 n.c.^1 1 1 2 0 n.c.^2 212	5 n.c.^1 n.c.^1 2 5 6 1 n.c.^2 205	2.00 n.c.^1 n.c.^1 0.75 1.50 2.00 0.25 n.c.^2 104.25	2.28 n.c.^1 n.c.^1 1.03 2.29 2.86 0.37 n.c.^2 160.38^s

^anumber of colonies 7 days after seeding about 2 * 10⁶cells/flask in selection medium

^bcorrection on the basis of the absolute cloning efficiency 2 at the end of the expression period

^smutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c.¹culture was not continued since a minimum of only four analysable concentrations is required

n.c.²culture was not continued since only one concentration beyond the solubility limit is required

Table 3: Cytotoxicity data (4-hour exposure)

Exp.	S9 mix	Test groups [µg/mL]	Cell density at the end of treatment [* 10^5/mL]	Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)					Relative survival [%]
				Number of colonies		Abs. [%]	Rel. [%]	Adjusted [%]
				Dish 1	Dish 2
1	- - - - - - - - -	Negative control 1.09 2.19 4.38 8.75 17.50 35.00 70.00 EMS 400.0	8.28 8.84 8.61 8.45 8.73 8.05 8.90 8.41 8.70	157 166 160 167 152 186 158 n.c.^2 130	164 151 172 149 161 179 153 n.c.^2 107	80.3 79.3 83.0 79.0 78.3 91.3 77.8 n.c.^2 59.3	100.0 98.8 103.4 98.4 97.5 113.7 96.9 n.c.² 73.8	132.9 140.1 142.9 133.5 136.6 146.9 138.4 n.c.² 103.1	100.0 105.4 107.5 100.5 102.8 110.5 104.1 n.c.² 77.6
1	+ + + + + + + + +	Negative control 1.09 2.19 4.38 8.75 17.50 35.00 70.00 DMBA 1.25	7.56 7.72 7.49 11.74 8.19 8.22 8.20 8.18 6.75	156 166 185 139 126 132 135 n.c.² 152	146 162 153 135 146 137 138 n.c.² 151	75.5 82.0 82.0 68.5 68.0 67.3 68.3 n.c.² 75.8	100.0 108.6 108.6 90.7 90.1 89.1 90.4 n.c.² 100.3	114.2 126.6 122.8 160.8 111.4 110.6 111.9 n.c.² 102.3	100.0 110.9 107.6 140.9 97.6 96.8 98.1 n.c.² 89.6
2	- - - - - - - - -	Negative control 1.56 3.13 6.25 12.50 25.00 50.00 70.00 EMS 400.0	10.60 11.92 11.83 12.57 11.74 11.46 11.21 11.22 12.86	196 188 177 180 191 194 181 n.c.² 123	192 192 182 185 196 189 197 n.c.² 135	97.0 95.0 89.8 91.3 96.8 95.8 94.5 n.c.² 64.5	100.0 97.9 92.5 94.1 99.7 98.7 97.4 n.c.² 66.5	205.6 226.5 212.3 229.4 227.2 219.5 211.9 n.c.² 165.9	100.0 110.1 103.3 111.6 110.5 106.7 103.0 n.c.² 80.7
2	+ + + + + + + + +	Negative control 1.56 3.13 6.25 12.50 25.00 50.00 70.00 DMBA 1.25	8.02 9.16 9.39 9.51 9.60 9.93 9.72 9.83 10.23	189 177 155 173 164 148 148 n.c.² 148	192 190 164 187 130 165 141 n.c.² 145	95.3 91.8 79.8 90.0 73.5 78.3 72.3 n.c.² 73.3	100.0 96.3 83.7 94.5 77.2 82.2 75.9 n.c.² 76.9	152.8 168.1 149.8 171.2 141.1 155.4 140.5 n.c.² 149.9	100.0 110.0 98.0 112.0 92.4 101.7 91.9 n.c.² 98.1

n.c.²culture was not continued since only one concentration beyond the solubility limit is required

Table 4: Historical Negative Control Data (with and without S9 mix, all vehicles*)

	Corrected Mutant Frequency**
Exposure period	4 hours
Mean	2.35
Minimum	0.00
Maximum	9.93
Standard deviation	2.21
95 % Lower Control Limit	0.00
95 % Upper Control Limit	6.84
No. of Experiments	50

* culture medium, water 10 % (v/v), DMSO 1 % (v/v), acetone 1 % (v/v)

** mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE₂)

Table 5: Historical Positive Control Data - without S9 mix, 400 µg/mL EMS

	Corrected Mutant Frequency*
Exposure period	4 hours
Mean	177.96
Minimum	42.47
Maximum	419.90
Standard deviation	110.66
95 % Lower Control Limit	0.00
95 % Upper Control Limit	410.86
No. of Experiments	25

* mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE₂)

Table 6: Historical Positive Control Data - with S9 mix, 1.25 µg/mL DMBA

	Corrected Mutant Frequency*
Exposure period	4 hours
Mean	120.59
Minimum	21.52
Maximum	270.48
Standard deviation	63.59
95 % Lower Control Limit	0.00
95 % Upper Control Limit	254.44
No. of Experiments	25

* mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE₂)

Conclusions:

In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital and ß-naphthoflavone induced rats (exogeneous metabolic activation).

