4-hydroxy-3,5-dimethoxybenzonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The objective of this assay was to assess the potential of Mediator SNP (4-hydroxy-3,5-dimethoxybenzonitrile) to induce point mutations (frame-shift and base-pair) in four strains of Salmonella typhimurium, TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA.

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:

2008-12-5 to 2008-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Composition of test material, percentage of components: syringonitrile 99.8%
- Expiration date of the lot/batch: May 2010
-Physical description: pale yellow powder
-solubility: soluble in water
-stability: test substance was expected to be stable for the duration of the testing.

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: his D 3052; rfa-; uvrB-; R-factor:

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: his G 46; rfa-; uvrB-; R-factor:

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: his G 46; rfa-; uvrB-:

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: his C 3076; rfa-; uvrB- :

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: WP2 uvrA: trp-; uvrA-:

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver; S-9 mix

Test concentrations with justification for top dose:

A preliminary toxicity test was performed in strain TA 98 and TA 100 with 8 concentrations ranging from 3.16 to 5000 μg/plate. Based on the results from the preliminary toxicity test, the maximum concentration used in the main experiments was 5000 μg/plate.
In two independent experiments several concentrations of Mediator SNP were used. The concentrations, including the controls, were tested in triplicate. Mediator SNP was prepared and used at: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in both presence and absence of S-9 mix.

Vehicle / solvent:

DMSO. The solvent was compatible with the survival of the bacteria and S9 activity.

Untreated negative controls:

yes

Remarks:

treated by addition of sterile saline solution

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

for assays without the metabolic activatio(S-9)

Positive control substance:

methylmethanesulfonate

Remarks:

for E. coli. For with S-9 mix, the positive control substance is 2-aminoanthracene

Untreated negative controls:

yes

Remarks:

treated by addition of sterile saline solution

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide & 4-nitro-o-phenylene diamine for S. typhimurium

Remarks:

for S. typhimurium. For with s-9 mix, the pos. control is 2-aminoanthracene.

Details on test system and experimental conditions:

Preparation of Bacteria
Samples of each tester strain were grown by culturing for 12 h at 38.5 °C in S. typhimurium medium (Nutrient broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL).

Experimental Performance
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
Test solution at each dose level, solvent or negative control or reference mutagen solution (positive control),
S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain), Overlay agar.
For the pre-incubation method: test item-preparation is preincubated with the tester strains and sterile buffer or the metabolic activation system for 60 minutes at 37°C prior to adding the overlay agar and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used. After solidification the plates were inverted and incubated at 37°C for at least 48 h in the dark.

Data Recording
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgerate GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation criteria:

Evaluation of Cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "B" in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately less than or equal to 0.5 in relation to the solvent control.

Evaluation of Mutagenicity
A test item is considered as mutagenic if: a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

Species / strain:

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

Remarks:

The S. typhimurium histidine (his) reversion system

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for TA 1537 and E. coli, at 2500 µg/plate and higher (no S-9), 5000 µg/plate with S-9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Additional information on cytotoxicity
Toxic effects of the test item were noted in several tester strains evaluated in experiment I and II.
In experiment I toxic effects of the test item were observed in tester strains TA 1537 and E. coli WP2 uvrA at doses of 2500 µg/plate and higher (without metabolic activation). In tester strains TA 1535 and E. coli WP2 uvrA toxic effects of the test item were noted at doses of 5000 µg/plate (with metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in tester strain TA 1535 at a dose of 316 µg/plate and in tester strain TA 1537 at a dose of 31.6 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship. In experiment II toxic effects of the test item were noted in tester strain E. coli WP2 uvrA at doses of 2500 µg/plate and higher (with metabolic activation).

Conclusions:

Under the conditions of this assay, Mediator SNP (4-hydroxy-3,5-dimethoxybenzonitrile) has not shown any evidence of mutagenic activity in the assay and is not a mutagen.

Executive summary:

The objective of this assay was to assess the potential of Mediator SNP to induce point mutations (frame-shift and basepair) in four strains of Salmonella typhimurium, TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA. The test material was tested both in the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver; S-9 mix). The tests were performed using the plate incorporation method.

