(2-hydroxypropyl)trimethylammonium formate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2-hydroxypropyl)trimethylammonium formate

EC Number:

263-503-6

EC Name:

(2-hydroxypropyl)trimethylammonium formate

Cas Number:

62314-25-4

Molecular formula:

C6H16NO.CHO2

IUPAC Name:

(2-hydroxypropyl)trimethylammonium formate

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Air Product & Chemicals, Inc- Corporate Toxicology Dept. / EHS, Allentown/790K13950
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, Dry, Ventilated Storage
- Stability under storage conditions: Stable
- Stability under test conditions: Stable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution: All strains were treated with the test substance at a concentration of 10 mg/ plate, since the test substance, based upon the results of the Range Finding Study, did not show toxicity at this concentration.

Method

Target gene:

his-, trp-

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Other phenotypic characteristics were verfied by using crystal violet sensitivity and resistance to ampicillin and tetracycline methods

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: post-mitochondrial S9 fraction of rat liver homogenate obtained from Aroclor 1254 treated Sprague Dawley rats
- method of preparation of S9 mix: the S9 activation system, prepared fresh on the day of the assay and kept refrigerated or on ice, contained the following per 10 mL:
0.4 M MgC12/1.65M KCL 0.20 mL
1 M glucose-6-phosphate 0.05 mL
0.1 M NADP 0.40 mL
0.2 M phosphate buffer Type and composition of metabolic activation system:
- source of S9: post-mitochondrial S9 fraction of rat liver homogenate obtained from Aroclor 1254 treated Sprague Dawley rats
- method of preparation of S9 mix: the S9 activation system, prepared fresh on the day of the assay and kept refrigerated or on ice, contained the following per 10 mL:
0.4 M MgC12/1.65M KCL 0.20 mL
1 M glucose-6-phosphate 0.05 mL
0.1 M NADP 0.40 mL
0.2 M phosphate buffer pH=7.4 5.00 mL
USP Water for Injection 3.35 mL
S9 Fraction 1.00 mL

Test concentrations with justification for top dose:

All strains were treated with the test substance at a concentration of 10 mg/plate, since the
test substance, based upon the results of the Range Finding Study, did not show toxicity at
this concentration.

Vehicle / solvent:

NaCl

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

E.coli WP2 (20 Mg/mL) without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Salmonella typhimurium TA1537 (800 µg/mL) without metabolic activation.

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Salmonella typhimurium TA100 (100 µg/mL); Salmonella typhimurium TA1535 (5 µg/mL) without metabolic activation

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Salmonella typhimurium TA98 (10 µg/mL), without metabolic activation

Controls 5

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Salmonella typhimurium TA98 (5 µg/mL); Salmonella typhimurium TA100 (10 µg/mL); Salmonella typhimurium TA1535 (20 µg/mL); Salmonella typhimurium TA1537 (30 µg/mL); E. coli WP2 (200 µg/mL) with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Plates were incubated at 37±2°C for 72 or 48 hours, checked for uniform background lawns, and revertant colonies counted.
- Test substance was administered in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Plates were incubated at 37±2°C for 72 or 48 hours, checked for uniform background lawns, and revertant colonies counted.

Evaluation criteria:

The test results were analyzed using a statistical program such as Tallarida, R. S. and R.B. Murray's Pharmacological Calculations Procedure, analysis of variance (ANOVA), and Newman-Keuls Test for confirmation of pairwise comparisons. The statistical method was determined if there was a significant (p < 0.05) increase in the mutation frequency of the test substance compared to the negative control substance. The results obtained for the mutation frequency at the various dose levels (wherever applicable) were analyzed by the method of Linear Regression using a program such as "Linear Regression I" by R.J. Tallarida and R.B. Murray, (Manual of Pharmacologic Calculations with Computer Programs, Springer-Verlag, New York, 1986, pp 10-13). This determined if there was a positive dose response.

Statistics:

Statistical analysis performed are : analysis of variance (ANOVA), and Newman-Keuls Test for confirmation of pairwise comparisons.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
No toxicity was observed with the test article in the Range Finding Assay
STUDY RESULTS
- Concurrent vehicle negative and positive control data:
All positive controls exhibited a statistically significant increase in the number of mutants as
compared to the corresponding negative control, demonstrating that the test system was
functional with known mutagens.

Any other information on results incl. tables

Table 4. Confirmatory test Reverse Mutation Assay without Microsomal Activation

Strain	Controls		Test Article
	Positive Control	Negative Control	Dose Level 10 mg/plate
TA 98	135.3 ± 9.3	27.0 ± 7.0	25.7 ± 3.5
TA 100	502.0± 52.4	109.3 ± 17.4	109.0 ± 7.8
TA 1535	117.0 ± 30.1	24.7 ± 11.6	16.7 ± 2.3
TA 1537	146.3 ± 18.3	10.7 ± 2.5	7.7 ± 2.1
WP2	394.7 ± 10.3	130.7 ± 6.1	123.0 ± 4.4

Table 3. Confirmatory test Reverse Mutation Assay with Microsomal Activation

Strain	Controls		Test Article
	Positive Control	Negative Control	Dose Level
TA 98	137.3 ± 26.0	27.3 ± 9.1	33.3 ± 5.9
TA 100	440± 85.0	121.3 ± 10.5	107.3 ± 19.3
TA 1535	144 ± 9.9	18.3 ± 3.8	17.3 ± 5.1
TA 1537	152 ± 24.5	14.0 ± 4.6	9.7 ± 1.5
WP2	428.7 ± 2.9	129.3 ± 4.0	124.7 ± 5.7

 

 

Table 2. Reverse Mutation Assay without Microsomal Activation

Strain	Controls		Test Article
	Positive Control	Negative Control	Dose Level
TA 98	135.3 ± 9.3	27.0 ± 7.0	20.7 ± 2.5
TA 100	502.0± 52.4	109.3 ± 17.4	100.7 ± 11.09
TA 1535	117.0 ± 30.1	24.7 ± 11.6	20.3 ± 7.4
TA 1537	146.3 ± 18.3	10.7 ± 2.5	11.7 ± 3.5
WP2	394.7 ± 10.3	130.7 ± 6.1	133.0 ± 4.6

Table 1. Reverse Mutation Assay with Microsomal Activation

Strain	Controls		Test Article
	Positive Control	Negative Control	Dose Level
TA 98	137.3 ± 26.0	27.3 ± 9.1	31.7 ± 7.1
TA 100	440± 85.0	121.3 ± 10.5	122.3 ± 26.0
TA 1535	144 ± 9.9	18.3 ± 3.8	16.0 ± 4.6
TA 1537	152 ± 24.5	14.0 ± 4.6	10.3 ± 4.9
WP2	428.7 ± 2.9	129.3 ± 4.0	126.7 ± 3.5

Applicant's summary and conclusion

Conclusions:

The Salmonella typhimurium and Escherichia coli Reverse Mutation Assay (Ames Assay) evaluated the potential of the test substance, Hydroxypropyl, 2-, trimethylammonium formate, to induce histidine (his)reversion (his- to his+) and tryptophan (tryp) reversion (tryp- to tryp+) in the genomes of these respective organisms. This direct plate incorporation assay was conducted with four strains of Salmonella typhimurium (S. typhimurium) and one strain of Escherichia coli (E. coli) in the presence and absence of an exogenous mammalian activation system. A confirmatory assay was performed in order to verify the results of the Reverse Mutation Assay. Based on the criteria of the study protocol, the test substance is considered non-mutagenic