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, the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 1.09; 2.19; 4.38; 8.75; 17.50; 35.00; 70.00 µg/mL

with S9 mix

0; 1.09; 2.19; 4.38; 8.75; 17.50; 35.00; 70.00 µg/mL

2nd Experiment

without S9 mix

0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 70.00 µg/mL

with S9 mix

0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 70.00 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, 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, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

Dose selection for genotoxicity testing based on the solubility properties of the test substance in culture medium. Test substance concentration from 1000.9 µg/mL onwards could be dissolved in culture medium, however, with a significant shift in the pH values towards acidity. Adjustment of the pH values led to test substance precipitation. Thus, test substance precipitation was observed from 35.0 µg/mL onwards at neutral pH in the 1st Experiment and from 50.0 µg/mL onwards at neutral pH in the 2nd Experiment.

In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolising 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation in bacteria

The Ames experiments with zirconium dinitrate oxide were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone induced rats. This test was performed according to OECD guideline 471 and in accordance with GLP and was thus scored as Klimisch 1.

The study included a preliminary compatibility test, a preliminary concentration range finding test (informatory toxicity test), and two independently performed main tests (initial mutation test (plate incorporation method) and confirmatory mutation test (pre-incubation method)).

Based on the available information and the results of a solubility test, the test item was formulated in distilled water. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the preliminary concentration range finding test. Based on the observed mutation factor values, solubility and cytotoxicity results of the preliminary experiment, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

In the initial mutation test and confirmatory mutation test, none of the observed revertant colony numbers were above the respective biological threshold value of 2 (Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA) or 3 (Salmonella typhimurium TA1535 and TA1537) either in the presence or absence of metabolic activation. There were no dose-related trends and no indication of any treatment effect. In all test item groups, the numbers of revertant colonies did not exceed the biological threshold value when compared to the vehicle control and were within the normal biological variability of the test system.

Precipitate / slight precipitate was detected on the plates in the main tests in all examined bacterial strains with and without metabolic activation, but the precipitate did not interfere with the scoring.

No inhibitory, cytotoxic effects of the test item were seen in the main tests (initial mutation test and confirmatory mutation test) in any examined strains with and without metabolic activation.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range and the mean number of revertant colonies of the positive control substances showed the expected increase. The viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests; the examined concentration range was considered to be adequate. The study was considered to be valid.

The data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial strains used. In conclusion, the test item zirconium dinitrate oxide had no mutagenic activity in the examined bacterial strains under the test conditions of this study.

In vitro cytogenicity in mammalian cells

An in vitro mammalian cell micronucleus test (Envigo, 2018) in human lymphocytes (OECD 487) was performed with zirconium oxynitrate hydrate (CAS 14985-18-3), the hydrated form of the reference substance zirconium dinitrate oxide.

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments.

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in the pre-experiment (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In Experiment I in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. The highest concentration, which showed precipitation on the slides by the unaided eye, however, was not evaluable for cytogenetic damage. In Experiment I in the presence of S9 mix and in Experiment II in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In both experiments in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating or to the highest evaluable concentrations.

In vitro gene mutation in mammalian cells

An in vitro gene mutation assay in mammalian cells (HPRT, BASF, 2018, OECD 476) was performed with zirconium oxynitrate hydrate (CAS 14985-18-3), the hydrated form of the reference substance zirconium dinitrate oxide.

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital and ß-naphthoflavone induced rats (exogeneous metabolic activation).

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, the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

- without S9 mix (0; 1.09; 2.19; 4.38; 8.75; 17.50; 35.00; 70.00 µg/mL)

- with S9 mix (0; 1.09; 2.19; 4.38; 8.75; 17.50; 35.00; 70.00 µg/mL)

2nd Experiment

- without S9 mix (0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 70.00 µg/mL)

- with S9 mix (0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 70.00 µg/mL)

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, 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, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. Test substance concentrations from 1000.9 µg/mL onwards could be dissolved in culture medium, however, with a significant shift in the pH values towards acidity. Adjustment of the pH values led to test substance precipitation. Test substance precipitation was observed from 35.0 µg/mL onwards at neutral pH in the 1st Experiment and from 50.0 µg/mL onwards at neutral pH in the 2nd Experiment.

In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolising 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.

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation, zirconium dinitrate oxide should not be classified as a mutagenic compound.