[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity in vitro (Ames) (Read-across from ytterbium trifluoride, Thompson, 2017):

The test material was considered to be non-mutagenic.

Genetic Toxicity in vitro (Ames) (Read-across from ytterbium oxide, Váliczkó, 2017):

The test material was considered to be non-mutagenic.

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:

07 December 2016 to 10 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark over silica gel

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Storage: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
MEDIA USED
- Type and identity of media: overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Storage: All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
MEDIA USED
- Type and identity of media: overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The the maximum recommended dose level was 5000 µg/plate.
Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate. The dose range used for Experiment 2 was determined by the results of Experiment 1. Six test material concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test material following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle. Sterile distilled water was not evaluated as a vehicle in this test system as information provided by the Sponsor suggested the test material is insoluble in water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene and 4-Nitroquinoline-1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

TEST FOR MUTAGENICITY: EXPERIMENT 1 – PLATE INCORPORATION METHOD
- Without Metabolic Activation
0.1 mL of the appropriate concentration of test material, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test material, appropriate positive, vehicle and negative control, and each bacterial strain, was assayed in triplicate.
- With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test material formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
- Incubation and Scoring
All of the plates were incubated at 37 ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

TEST FOR MUTAGENICITY: EXPERIMENT 2 – PRE-INCUBATION METHOD
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
- Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test material formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
- With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test material formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
- Incubation and Scoring
All of the plates were incubated at 37 ± 3°C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows; TA1535: 7 to 40, TA100: 60 to 200, TA1537: 2 to 30, TA98: 8 to 60 and WP2uvrA: 10 to 60.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

EVALUATION CRITERIA
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentration.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium, other: TA100, TA1535, TA98, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test material formulation was also shown to be sterile.

- Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

- A summary of the results for both experiments, with and without metabolic activation are presented in Tables 1 and 2.

- The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

- There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 2 (pre incubation method).

- The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Table 1: Summary of Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 1.5 5 15 50 150 500 1500 5000	69 68 64 68 72 69 65 69 63	28 29 28 33 31 30 30 29 29	19 20 25 17 18 22 23 24 21	15 13 17 20 19 16 16 11 22	12 7 10 11 13 14 10 13 13
+	Solvent 1.5 5 15 50 150 500 1500 5000	76 73 78 77 76 80 78 67 69	29 26 29 27 30 27 26 27 31	23 31 22 23 28 22 26 24 27	21 24 25 14 19 30 25 23 24	8 8 8 11 11 8 10 11 12
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	280	566	234	132	530
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	2222	265	332	88	305

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Table 2: Summary of Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 15 50 150 500 1500 5000	97 109 106 101 99 99 97	9 12 10 13 12 11 11	16 13 16 17 16 17 13	17 13 15 13 15 17 15	9 8 11 7 12 10 7
+	Solvent 15 50 150 500 1500 5000	99 94 104 104 122 107 115	12 8 9 8 8 12 7	20 24 19 16 22 20 22	22 20 21 15 21 17 17	12 9 13 10 13 15 13
Positive Controls
-	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentration (µg/plate)	3	5	2	0.2	80
	Mean no. colonies/plate	1224	1021	373	272	483
+	Name	2AA	2AA	2AA	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	1789	231	173	108	506

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

Conclusions:

Under the conditions of this study the test material was considered to be non-mutagenic.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B13/14, EPA OCSPP870.5100 and the major Japanese Regulatory Authorities including METI, MHLW and MAFF. The reverse mutation assay was performed under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test material concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test material following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 2 (pre incubation method). 

Under the conditions of this study the test material was considered to be non-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 July 2016 to 12 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

No correction for purity of the test material was applied.

Target gene:

histidine and tryptophan

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:

Type and composition of metabolic activation system:
- source of S9: cofactor-supplemented post-mitochondrial S9 fraction (rat liver)
- composition of S9 mix: 400 mL Salt solution for S9 Mix (NADP Na: 7.66 g, D-glucose-6 phosphate Na: 3.53 g, MgCl2 x 6 H2O: 4.07 g, KCl: 6.15 g, Distilled water q.s. ad 1000 mL and filter sterilised at 0.22 µm); 500 mL ice cold 0.2 M sodium phosphate buffer (pH 7.4) and 100 mL rat liver homogenate (S9).

