Serine, 2-[(2,3,4-trihydroxyphenyl)methylene]hydrazide, hydrochloride (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-3-hydroxy-N'-[(E)-(2,3,4-trihydroxyphenyl)methylidene]propanehydrazide hydrochloride

EC Number:

940-411-0

Cas Number:

1353749-74-2

Molecular formula:

C10H14ClN3O5

IUPAC Name:

2-amino-3-hydroxy-N'-[(E)-(2,3,4-trihydroxyphenyl)methylidene]propanehydrazide hydrochloride

Details on test material:

- Purity: 100%

Method

Target gene:

histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9.

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.
Confirmatory mutagenicity assay: 50, 150, 500, 1500, 2000 and 5000 μg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: incubated for 48 to 72 hours at 37±2°C
- Fixation time (start of exposure up to fixation or harvest of cells): 48 to 72 hours

NUMBER OF REPLICATIONS: Initial: 2 reps; Confirmatory: 3 reps

DETERMINATION OF CYTOTOXICITY: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope.

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

The following criteria must be met for the initial toxicity-mutation and confirmatory mutagenicity assays to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10e9 cells/mL. The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate assay data.

Statistics:

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg per plate (all strains only in the absence of S9)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg per plate

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg per plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the Initial Toxicity-Mutation Assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. Positive mutagenic responses (ranging from 2.4- to 6.4-fold maximum increases) were observed with all tester strains in the absence of S9 activation and with tester strains TA98, TA100, TA1535 and WP2 uvrA in the presence of S9 activation. A dose-responsive increase (2.8-fold maximum increase) was observed with tester strain TA1537 in the presence of S9 activation; however, this increase did not meet all criteria required for evaluation as positive (i.e., a maximum response of at least 3.0-times the mean vehicle control value). No precipitate was observed. Toxicity was observed at 5000 μg per plate with all tester strains in the absence of S9 activation and with tester strain WP2 uvrA in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the Confirmatory Mutagenicity Assay, positive mutagenic responses (ranging from 2.6- to 6.7-fold maximum increases) were observed with all tester strains in the presence of S9 activation and all Salmonella tester strains in the absence of S9 activation. A non-dose responsive increase (1.7-fold maximum increase) was observed with tester strain WP2 uvrA in the absence of S9 activation. Toxicity was observed at 5000 μg per plate with all tester strains in the absence of S9 activation and with tester strain WP2 uvrA in the presence of S9 activation.

