Amidinourea phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse Mutation Assay using Bacteria (Salmonella typhimurium)

In this bacterial reverse mutation assay (Ames test) and under the experimental conditions reported, GUP did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, GUP is considered to be non-mutagenic in this bacterial reverse mutation assay.

In vitro Mammalian Chromosome Aberration Test in Chinese Hamster V79 cells

GUP in this in vitro chromosome aberration test, under the experimental conditions reported, did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line in the experiments

without and with metabolic activation. Therefore, the test item GUP is considered to be non-clastogenic in this chromosome aberration test.

In vitro Mammalian Cell Gene Mutation Assay (Thymidine Kinase Locus/TK+/') in Mouse Lymphoma L5178Y

In this mutagenicity test, under the experimental conditions reported, the test item GUP is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in

mouse lymphoma L5178Y cells.

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:

13-03-2017 to 11-05-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial
Reverse Mutation Test", adopted 21st July, 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:

Batch No.: 16VL8189
Expiry Date: 03 August 2017
Storage Conditions: room temperature, protected from light

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 μg/plate was selected as
the maximum concentration. The concentration range covered two logarithmic decades.

Pre-Experiment for Toxicity: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Exposure Concentrations: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

Water or DMSO - dimethylsulfoxide.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD; 4-nitro-o-phenylene-diamine; 2-AA; 2-aminoanthracene

Details on test system and experimental conditions:

Five strains of S. typhimurium with the following characteristics were used:
TA 98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations

TA 100:
his G 46; rfa-; uvrB-; R-factor: base-pair substitutions

TA 1535:
his G 46; rfa-; uvrB-: base-pair substitutions

TA 1537:
his C 3076; rfa-; uvrB-: frame shift mutations

TA 102:
his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

Pre-Experiment for Toxicity

The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Exposure Concentrations

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see chapter 12.1.1 Pre-Experiment). 5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate
As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.

Experimental Performance

For the plate incorporation method, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 μL Overlay agar.
For the pre-incubation method 100 μL of the test item preparation was pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Rationale for test conditions:

See section above

Evaluation criteria:

Evaluation of Cytotoxicity

Cytotoxicity was detected by a diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approx. ≤ 0.5 in relation to the solvent control.

Criteria of Validity
A test was considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- negative control plates (water) =/- S9 mix were within the mean values of the spontaneous reversion frequency for the laboratory’s historical control data range (2014 -2016)
- corresponding background growth on negative control, solvent control and test plates was observed
- positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity
Mutation Factor was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control.
A test item was considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain +/- metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups was non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 μg/plate (without metabolic activation)

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

See attached results tables in attached background material section

Conclusions:

GUP was considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In this bacterial reverse mutation assay (Ames test) and under the experimental conditions reported, GUP did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, GUP is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-03-2017 to 13-11-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Specific details on test material used for the study:

Batch No.: 16VL8189
Expiry Date: 03 August 2017
Storage Conditions: room temperature, protected from light

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells in vitro are widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the Eurofins Munich V79 in stock cultures: 12 - 14 h) and a high plating efficiency of untreated cells (normal

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Homogenate

Test concentrations with justification for top dose:

Experiment I:
without and with metabolic activation: 50, 100 and 250 μg/mL

Experiment II:
without metabolic activation: 100, 250 and 500 μg/mL

A pre-experiment was conducted under identical conditions as described for the experiment I. The following concentrations were tested without and with S9 mix:
5, 10, 25, 50, 100, 250, 500, 1000, 1500 and 2000 μg/mL

Cytotoxicity was characterised by the relative increase in cell count (RICC) in comparison with the controls. In general the culturing and experimental conditions were the same as described for the experiment I.

Vehicle / solvent:

Minimum Essential Medium cell culture medium (MEM + 10% Fetal Bovine Serum).

Untreated negative controls:

yes

Remarks:

MEM cell culture medium (MEM + 10% FBS).

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

V79 cells in vitro are widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are chosen because of their relatively small number of chromosomes (diploid number, 2n = 22), their high proliferation rate (doubling time of the Eurofins Munich V79 in stock cultures: 12 - 14 h) and a high plating efficiency of untreated cells (normally more than 50%). These facts are necessary for the appropriate performance of the study.

The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections was carried out before freezing.
For the experiment thawed cultures were set up in 75 cm2 cell culture plastic flasks at 37 °C in a 5%
carbon dioxide atmosphere (95% air). 5 x 105 cells per flask were seeded in 15 mL of MEM
(minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures
were made 3-4 days after seeding.

Rationale for test conditions:

The chromosomal aberration assay is considered acceptable if it meets the following criteria:

- the number of aberration found in the negative and/or solvent controls falls within the range of historical laboratory control data / is considered acceptable for addition to the laboratory historical negative control database.

- concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase
compared with the concurrent negative control

- the proliferation criteria in the solvent control should be similar to the corresponding negative control (where applicable)

- All three experimental conditions were tested unless one resulted in positive results

- Adequate number of cells and concentrations are analysable

- The criteria for the selection of top concentration are consistent with those described earlier

Evaluation criteria:

Evaluation of Results

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

a) at least one of the test concentrations exhibits a statistically significant increase compared with
the concurrent negative control,

b) the increase is dose-related when evaluated with an appropriate trend test,

c) any of the results are outside the distribution of the historical negative control data

When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,

b) there is no concentration-related increase when evaluated with an appropriate trend test,

c) all results are inside the distribution of the historical negative control data.

The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

The Fisher´s exact test was performed to verify the results in the experiment.
The Chi ² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment I without metabolic activation, cytotoxic effects of the test item were noted at a concentration of 2000 μg/mL. With metabolic activation, cytotoxic effects of the test item were noted at concentrations of 250 μg/mL and higher.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

See any other information on results.

Summary: Experiment I, without and with metabolic activation

	Dose Group	Concentration mg/mL	RICC [%]	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipitation^a	Statistical Significance^b
				Inc.Gaps	Excl. Gaps
without 4 h treatment, 21 h preparation interval	C	0	100	4.7	2.7	-0.28% - 3.70% aberrant cells, excl. gaps	-	-
	2	50	112	3	1.3		-	-
	3	100	126	4	2.7		-	-
	4	250	105	6	3.7		+	-
	EMS	600	91	11	8.3		-	+
with 4 h treatment, 21 h preparation interval	C	0	100	5	2.7	-0.23% - 3.95% aberrant cells, excl. gaps	-	-
	2	50	91	5	3.7		-	-
	3*	100	90	5.5	3.3		-	-
	4**	250	45	7.8	4.8		+	-
	CPA	0.83	76	10.7	8		-	+

C:       Negative Control (Culture Medium)

EMS: Ethylmethanesulfonate CPA: Cyclophosphamide

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the negative control groups. The cell count was determined by a cell counter per culture for each test group. a:- without precipitation, + with precipitation

b:        statistical significant increase compared to negative controls (Fisher's exact test, p< 0.05),

+: significant; -not significant

*:         in dose group 3, 600 metaphases scored on four slides were evaluated to increase significance

**:       in dose group 4, the number of evaluated metaphases could not be increased as already four slides were

scored. However, the slides were microscopically evaluated a second time by different persons and the mean value of both evaluations was reported.

Summary: Experiment II, without metabolic activation

	Dose Group	Concentration mg/mL	RICC %	Mean % Aberrant Cells		Historical Laboratory Negative Control Range	Precipitation^a	Statistical significance^b
				incl.	excl.
				Gaps	Gaps
Experiment II 21 h treatment, 21 h preparation interval	C	0	1 00	0.3	0.3	-0.20% - 2.71% aberrant cells, excl. gaps	-	-
	4	100	105	1.7	1		-	-
	5	250	101	1.3	1.3		-	-
	6	500	85	1.3	0.3		+	-
	EMS	400	70	11.6	8.8		-	+

C:       Negative Control (Culture Medium)

EMS: Ethylmethanesulfonate

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the control groups. The cell count was determined by a cell counter per culture for each test group. a:               - without precipitation, + with precipitation

b:        statistical significant increase compared to negative controls (Fisher's exact test, p< 0.05),

+: significant; -not significant

Conclusions:

In conclusion, in an in vitro chromosome aberration test and under the experimental conditions reported, the test item GUP did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line in the experiments without and with metabolic activation. Therefore, the test item GUP is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

In a chromosome aberration study with the test substance, two experiments were conducted at the following concentrations of the test substance with and without metabolic activation:

Experiment I: without and with metabolic activation: 50, 100 and 250 μg/mL

Experiment II: without metabolic activation: 100, 250 and 500 μg/mL

GUP in this in vitro chromosome aberration test, under the experimental conditions reported, did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line in the experiments without and with metabolic activation. Therefore, the test item GUP is considered to be non-clastogenic in this chromosome aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-03-2017 to 30-05-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Section 4, No. 490, "In vitro Mammalian Cell Gene
Mutation Tests Using the Thymidine Kinase Gene" adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Specific details on test material used for the study:

Batch No.: 16VL8189
Expiry Date: 03 August 2017
Storage Conditions: room temperature, protected from light

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

The test item was investigated at the following concentrations:

without metabolic activation:
150, 500, 1200, 1400, 1500, 1550, 1600 and 1700 μg/mL

and with metabolic activation:
250, 500, 1000, 1250, 1500, 1750, 1800 and 2000 μg/mL

The selection of the concentrations used in the main experiment was based on data from the pre-experiment and considered the cytotoxicity of the test substance.

