Potassium (R)-(4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-enyl)amino]acetate

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:

end on 05-JUL-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed according to OECD guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Potassium (R)-(4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-enyl)amino]acetate

EC Number:

273-992-8

EC Name:

Potassium (R)-(4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-enyl)amino]acetate

Cas Number:

69416-61-1

Molecular formula:

C13H15NO5.K

IUPAC Name:

potassium (4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-en-1-yl)amino]acetate

Details on test material:

- Name of test material (as cited in study report): D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS
- Substance type: monoconstituent substance
- Physical state: data not available
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature, light and moisture protected

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 is used as the metabolic activation system

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9 mix: sodium azide (NaN3, 10µg/p) in TA1535 & TA100, 4-nitro-o-phenylene-diamine (4-NOPD, 10-50µg/p) in TA1537 & TA98, methyl methane sulfonate (MMS, 3µg/p) in TA 102 ; +S9 mix: 2-aminoanthracene (2-AA, 2.5-10µg/p)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 hours at 37 °C in the dark

NUMBER OF REPLICATES: For each strain and dose level, including the controls three plates were used

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item occurred up to the highest investigated dose
- Effects of pH, Effects of osmolality, Evaporation from medium, Water solubility:


RANGE-FINDING/SCREENING STUDIES:
The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I and II with metabolic activation, the data in the solvent control of strain TA 102 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Results Pre-Experiment and Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	Deionised water			15 ± 7	11 ± 1	31 ± 3	166 ± 20	472 ± 18
	Untreated			16 ± 4	7 ± 1	31 ± 6	166 ± 9	492 ± 22
	HPG - DS	3 µg		15 ± 8	11 ± 3	27 ± 3	160 ± 5	447 ± 46
		10 µg		15 ± 4	15 ± 2	25 ± 4	154 ± 5	460 ± 12
		33 µg		13 ± 4	14 ± 3	32 ± 7	158 ± 10	492 ± 42
		100 µg		14 ± 4	13 ± 3	26 ± 5	196 ± 21	450 ± 14
		333 µg		15 ± 3	10 ± 4	29 ± 5	171 ± 21	431 ± 11
		1000 µg		16 ± 3	11 ± 2	33 ± 6	162 ± 7	463 ± 9
		2500 µg		12 ± 3	8 ± 2	29 ± 5	170 ± 12	450 ± 34
		5000 µg		14 ± 1	12 ± 2	28 ± 1	163 ± 15	357 ± 114
	NaN3	10 µg		2059 ± 98			2165 ± 86
	4-NOPD	10 µg				294 ± 4
	4-NOPD	50 µg			81 ± 2
	MMS	3.0 µL						3498 ± 218
With Activation	Deionised water			20 ± 4	16 ± 5	37 ± 3	174 ± 14	695 ± 24
	Untreated			18 ± 7	21 ± 4	36 ± 7	199 ± 23	644 ± 11
	HPG - DS	3 µg		16 ± 1	15 ± 7	35 ± 6	183 ± 11	678 ± 30
		10 µg		15 ± 5	16 ± 2	40 ± 6	184 ± 13	687 ± 21
		33 µg		19 ± 5	9 ± 5	41 ± 3	189 ± 9	682 ± 31
		100 µg		18 ± 4	10 ± 2	38 ± 7	196 ± 21	607 ± 65
		333 µg		23 ± 7	18 ± 3	35 ± 8	190 ± 11	602 ± 32
		1000 µg		17 ± 3	18 ± 3	46 ± 7	191 ± 19	650 ± 47
		2500 µg		19 ± 11	11 ± 3	36 ± 4	206 ± 3	649 ± 6
		5000 µg		22 ± 5	13 ± 1	36 ± 9	203 ± 6	646 ± 34
	2-AA	2.5 µg		497 ± 57	669 ± 44	3679 ± 223	4964 ± 175
	2-AA	10.0 µg						2736 ± 318

Table 2: Summary of Results Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	Deionised water			11 ± 3	10 ± 4	35 ± 2	175 ± 16	432 ± 17
	Untreated			13 ± 0	15 ± 4	38 ± 4	150 ± 8	414 ± 16
	HPG - DS	33 µg		12 ± 3	14 ± 1	33 ± 6	169 ± 11	365 ± 42
		100 µg		11 ± 4	7 ± 1	33 ± 2	167 ± 7	415 ± 48
		333 µg		11 ± 3	12 ± 2	32 ± 3	168 ± 19	401 ± 36
		1000 µg		13 ± 1	13 ± 4	38 ± 12	157 ± 11	437 ± 40
		2500 µg		12 ± 2	9 ± 3	32 ± 6	188 ± 40	439 ± 18
		5000 µg		9 ± 3	12 ± 6	34 ± 2	169 ± 6	447 ± 34
	NaN3	10 µg		1857 ± 126			2063 ± 192
	4-NOPD	10 µg				392 ± 8
	4-NOPD	50 µg			85 ± 13
	MMS	3.0 µL						2160 ± 628
With Activation	Deionised water			25 ± 4	14 ± 2	44 ± 8	199 ± 18	719 ± 50
	Untreated			26 ± 7	15 ± 2	57 ± 6	192 ± 13	624 ± 40
	HPG - DS	33 µg		27 ± 7	14 ± 1	48 ± 8	186 ± 29	660 ± 37
		100 µg		21 ± 7	12 ± 3	43 ± 5	216 ± 14	680 ± 64
		333 µg		26 ± 3	15 ± 2	49 ± 1	211 ± 24	603 ± 73
		1000 µg		24 ± 4	15 ± 1	53 ± 2	204 ± 3	650 ± 25
		2500 µg		20 ± 6	14 ± 2	48 ± 4	195 ± 30	636 ± 43
		5000 µg		22 ± 13	8 ± 2	47 ± 11	205 ± 19	501 ± 116
	2-AA	2.5 µg		454 ± 31	541 ± 36	3619 ± 270	3505 ± 257
	2-AA	10.0 µg						2420 ± 133

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471, GLP), strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 of S. typhimurium were exposed to D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS in deionised water in the presence and absence of mammalian metabolic activation at the following concentrations:

- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate (plate incorporation)

- Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate (pre-incubation)

D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS was tested up to limit concentration (5000 µg/plate).

 

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with D(-)-P-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.