A preliminary toxicity test was performed in strain TA 98 and TA 100 with 8 concentrations ranging from 3.16 to 5000 μg/plate. Based on the results from the preliminary toxicity test, the maximum concentration used in the main experiments was 5000 μg/plate.

In two independent experiments several concentrations of Mediator SNP were used. The concentrations, including the controls, were tested in triplicate. Mediator SNP was prepared and used at: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in both presence and absence of S-9 mix. The highest dose level tested (5000 μg/plate) is the maximum required by the OECD guideline. The positive controls used for assays without S-9 mix were sodium azide and 4-nitro-o-phenylenediamine for S. typhimurium and methyl methane sulfonate for E. coli.The positive controls used for assays with S-9 mix was 2-aminoanthracene. Negative control plates were treated by the addition of sterile saline solution. This assay was conducted in accordance with OECD guideline No. 471 adopted July 21, 1997, the US EPA Test Guidelines, OPPTS 870.5100 and complied with OECD Principles on GLP (as revised in 1997) and all subsequent OECD consensus documents.

In the screening assay, Mediator SNP was toxic to the test bacteria at concentrations of 2500 μg/plate and higher in the absence of S-9 mix and at 5000 μg/plate in the presence of S-9 mix. Therefore, the 5000 μg/plate was selected as the highest dose level for the main tests.

In the main tests, no biologically significant increases in the number of revertant colonies were observed in any S. typhimurium tester strain after treatment with Mediator SNP at any dose level either in the presence or absence of S-9 mix. No biologically or statistically significant increases in the number of revertant colonies were observed in E. coli WP2 urvA at any dose level either in the presence or absence of S-9 mix. Statistical increases in the number of revertant colonies were noted with the positive controls in both the presence and absence of metabolic activation substantiating the sensitivity of the treat and plate assay and the efficacy of the metabolic activation mixture.

Under the conditions of this assay, Mediator SNP has not shown any evidence of mutagenic activity in the assay and is not a mutagen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The objective of this assay was to assess the potential of Mediator SNP to induce point mutations (frame-shift and basepair) in four strains of Salmonella typhimurium, TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA. The test material was tested both in the presence and absence of a metabolic activation system (Aroclor 1254-induced rat liver; S-9 mix). The tests were performed using the plate incorporation method.

A preliminary toxicity test was performed in strain TA 98 and TA 100 with 8 concentrations ranging from 3.16 to 5000 μg/plate. Based on the results from the preliminary toxicity test, the maximum concentration used in the main experiments was 5000 μg/plate.

In two independent experiments several concentrations of Mediator SNP were used. The concentrations, including the controls, were tested in triplicate. Mediator SNP was prepared and used at: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in both presence and absence of S-9 mix. The highest dose level tested (5000 μg/plate) is the maximum required by the OECD guideline. The positive controls used for assays without S-9 mix were sodium azide and 4-nitro-o-phenylenediamine for S. typhimurium and methyl methane sulfonate for E. coli. The positive controls used for assays with S-9 mix was 2-aminoanthracene. Negative control plates were treated by the addition of sterile saline solution. This assay was conducted in accordance with OECD guideline No. 471 adopted July 21, 1997, the US EPA Test Guidelines, OPPTS 870.5100 and complied with OECD Principles on GLP (as revised in 1997) and all subsequent OECD consensus documents.

In the screening assay, Mediator SNP was toxic to the test bacteria at concentrations of 2500 μg/plate and higher in the absence of S-9 mix and at 5000 μg/plate in the presence of S-9 mix. Therefore, the 5000 μg/plate was selected as the highest dose level for the main tests.

In the main tests, no biologically significant increases in the number of revertant colonies were observed in any S. typhimurium tester strain after treatment with Mediator SNP at any dose level either in the presence or absence of S-9 mix. No biologically or statistically significant increases in the number of revertant colonies were observed in E. coli WP2 urvA at any dose level either in the presence or absence of S-9 mix. Statistical increases in the number of revertant colonies were noted with the positive controls in both the presence and absence of metabolic activation substantiating the sensitivity of the treat and plate assay and the efficacy of the metabolic activation mixture.

Under the conditions of this assay, Mediator SNP has not shown any evidence of mutagenic activity in the assay and is not a mutagen.