Vehicle / solvent:

RPMI cell culture medium [RPMI = Roswell Park Memorial Institute]

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

Pre-Experiment for Toxicity

The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 2 mg/mL. For the experiment eight concentrations [10, 25, 50, 150, 500, 1000, 1500 and 2000 μg/mL] were tested without and with metabolic activation. The experimental conditions in this pre-experiment were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures is calculated according to the method of Clive and Spector.


Exposure Concentrations

The selection of the concentrations used in the main experiment was based on data from the preexperiment. In the main experiment without metabolic activation 1700 μg/mL and with metabolic activation 2000 μg/mL were selected as the highest concentrations. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation:
150, 500, 1200, 1400, 1500, 1550, 1600 and 1700 μg/mL

and with metabolic activation:
250, 500, 1000, 1250, 1500, 1750, 1800 and 2000 μg/mL

According to OECD Guidelines at least 8 concentrations of the test item were set up in the
experiments without and with metabolic activation.


For a short-term exposure experiment 1 x 107 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of 2 days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3 x 105 cells/mL in a total culture volume of 20 mL, if necessary.

After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells) and those without cell growth (negative wells) compared to the total number of cultures seeded. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 μL selective medium (see below) with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air.

The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to –ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in non-selective medium)). The mutant frequency is usually expressed as “mutants per 106 viable cells”.

Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density. When carrying out a short-term treatment (4 h) a 2-day growth period was considered. The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100. The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup) analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.

Rationale for test conditions:

A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:

- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable.

- The cloning efficiency of the negative and/or solvent controls is in the range 65% -120%.

- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 106 cells.

- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period (short-term treatment)

- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 106 cells. The RTG must be
greater than 10%

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met :

- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10 6 cells and

- a dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small chromosomal aberrations.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.

The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup) analyzed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 for the microwell method.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Summary: Main Experiment, without and with metabolic activation

	Test Group	Conc. Mg/mL	RCE^a%	RTG^b%	MF^c[mutants/ 10^6cells]	IMF^d[mutants/ 10^6cells]	GEF^eexceeded	Stat. Sig. Increase^f	Precipitate
Expt. 1 without metabolic activation	C1	0	100	100	80	/	/	/	-
	C2					/	/	/	-
	2	150	82.7	84.6	83.2	3.1	-	-	-
	3	500	108.4	79.8	68.5	-11.6	-	-	-
	5	1200	89.5	48.1	87.2	7.2	-	-	-
	6	1400	94.1	36.4	81.4	1.3	-	-	-
	7	1500	102.6	29.8	63.2	-16.8	-	-	-
	8	1550	79	25	105.7	25.7	-	-	-
	9	1600	75.5	18.7	91.1	11	-	-	-
	11	1700	106.4	13.5	68	-12	-	-	-
	EMS	300	88.1	81.3	677.6	597.6	+	+	-
	MMS	10	62.5	47.3	623.8	543.7	+	+	-
Expt. 1 with metabolic activation	C1	0	100	100	69.7	/	/	/	-
	C2					/	/	/	-
	5	250	93	99.5	55.7	-13.9	-	-	-
	6	500	80	85.5	80.1	10.5	-	-	-
	7	1000	100.7	103.1	56	-13.7	-	-	-
	8	1250	94.5	86.5	61.4	-8.2	-	-	-
	9	1500	93	56.2	82.7	13	-	-	-
	10	1750	94.5	49.1	65.8	-3.9	-	-	-
	11	1800	88.8	54.4	81.7	12.1	-	-	-
	12	2000	86.2	41.5	72.2	2.5	-	-	-
	B[a]P	2.5	77.7	46.2	672.7	603.1	+	+	-

C: Negative Controls

a: Relative Cloning Efficiency, RCE = [(CE dose group / CE of corresponding controls) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test , p<0.05).

+: significant; -not significant

EMS: Ethylmethanesulfonate [300 μg/mL]

MMS: Methylmethanesulfonate [10 μg/mL]

B[a]P: Benzo[a]pyrene [2.5 μg/mL]

Conclusions:

In this mutagenicity test, under the experimental conditions reported, the test item GUP is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item GUP was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The test item was investigated at the following concentrations:

without metabolic activation: 150, 500, 1200, 1400, 1500, 1550, 1600 and 1700 μg/mL

and with metabolic activation: 250, 500, 1000, 1250, 1500, 1750, 1800 and 2000 μg/mL

No precipitation of the test item was noted in the experiment. Growth inhibition was observed in main experiment without and with metabolic activation.

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item GUP is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In vitro genotoxicity tests

All three in vitro genotoxicity tests (see above) were negative. Thus, no classification is proposed (Annex I of Regulation (EC) 1272/2008).