Test concentrations with justification for top dose:

100 mg/mL (5000 μg/plate), 31.62 mg/mL (1581 μg/plate), 10 mg/mL (500 μg/plate), 3.162 mg/mL (158.1 μg/plate), 1 mg/mL (50 μg/plate), 0.3162 mg/mL (15.81 μg/plate) and 0.1 mg/mL (5 μg/plate)

The selection of the doses was based on the results of a range finding study, in which doses of 10, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate were used.
The observed number of revertant colonies was in the normal range. Minor differences compared to the solvent control numbers were observed.
However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
Slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate.
Inhibitory or toxic effects of the test material were not detected in the Preliminary Range Finding Test.

Based on the results of the preliminary tests, a 100 mg/mL stock formulation was prepared in dimethyl sulfoxide (DMSO). Seven test concentrations were prepared by successive dilutions of the stock formulation, to obtain lower doses. The maximum test concentration was 5000 μg test material/plate.
Dimethyl sulfoxide (DMSO) was used as solvent to prepare the stock formulation of the test material. Test suspensions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock formulation using the selected solvent and were used within 4 hours after preparation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The appropriate vehicle (solvent) and the behaviour of the test material formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. The solubility of the test material was examined using distilled water, dimethyl sulfoxide (DMSO), ethanol, acetone and N,N-dimethylformamide (DMF). The formulations at 100 mg/mL concentration using distilled water, ethanol or acetone as vehicle were suspensions with fast sedimentation. Slower sedimentation was observed in the formulations using DMSO or DMF as vehicle. Due to the better biocompatibility, DMSO was selected as vehicle for the test with continuous stirring of the formulations.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (TA98: without metabolic activation), 2-aminoanthracene (WP2 uvrA and TA98, TA100, TA1535 and TA1537: with metabolic activation)

Details on test system and experimental conditions:

INITIAL MUTATION TEST
- Method of application: in agar (plate incorporation)
- Bacteria (cultured in Nutrient Broth No.2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly and added to the overlay (45°C).
- The content of the tubes: top agar 2000 μL; vehicle or test material formulation (or reference controls) 50 μL; overnight culture of test strain 100 μL; phosphate buffer (pH 7.4) or S9 mix 500 μL.
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.

CONFIRMATORY MUTATION TEST (pre-incubation method)
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since no biologically relevant increase in the number of revertant colonies was observed in the Initial Mutation Test.
- Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
- Before the overlaying, the test material formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test material (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator.
- After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of nonactivated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.

EVALUATION OF EXPERIMENTAL DATA
- The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test material and for the controls using Microsoft Excel TM software.
* Mutation factor (MF): mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate.

In the main tests each sample (including the controls) was tested in triplicate.

VALIDITY CRITERIA
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests;
- the selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate;
- no more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Evaluation criteria:

A test material was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

A test material was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.

Statistics:

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

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:

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:

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:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
- The observed number of revertant colonies was in the normal range. Minor differences compared to the solvent control numbers were observed. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
- Slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate.
- Inhibitory or toxic effects of the test material were not detected in the Preliminary Range Finding Test.
- Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

INITIAL AND CONFIRMATORY MUTATION TESTS
- Slight precipitate was observed at 5000 μg/plate with and without metabolic activation in all tester strains in the Initial Mutation Test and in all Salmonella typhimurium strains in the Confirmatory Mutation Test.
- Slight decreases of the revertant counts were observed compared to the solvent control sporadically in the study. However, the mean number of revertant colonies was within the historical control range, thus they were considered as biological variability of the test system.
- No signs of inhibitory, cytotoxic effect of the test material (such as reduced background lawn development and/or reduced number of revertant colonies) were observed in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any concentrations with or without metabolic activation.
- In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
- In the Initial Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain with metabolic activation at the concentration of 50 μg/plate. The mutation factor value was 1.52. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
- In the Confirmatory Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 500 μg/plate concentration with metabolic activation. The calculated mutation factor value at this dose level was 1.60. However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
- Slight increases in the numbers of revertant colonies were detected compared to the solvent control during the study in some sporadic cases. However, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and were within the historical control range. They were considered as reflecting the biological variability of the test.

