L-Glutamic acid, N-acetyl-, compd. with (.alpha.S)-.alpha.-(2-methylpropyl)-2-(1-piperidinyl)benzenemethanamine (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

Study period:

25 June - 4 July 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 471) and in complicane with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

AG-EE 623 Glutamate

IUPAC Name:

AG-EE 623 Glutamate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): AG-EE 623 glutamate: (S)-3-methyl-1-(2-piperidinophenyl)-1-butylamino-N-acetyl-L-glutamate
- Purity test date: 1995-11-29
- Lot/batch No.: WB 02
- Expiration date of the lot/batch: 1996-11-30
- Storage condition of test material: At room temperature in the dark ( ambient humidity)

Method

Target gene:

TA 1537, hisC3076, rfa, uvrB, frameshift mutations
TA 98, hisD3052, rfa, uvrB, pKM101, frameshit mutations
TA 100, hisG46, rfa, uvrB, pKM101, base-pair substitution
TA 1535, hisG46, rfa, uvrB, base-pair substitution

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

10, 50, 100, 250, 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

Evaluation criteria:

A concentration-dependent increase in the number of revertants of at least one tester strain over the vehicle control value and/or outside the historical control range is indicative of genotoxic activity.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and Toxicity:
The mean number of bacteria tested varied between 0.54 and 1.08 x E8 /plate. There were no signs for microcrystalline precipitation in the soft agar. A strong microbial toxicity, as seen by a reduced background lawn and/or a decrease of absolute revertant numbers, was observed in all strains of
S. typhimurium at concentration Ievels of 250 ,ug/plate (in some strains at 100 ,ug/plate) and higher in the presence and absence of liver enzymes.

Mutagenicity:
Contra! plates (vehicle control) showed spontaneaus revertant colanies for the tester strains of S. typhimurium at frequencies similar to those described in the Iiterature and within the historical control range for our laboratory. Number of revertants in different S. typhimurium strains
with increasing concentrations of AG-EE 623 glutamate were of the same order of magnitude as observed with the controls. Addition of liver
homogenates from rats pretreated with Aroclor 1254 bad no influence on mutation induction. The negative results were qualitatively confirmed
by the repeated experiment.

Positive Controls:
The positive control substances used in these experiments NaN3 and 2-NF in the absence and 2-AA in the presence of mammalian liver enzymes
showed the expected strain specific responses. The results with the indirect mutagen confirmed the metabolic activation capacity of the S9 fractions used.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

without metabolic activation

Compound (µg/plate)	Mean Revertants/plate (S.typhimurium)
	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
negative control
DMSO	10	10	6	9	23	32	123	105
positive control
sodium azide	913	945					1105	1092
2 -nitrofluorene			45	45	826	891
AG-EE 623 glutamate
2		11		6		27		102
10	10	13	6	9	31	31	108	116
50	11	9	7	7	19	25	99	93
100	10	9	5	6	17	14T	72	77T
250T	9	5	7	5	12	6	47	46
500T	3		0		3		6

with metabolic activation

Compound (µg/plate)	Mean Revertants/plate (S.typhimurium)
	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
negative control
DMSO	14	12	9	9	27	32	125	118
positive control
2 -AA	198	215	221	199	1304	1316	1757	1836
AG-EE 623 glutamate
2		13		13		29		113
10	14	8	7	6	36	27	110	114
50	13	10	8	7	21	19	105	115
100	7	8	8T	6	21	28	109	90T
250T	7	7	5	8	19	14	87	88
500T	8		4		20		67

T: Toxicity

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on these results it is concluded, that AGEE 623 glutamate, when tested up to clearly bacteriotoxic concentration, caused neither base-pair substitution nor frameshift mutations in bacteria. No evidence of genotoxic activity was observed in S.typhimurium in the absence and presence of metabolic activation. The test compound is, therefore, classified as 'Ames negative'.