Confirmatory Mutagenicity Assay Without S9 Activation
Strain	Substance	Dose level per plate	Mean revertants per plate	Standard Deviation	Ratio treated/ solvent	Individual revertant colony counts and background codes
TA98	H-30744	5000 µg	0	0	0.0	0^M 4, 0^M 4, 0^M 4
		2000µg	49	6	2.6	49^M, 55^M, 44^M
		1500µg	36	2	1.9	33^M, 37^M, 37^M
		500µg	22	8	1.2	18^M, 31^M, 18^M
		150µg	17	2	0.9	19^M, 16^M, 17^M
		50µg	18	3	0.9	21^M, 16^M, 17^M
	Water	100µL	19	7		27^M, 14^M, 17^M
TA100	H-30744	5000 µg	0	0	0.0	0^M 4, 0^M 4, 0^M 4
		2000µg	559	23	5.9	533^A, 565^A, 578^A
		1500µg	428	46	4.5	395^A, 408^A, 481^A
		500µg	350	14	3.7	335^A, 351^A, 363^A
		150µg	186	4	2.0	189^A, 183^A, WDN#
		50µg	117	12	1.2	126^A, 103^A, 122^A
	Water	100µL	95	7		102^A, 88^A, 96^A
TA1535	H-30744	5000 µg	0	0	0.0	0^M 4, 0^M 4, 0^M 4
		2000µg	57	7	3.6	52^A, 55^A, 65^A
		1500µg	38	5	2.4	42^A, 40^A, 33^A
		500µg	39	6	2.4	45^A, 33^A, 38^A
		150µg	16	4	1.0	20^A, 13^A, 15^A
		50µg	16	1	1.0	17^A, 15^A, WDN#
	Water	100µL	16	6		19^A, 19^A, 9^A
TA1537	H-30744	5000 µg	0	0	0.0	0^M 4, 0^M 4, 0^M 4
		2000µg	32	5	3.2	37^A, 33^A, 27^A
		1500µg	34	6	3.4	29^A, 31^A, 41^A
		500µg	17	3	1.7	14^A, 20^A, 18^A
		150µg	14	4	1.4	10^A, 15^A, 17^A
		50µg	11	3	1.1	8^A, 10^A, 14^A
	Water	100µL	10	3		8^A, 13^A, 9^A
WP2urvA	H-30744	5000 µg	0	0	0.0	0^M 5, 0^M 5, 0^M 5
		2000µg	35	9	1.0	29^A, 45^A, 32^A
		1500µg	41	6	1.2	45^A, 34^A, 43^A
		500µg	52	5	1.5	57^A, 51^A, 48^A
		150µg	58	5	1.7	52^A, 59^A, 62^A
		50µg	41	12	1.2	37^A, 31^A, 54^A
	Water	100µL	34	7		34^A, 41^A, 28^A
TA98	2NF	1.0µg	202	6	10.6	209^A, 199^A, 199^A
TA100	SA	1.0µg	522	27	5.5	516^A, 499^A, 552^A
TA1535	SA	1.0µg	474	50	29.6	525^A, 425^A, 473^A
TA1537	9AAD	75µg	160	49	16.0	122^A, 143^A, 216^A
WP2urvA	MMS	1000µg	317	21	9.3	332^A, 293^A, 326^A
Key to Positive Controls   2NF – 2-Nitrofluorene SA – Sodium azide 9AAD – 9-Aminoacridine MMS – Methyl methanesulfonate			Key to Automatic & Manual Count Flags   M – Manual count A – Automatic count		Key to Plate Postfix Codes   4 – Extremely reduced background 5 – Absent background WD – Water damaged plate N# - Not counted

 

 

For the Confirmatory Assay With S9 Activation results see below in Overall remarks, attachments

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The test substance was concluded to be positive overall in this assay.

Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.

In the initial toxicity-mutation assay positive mutagenic responses (ranging from 2.4- to 6.4-fold maximum increases) were observed with all tester strains in the absence of S9 activation and with tester strains TA98, TA100, TA1535 and WP2 uvrA in the presence of S9 activation. A dose-responsive increase (2.8-fold maximum increase) was observed with tester strain TA1537 in the presence of S9 activation; however, this increase did not meet all criteria required for evaluation as positive (i.e., a maximum response of at least 3.0-times the mean vehicle control value).

In the confirmatory mutagenicity assay, positive mutagenic responses (ranging from 2.6- to 6.7-fold maximum increases) were observed with all tester strains in the presence of S9 activation and all Salmonella tester strains in the absence of S9 activation. A non-dose responsive increase (1.7-fold maximum increase) was observed with tester strain WP2 uvrA in the absence of S9 activation. The dose levels tested were 50, 150, 500, 1500, 2000 and 5000 μg per plate. No precipitate was observed. Toxicity was observed at 5000 μg per plate with all tester strains in the absence of S9 activation and with tester strain WP2 uvrA in the presence of S9 activation.

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance did exhibit reproducible mutagenic responses with tester strains TA98, TA100, TA1535 and TA1537 in the absence of Aroclor-induced rat liver S9 and with tester strains TA98, TA100, TA1535 and WP2 uvrA in the presence of Aroclor-induced rat liver S9. Therefore, the test substance was concluded to be positive with those test conditions in this assay. Since reproducible mutagenic responses were not observed with tester strain WP2 uvrA in the absence of S9 activation and with tester strain TA1537 in the presence of S9 activation, the test substance was concluded to be equivocal with those test conditions. Therefore, the test substance was concluded to be positive overall in this assay.