VALIDITY OF TESTS
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range-finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event.

The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Conclusions:

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B13/14, and EPA OPPTS 870.5100. The reverse mutation assay was performed under GLP conditions.

The test material was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments 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/β-naphthoflavoneinduced rats. The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method). Based on the results of the Compatibility Test, the test material was dissolved in DMSO at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material was tested in the Initial Mutation Test and Confirmatory Mutation Test in tester strains Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA, in the absence and presence of metabolic activation at concentrations of 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

Slight precipitate was observed at 5000 μg/plate with and without metabolic activation in all tester strains in the Initial Mutation Test and in all Salmonella typhimurium strains in the Confirmatory Mutation Test. There were no signs of inhibitory, cytotoxic effect of the test material in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any of the concentrations tested with or without metabolic activation. In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the solvent control plates were within the historical control range and the reference mutagens showed the expected increase in the number of revertant colonies, indicating that the test conditions were adequate. In addition, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range-finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The substances are structurally similar, both ytterbium compounds are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA100, TA1535, TA98, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions of this study the test material was considered to be non-mutagenic.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B13/14, EPA OCSPP870.5100 and the major Japanese Regulatory Authorities including METI, MHLW and MAFF. The reverse mutation assay was performed under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test material concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test material following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 2 (pre incubation method). 

Under the conditions of this study the test material was considered to be non-mutagenic.

 

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The substances are structurally similar, both ytterbium compounds are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed.

Reason / purpose for cross-reference:

read-across source

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:

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:

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

Conclusions:

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B13/14, and EPA OCSPP870.5100. The reverse mutation assay was performed under GLP conditions.

The test material was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments 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/β-naphthoflavoneinduced rats. The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method). Based on the results of the Compatibility Test, the test material was dissolved in DMSO at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material was tested in the Initial Mutation Test and Confirmatory Mutation Test in tester strains Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA, in the absence and presence of metabolic activation at concentrations of 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

Slight precipitate was observed at 5000 μg/plate with and without metabolic activation in all tester strains in the Initial Mutation Test and in all Salmonella typhimurium strains in the Confirmatory Mutation Test. There were no signs of inhibitory, cytotoxic effect of the test material in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any of the concentrations tested with or without metabolic activation. In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the solvent control plates were within the historical control range and the reference mutagens showed the expected increase in the number of revertant colonies, indicating that the test conditions were adequate. In addition, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range-finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Read-across argument

The read across substances are structurally similar, both are ytterbium compounds that are in their +3 oxidation state. As the metal cation governs toxicity, it is concluded that the same effect will be observed for the read across substances as for the substance object of this registration.

An overview of the available information on the read across substances is provided below.

Genetic Toxicity in vitro (Ames) (Read-across from ytterbium triflouride, Thompson, 2017):

The genetic toxicity of the test material was determined in accordance with the standardised guidelines OECD 471, EU Method B13/14, EPA OCSPP870.5100andthe major Japanese Regulatory Authorities including METI, MHLW and MAFF. The reverse mutation assay was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test material concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test material following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test material in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix), in Experiment 2 (pre incubation method). 

Under the conditions of this study the test material was considered to be non-mutagenic.

 

Genetic Toxicity in vitro (Ames) (Read-across from ytterbium oxide, Váliczkó, 2017):

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471, EU Method B13/14, and EPA OCSPP870.5100. The reverse mutation assay was performed under GLP conditions.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997)

The test material was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments 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/β-naphthoflavoneinduced rats. The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method). Based on the results of the Compatibility Test, the test material was dissolved in DMSO at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material was tested in the Initial Mutation Test and Confirmatory Mutation Test in tester strains Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA, in the absence and presence of metabolic activation at concentrations of 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

Slight precipitate was observed at 5000 μg/plate with and without metabolic activation in all tester strains in the Initial Mutation Test and in all Salmonella typhimurium strains in the Confirmatory Mutation Test. There were no signs of inhibitory, cytotoxic effect of the test material in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any of the concentrations tested with or without metabolic activation. In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect. The mean values of revertant colonies of the solvent control plates were within the historical control range and the reference mutagens showed the expected increase in the number of revertant colonies, indicating that the test conditions were adequate. In addition, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range-finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.