D-(-)-α-phenylglycine

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 08-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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

D-(-)-α-phenylglycine

EC Number:

212-876-3

EC Name:

D-(-)-α-phenylglycine

Cas Number:

875-74-1

Molecular formula:

C8H9NO2

IUPAC Name:

(2R)-2-amino-2-phenylacetic acid

Details on test material:

- Name of test material (as cited in study report): D(-)alpha-Phenylglycine; Code Name: PG
- Substance type: monoconstituent substance
- Physical state: data not available
- Stability under test conditions: Not indicated by the sponsor
- 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/β-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: DMSO
- 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: not indicated

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.

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	DMSO			14 ± 5	10 ± 4	31 ± 10	126 ± 13	371 ± 10
	Untreated			15 ± 3	11 ± 3	29 ± 7	150 ± 8	377 ± 12
	D(-)ALPHA-	3 µg		11 ± 4	10 ± 2	27 ± 4	111 ± 2	379 ± 18
	PHENYLGLYCINE;	10 µg		11 ± 2	9 ± 2	27 ± 8	117 ± 13	398 ± 11
	CODE NAME: PG	33 µg		17 ± 2	10 ± 3	30 ± 6	127 ± 6	406 ± 17
		100 µg		12 ± 3	11 ± 3	30 ± 7	126 ± 9	377 ± 12
		333 µg		9 ± 1	7 ± 2	28 ± 4	143 ± 11	361 ± 24
		1000 µg		14 ± 0	10 ± 1	27 ± 7	131 ± 14	394 ± 13
		2500 µg		13 ± 4	8 ± 2	34 ± 1	123 ± 19	401 ± 17
		5000 µg		11 ± 3	6 ± 2	27 ± 2	127 ± 18	397 ± 29
	NaN3	10 µg		1755 ± 24			1921 ± 49
	4-NOPD	10 µg				272 ± 15
	4-NOPD	50 µg			64 ± 2
	MMS	3.0 µL						3009 ± 137
With Activation	DMSO			17 ± 6	18 ± 5	35 ± 5	150 ± 9	516 ± 16
	Untreated			20 ± 1	18 ± 4	37 ± 9	161 ± 4	584 ± 9
	D(-)ALPHA-	3 µg		17 ± 4	17 ± 5	34 ± 4	164 ± 2	548 ± 14
	PHENYLGLYCINE;	10 µg		17 ± 3	17 ± 6	37 ± 1	154 ± 11	573 ± 27
	CODE NAME: PG	33 µg		20 ± 3	19 ± 5	35 ± 4	144 ± 19	586 ± 20
		100 µg		21 ± 2	21 ± 4	30 ± 6	150 ± 8	547 ± 44
		333 µg		14 ± 5	12 ± 3	31 ± 2	158 ± 13	549 ± 23
		1000 µg		15 ± 6	13 ± 0	39 ± 4	150 ± 5	565 ± 25
		2500 µg		15 ± 1	14 ± 2	35 ± 6	156 ± 10	590 ± 13
		5000 µg		15 ± 7	13 ± 1	33 ± 5	161 ± 16	581 ± 37
	2-AA	2.5 µg		423 ± 37	533 ± 40	2952 ± 346	3727 ± 199
	2-AA	10.0 µg						2806 ± 184

 

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	DMSO			16 ± 1	11 ± 3	29 ± 5	164 ± 12	432 ± 17
	Untreated			15 ± 4	10 ± 4	28 ± 7	169 ± 12	456 ± 19
	D(-)ALPHA-	33 µg		14 ± 3	9 ± 2	32 ± 5	144 ± 9	414 ± 14
	PHENYLGLYCINE;	100 µg		16 ± 3	13 ± 5	20 ± 5	136 ± 20	435 ± 11
	CODE NAME: PG	333 µg		14 ± 4	12 ± 4	20 ± 1	159 ± 18	423 ± 17
		1000 µg		16 ± 6	8 ± 1	30 ± 5	160 ± 6	408 ± 16
		2500 µg		12 ± 4	10 ± 3	28 ± 6	159 ± 18	401 ± 10
		5000 µg		16 ± 1	11 ± 2	32 ± 8	155 ± 22	411 ± 9
	NaN3	10 µg		2235 ± 57			2350 ± 47
	4-NOPD	10 µg				375 ± 18
	4-NOPD	50 µg			61 ± 9
	MMS	3.0 µL						1694 ± 467
With Activation	DMSO			18 ± 3	8 ± 2	36 ± 5	166 ± 12	535 ± 17
	Untreated			15 ± 6	14 ± 2	39 ± 6	192 ± 9	613 ± 37
	D(-)ALPHA-	33 µg		20 ± 6	16 ± 5	30 ± 6	158 ± 13	596 ± 14
	PHENYLGLYCINE;	100 µg		18 ± 3	12 ± 3	35 ± 4	162 ± 12	556 ± 45
	CODE NAME: PG	333 µg		16 ± 3	14 ± 1	34 ± 6	163 ± 7	505 ± 25
		1000 µg		20 ± 3	16 ± 6	39 ± 5	159 ± 3	565 ± 14
		2500 µg		18 ± 5	14 ± 5	30 ± 5	182 ± 17	573 ± 29
		5000 µg		19 ± 3	13 ± 1	55 ± 37	169 ± 8	517 ± 48
	2-AA	2.5 µg		582 ± 37	436 ± 52	3696 ± 166	4296 ± 298
	2-AA	10.0 µg						2838 ± 510

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, D(-)alpha-Phenylglycine; Code Name: PG 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(-)alpha-Phenylglycine; Code Name: PG in DMSO 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(-)alpha-Phenylglycine; Code Name: PG 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(-)alpha-Phenylglycine; Code Name: PG at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

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.