DL-serinohydrazide monohydrochloride

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

Study period:

29 Jan - 06 Feb 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, non-GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH S2A,B

Deviations:

no

GLP compliance:

no

Remarks:

This study was not performed for REACH purposes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

DL-serinohydrazide monohydrochloride

EC Number:

259-839-8

EC Name:

DL-serinohydrazide monohydrochloride

Cas Number:

55819-71-1

Molecular formula:

C3H9N3O2.ClH

IUPAC Name:

2-amino-3-hydroxy-propanehydrazide;hydrochloride

Details on test material:

- Molecular weight: 155.5850
- Storage condition of test material: room temperature

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

other: S. typhimurium TA1535, TA97, TA98, TA100, TA102

Additional strain / cell type characteristics:

other: all strains except TA102 are deficient in excision repair (uvrB mutation); TA97, TA98, TA100, TA102 contain pKM101 plasmid for error prone DNA repair system; TA102 reverting gene on multicopy plasmid pAQ1 (a pBR322 derivative)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, Moltox post mitochondrial supernatant

Test concentrations with justification for top dose:

50, 158, 500, 1580, 5000 µg/plate with and without S9-mix

Vehicle / solvent:

solvent for test item: deionised water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: ICR 191; 2-Aminoanthracene (2-AA)

Remarks:

-S9 mix: Sodium azide in DMSO (TA1535, TA100) and ICR 191 (TA97): 1.0 µg/plate; -S9 mix: MMC in aqua dest. (TA102): 0.2 µg/plate; -S9 mix: 2-Nitrofluorene in DMSO (TA98): 0.5 µg/plate; +S9 mix: 2-AA in DMSO (all strains): 4.0 and 10.0 (for TA102) µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- Three replicate plates for the test item and negative control
- two replicate plates for the positive controls

DETERMINATION OF CYTOTOXICITY
- Method: determination of the growth of the background lawn and/or frequency of spontaneous revertants

Evaluation criteria:

A positive result is often defined as a reproducible, dose-related increase in the number of his+ revertants reaching at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535 and TA98 or a 1.5 - fold increase for strains TA97, TA100 and TA102. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains).

A negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

A positive response in one strain, under one treatment condition, will usually be sufficient to call a test item mutagenic. However, concordant results under variable conditions and/or in several strains usually carry more weight than singular findings. Strong increases (in absolute terms or as fold-increase) and increases at low concentrations are also usually considered to be more relevant than weak increases at high concentrations although it has to be noted that there is no quantitative relationship between mutagenic potency in the Ames tests and carcinogenic potency in mammals.

The described rules of thumb have a questionable scientific foundation and biological relevance should always be taken into account. Since the shape of the dose response curve, toxicity, precipitation, and the observed plate to plate variability of the counts may all influence the final call, it is impossible to predefine unequivocal criteria for positive or negative responses which would apply to every configuration of the data generated by the mutation assay.

The study director is ultimately responsible for the final assessment.

Statistics:

There is no generally accepted statistical treatment of Ames test data.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA1535, TA97, TA98, TA100, TA102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test item was soluble in deionised water
- Precipitation: no precipitation observed up to the maximal dose

RANGE-FINDING/SCREENING STUDIES:
- range finder assay with strain TA100 (plate incorporation version, +S9) was performed
- no precipitation and no toxic effects observed

COMPARISON WITH HISTORICAL CONTROL DATA:
- mutant frequencies of the controls were in the range of historical control values and the data published in literature

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- no cytotoxic effects were apparent up to the maximal dose

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test item induces dose related increases in the number of revertant colonies in several strains. The effect was most obvious in strain TA1535 (-S9), was also apparent in strains TA1535 (+S9), TA97 (+/-S9) and TA100 (-S9). For strain TA98 the plates with S9 were not evaluable but a repeat with this strain was not considered necessary as it would not change the test outcome.

Table 1 Summary Data of Results of Plate Incorporation Assay

Summary Data: Plate Incorporation Assay
S9-Mix	Without
Test item (µg/plate)	TA 1535	TA 97	TA 98	TA 100	TA 102
Solvent Control (deionised water)	16 ± 2	295 ± 20	20 ± 9	98 ± 8	521 ± 57
Test item (50)	24 ± 7	323 ± 43	17 ± 1	102 ± 3	492 ± 7
Test item (158)	27 ± 8	329 ± 34	21 ± 8	97 ± 14	502 ± 46
Test item (500)	56 ± 9	383 ± 38	27 ± 5	105 ± 15	430 ± 29
Test item (1580)	116 ± 13	405 ± 32	27 ± 5	169 ± 26	454 ±24
Test item (5000)	161 ± 20	581 ± 37	23 ± 4	182 ± 18	487 ± 30
Na azide (1.0)	856 ±15			688 ± 69
ICR 191 (1.0)		1605 ± 601
2-NF (0.5)			93 ± 7
MMC (0.2)					1151 ± 54
S9-Mix	With
Test item (µg/plate)	TA 1535	TA 97	TA 98	TA 100	TA 102
Solvent Control (deionised water)	11 ± 5	439 ± 23	^e	118 ± 4	314 ± 188
Test item (50)	14 ± 4	423 ± 36	^e	124 ± 12	425 ± 32
Test item (158)	12 ± 5^c	441 ± 36	^e	115 ± 7	436 ± 34
Test item (500)	14 ± 6	476 ± 0^c	^e	125 ± 1	378 ± 50
Test item (1580)	26 ± 4	531 ± 11	^e	123 ± 2	356 ± 7
Test item (5000)	39 ± 6	663 ± 62	^e	145 ± 14	265 ± 42
2-AA (4.0)	234 ± 24	984 ± 108	^e	496 ± 40
2-AA (10.0)			^e		3043 ± 23
Viability
	TA 1535	TA 1537	TA 98	TA 100	TA 102
	187 ± 11	178 ± 28	45 ± 7	149 ± 34	136 ± 49
Key to Positive Controls Na azide: Sodium Azide 2-AA: 2-Aminoanthracen MMC: Mitomycin C ICR 19: ICR 191 2NF: 2-Nitrofluorene			Key to Plate Postfix Codes c: contaminated plate e: plate not evaluable (colonies not countable)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

The test item induced weak but dose related increases of the number of revertant colonies in three of the five tester strains. The effect was most apparent in strain TA1535 but also recognizable in strains TA97 and TA100.
Thus, it has to be concluded that the test item possesses a weak mutagenic activity in the Ames test under the described experimental conditions with and without metabolic activiation.

Executive summary:

The test item was evaluated for mutagenic activity in the Ames test. The test was performed in accordance with the OECD Guideline 471 for the Testing of Chemicals (Bacterial Reverse Mutation Test, Adopted 21st July 1997). A standard plate incorporation assay was performed in absence and in presence of an exogenous metabolic activation system (S9). Five Salmonella typhimurium tester strains (TA1535, TA97, TA98, TA100, and TA102) were employed. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment.

The test item was dissolved in deionised water. Concentrations ranging from 50 to 5000 µg/plate were selected for evaluation including

the generally recommended highest test concentration for non toxic compounds. No toxicity was observed in this dose range.

The test item induced weak but dose related increases of the number of revertant colonies in three of the five tester strains. The effect was most apparent in strain TA1535 but also recognizable in strains TA97 and TA100.

Thus, it has to be concluded that the test item possesses a weak mutagenic activity in the Ames test under the described experimental conditions with and without metabolic activation.