9-Anthracenecarboxylic acid, (triethoxysilyl)methyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-11-20 to 2002-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

historical control data of solvent/vehicle was missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the freezer protected from light, under nitrogen

Target gene:

TA98: his D3052
TA100 and TA1535: his G 46
TA1537: his C 3076

Species / strain / cell type:

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

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat liver microsomal enzymes were prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany. The rats were injected intraperitoneally with a solution (20% (w/v)) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed. The livers of the rats were removed, and washed in cold (0'C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na,-EDTA. Subsequently the livers were minced and homogenized. The homogenate was centrifuged for 15 minutes at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196'C).

- method of preparation of S9 mix :
S9-mix was prepared immediately before use and kept on ice. S9-mix contained·per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCI, solution; 1 mL 0.33 M KCI solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

- quality controls of S9:
Before use, all S9-batches were characterized with the metabolic activation requiring positive control; benzo[a]pyrene (Sigma) in tester strain TA98 at the concentration of 5 μg/plate.

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1: 3 (TA100 & WP2uvrA only), 10, 33, 100, 333, 1000, 3330 (TA100 & WP2uvrA only), and 5000 (TA100 & WP2uvrA only) µg/plate (with and without metabolic activation)
Experiment 2: 10, 33, 100, 333 & 1000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
The test item was dissolved in ethanol. The stock solution was treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were prepared directly prior to use, protected from light with aluminium foil and used within 30 minutes after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin (without metabolic activation) & 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)(both experiments)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicates
- Number of independent experiments : 2

TREATMENT:
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). Since the test substance was difficult to dissolve, 0.15 mL of a dilution of the test substance in ethanol was added to the top agar to reach the highest dose level of 5000 μg/plate in the dose range finding study. However, since the mutation frequencies of this dose level were within the limits of the historical control data range, the validity of the test was considered to be not affected. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1 °C for 48 hours. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.

The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Prolos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: to determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

ACCEPTABILITY CRITERIA:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
c) The selected dose·range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) it induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

A statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT/EXPERIMENT 1:
The test item was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix.
This dose range finding test is reported as a part of the first experiment of the mutation test.

The test substance precipitated in the top agar at concentrations of 100 μg/plate and upwards. Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 100 μg/plate and upwards and at the end of the incubalion period at 1000 μg/plate and upwards.

No reduction of lhe bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Based on the results of the dose range finding test, the test item was tested up to concentrations of 1000 μg/plate in the absence and presence of sg-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

The test item precipitated in the top agar at concentrations of 100 μg/plate and upwards. Precipitalion of TESAC on the plates was observed at the start of the incubation period at concentrations of 333 and 1000 μg/plate and at lhe end of the incubation period at 1000 μg/plate.

The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in lhe number of revertants was observed.

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-relaled, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating !hat the lest conditions were adequate and !hat the
metabolic activation system functioned properly.

Please also refer for results to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Any other information on results incl. tables" below.

Table 1: Historical control data (negative control; number of spontaneous revertants)

Strain	With and without metabolic activation	Minimum value	Maximum value	Mean±3 S.D.
TA1535	- S9-mix	3	25	12±12
	+ S9-mix	3	28	12 ± 12
TA1537	- S9-mix	3	26	6±9
	+ S9-mix	3	28	7±10
TA98	- S9-mix	12	45	18±17
	+ S9-mix	12	54	24±22
TA100	- S9-mix	60	188	110±95
	+ S9-mix	59	195	97±81
WP2uvrA	- S9-mix	4	32	13±15
	+ S9-mix	4	31	14±15

Table 2: Historical control data (positive control)

Strain	With and without metabolic activation	Minimum value	Maximum value	Mean±3 S.D.
TA1535	- S9-mix	90	1308	421±876
	+ S9-mix	60	1028	183±279
TA1537	- S9-mix	77	1897	374±626
	+ S9-mix	58	1794	288±553
TA98	- S9-mix	100	1855	576±973
	+ S9-mix	169	2597	690±932
TA100	- S9-mix	279	2099	851±795
	+ S9-mix	197	2753	804±1210
WP2uvrA	- S9-mix	67	1399	657±879
	+ S9-mix	59	1053	229±444

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance should not be considered to have a mutagenic potential based on a bacterial reverse mutation assay (OECD 471). The substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.