Propanoic acid, 2-hydroxy-, ammonium salt (1:1), (2S)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2005-07-15 to 2005-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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:

Propanoic acid, 2-hydroxy-, ammonium salt (1:1), (2S)-

EC Number:

604-012-2

Cas Number:

137296-15-2

Molecular formula:

C3H9O3N

IUPAC Name:

Propanoic acid, 2-hydroxy-, ammonium salt (1:1), (2S)-

Specific details on test material used for the study:

Name of test material (as cited in study report): Ammonium Lactate, PURASAL® NH
- Physical state: not reported
- Analytical purity: not reported
- Lot/batch No.: 0503002615
- Expiration date of the lot/batch: 2007-09-28
- Storage condition of test material: room temperature (20 ± 5 °C) in the dark
- Other: Reception date: 2005-04-01

Method

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test: 100, 50, 25, 12.5, 6.25 and 3.125 µL/plate (with and without metabolic activation)
Main test: 50, 16.67, 5.56, 1.85, 0.62 and 0.21 µL/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
The solvent used for the test item was distilled water. No solubility tests were done because the sponsor defined the test item as completely soluble in water. The maximum concentration of 100 µL/plate, i.e. undiluted substance, was used for the performance of the preliminary test. The subsequent concentrations were prepared by 1/2 dilutions with sterile distilled water.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, 1 µg/plate, all strains used

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, 1 µg/plate, TA 1535 and TA 100

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, 50 µg/plate, TA 1537

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9, 1 µg/plate, E. coli WP2

Details on test system and experimental conditions:

PREINCUBATION METHOD:
The amount of 0.1 mL of the test item/test solvent was preincubated with 0.1 mL of the test strain culture and 0.5 mL of sterile buffer or metabolic activation system for 20 minutes at 37 ± 1 °C prior to mixing with 2.0 mL of the overlay agar and pouring onto the surface of a minimal agar plate. During pre-incubation the tubes were aired using a shaker.
This overlay melted at 45 ± 1 °C contained a sterile solution of 0.5 mM L-histidine/0.5 mM biotin in the proportion of 1:10 for Salmonella typhimurium and 0.25 mL of a sterile solution of tryptophan and 5 mL of nutrient broth for every 100 mL of top agar for Escherichia coli. The metabolising enzymes in the S-9 mixture are not stable at 45 °C and so the contents of the test tubes were mixed rapidly in a rotamixer and poured into plates containing a base layer of minimal agar. The added agar was allowed to settle down and the plates were incubated at 37 ± 1 °C for 48-72 hours. After this incubation period, the number of revertant colonies that had grown on each plate was counted. The experiment was repeated on another day using fresh bacterial cultures.

Evaluation criteria:

According to OECD 471

Statistics:

The comparisons between the results for the standard products and the control were made using Student's t-test. The statistical comparison of the different test-item concentrations was carried out, for all the bacterial strains, both with and without metabolic activation, using a one-way analysis of variance with a level of significance of p < 0.05.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: see box "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The maximum concentration of 100 µL/plate, i.e. undiluted substance, was used for the performance of the preliminary test. At the concentration of 100 µL/plate a slight decrease was observed in the number of revertants in strain TA-100 without S-9. In accordance with the OECD, which suggests 5 µL/plate as the high concentration for liquids, lower concentration were tested in the main test. 50 µL/plate was taken as the maximum concentration and 1/3 dilutions were prepared.

RESULTS (Experiment 1):
Positive controls: All the bacterial strains responded positively.
Negative controls: Normal values (within the range of historical data for negative controls) were obtained in the revertant colony counts on all the dishes treated only with the solvent.
Test item: The test item was toxic for TA-100 with and without S-9 and for TA-1535 with S-9 at the concentration of 50 µL/plate. No mutagenic response was observed in any of the tested strains, with or without S-9.

RESULTS (Experiment 2):
Positive controls: All the bacterial strains responded positively.
Negative controls: Normal values (within the range of historical data for negative controls) were obtained in the revertant colony counts on all the dishes treated only with the solvent. The mean of the control for the strain TA-1537 without S-9 fell slightly above the upper limit established by historical data although it fell within the range given in literature.
Test item: The test item was toxic for TA-100 without S-9 and for TA-1535 with S-9 at the concentration of 50 µL/plate.
No mutagenic response was observed in any of the tested strains, either with or without S-9.

Any other information on results incl. tables

Table 2: Summary of the results obtained with the test item (experiment 1)
		Mean revertants per plate (µL/plate)
Test strain		% S-9	Control	0.21	0.62	1.85	5.56	16.67	50
TA 1535		0.0	21.7	20.3	21.7	20.7	20.3	22.3	21.0
TA 1537		0.0	19.7	19.0	19.0	19.0	18.7	20.7	19.3
TA 98		0.0	31.0	31.0	31.0	30.3	30.7	31.7	32.0
TA 100		0.0	133.3	132.3	132.7	131.0	133.0	132.7	-
E. coli WP2		0.0	143.3	143.0	142.3	141.3	142.3	141.0	141.7
TA 1535		10.0	21.0	20.0	20.7	19.0	20.7	20.7	-
TA 1537		10.0	20.3	19.0	19.3	18.7	20.7	19.7	19.7
TA 98		10.0	35.7	36.3	36.7	35.3	35.7	36.0	37.3
TA 100		10.0	120.7	121.3	119.3	120.7	119.7	120.0	-
E. coli WP2		10.0	143.0	141.7	142.3	142.0	142.0	141.3	142.0

Table 3: Summary of the results obtained with the test item (experiment 2)
		Mean revertants per plate (µL/plate)
Test strain		% S-9	Control	0.21	0.62	1.85	5.56	16.67	50
TA 1535		0	25.0	24.3	22.3	25.3	23.3	22.0	23.3
TA 1537		0	24.0	22.3	25.0	22.3	25.0	23.3	22.7
TA 98		0	29.7	30.0	32.0	29.0	28.7	28.7	28.7
TA 100		0	140.3	140.7	140.3	139.0	139.7	141.0	-
E. coli WP2		0	140.0	139.7	140.3	140.0	141.0	141.0	140.0
TA 1535		10	22.7	22.7	21.0	21.3	20.7	22.0	-
TA 1537		10	25.7	25.7	25.3	26.0	26.7	26.3	25.3
TA 98		10	32.3	34.3	33.7	34.3	33.7	32.7	33.7
TA 100		10	141.0	142.0	140.3	140.3	140.3	141.3	141.3
E. coli WP2		10	141.7	141.3	140.7	140.3	141.7	140.7	141.3

Applicant's summary and conclusion

Conclusions:

The test item showed no evidence of mutagenic activity in a bacterial reverse mutation assay (OECD 471).

Executive summary:

In a bacterial reverse mutation assay (according to OECD 471) Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one strain of Escherichia coli (WP2 uvrA) were exposed to the test item at concentrations of 0, 0.21, 0.62, 1.85, 5.56, 16.67 and 50 µL per plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies above background.

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