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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 March 2016 to 24 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Benzaldehyde, hydroxy-, polymer with phenol
Cas Number:
106466-55-1
Molecular formula:
(C7H6O2.C6H6O)x
IUPAC Name:
Benzaldehyde, hydroxy-, polymer with phenol
Test material form:
solid: flakes
Details on test material:
- Appearance: Red – reddish brown solid flake
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH %)

Method

Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Source: 21 April 2015, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA.
- Storage: The strains are stored at -80 ± 10 °C in the Culture Collection of the Microbiological Laboratory of the testing facility. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Confirmation of Phenotypes of Tester Strains: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
- Spontaneous Reversion of Tester Strains: Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2014 were as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24.
- Procedure for Growing Cultures: The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.
- Viability of the Testing Cultures: The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.
- Media: Nutrient Broth No.2 containing: Nutrient Broth No.2 25.0 g and Distilled water q.s. ad 1 000 mL. Sterilisation was performed at 121 °C in an autoclave.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Source: 21 April 2015, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Storage: The strains are stored at -80 ± 10 °C in the Culture Collection of the Microbiological Laboratory of the testing facility. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Confirmation of Phenotypes of Tester Strains: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
-Spontaneous Reversion of Tester Strains: Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to tryptophan independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2014 were as follows: Escherichia coli WP2 uvrA: 11-82.
- Procedure for Growing Cultures: The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.
- Viability of the Testing Cultures: The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.
- Media: Nutrient Broth No.2 containing: Nutrient Broth No.2 25.0 g and Distilled water q.s. ad 1 000 mL. Sterilisation was performed at 121 °C in an autoclave.
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
- Range Finding Test: 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate (Salmonella typhimurium TA98 and TA100 only)
- Initial Mutation Test and Confirmatory Mutation Test: Salmonella typhimurium strains were 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate and Escherichia coli WP2 uvrA strain were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
- Concentrations were selected based on the results of the preliminary tests.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected after a preliminary compatibility test was performed to examine the behaviour of the test material formulations with the solution of top agar and phosphate buffer.

PRELIMINARY COMPATABILITY TEST
-The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. The test material was insoluble at 100 mg/mL concentration using Distilled water, however the formulation at the same concentration using DMSO (after approximately 3 minutes stirring) or Acetone were solutions and were suitable for the test. Due to the better biocompatibility DMSO (with the appropriate time of stirring) was selected as vehicle for the test.
The obtained stock solution (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

- Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.
- Analytical determination of the test material concentration, stability and homogeneity was not performed because of the character and the short period of study.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine (Without activation, Salmonella TA98 at 4 µg/plate) and 2-aminoanthracene (With activation, 2 µg/plate for all Salmonella strains and 50 µg/plate for E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

INITIAL MUTATION TEST
- A standard plate incorporation procedure was performed, as an Initial Mutation Test. Bacteria were exposed to the test material both in the presence and absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).
- The content of the tubes: top agar 2 000 μL, vehicle or test material formulation (or reference controls) 50 μL, overnight culture of test strain 100 μL and phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

CONFIRMATORY TEST
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed. Bacteria were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
- Before the overlaying, the test material formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer (pH 7.4) was added into appropriate tubes to provide direct contact between bacteria and the test material (in its vehicle/solvent).
- The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

NUMBER OF REPLICATIONS: In the test each sample (including the controls) was tested in triplicate.

EVALUATION OF THE EXPERIMENTAL DATA
- The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported.
Evaluation criteria:
CRITERIA FOR VALIDITY
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests
- at least five analysable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE:
A test material was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than twice higher than the reversion rate of the negative (solvent) control.

CRITERIA FOR A NEGATIVE RESPONSE:
- A test material was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using Microsoft ExcelTM software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
- Inhibitory, cytotoxic effects of the test material (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the Preliminary Range Finding Test in both tested strains without metabolic activation at 5 000, 2 500, 1 000 and 316 μg/plate concentrations and in both tested strains with metabolic activation at 5 000, 2 500 and 1 000 μg/plate concentrations.
- No precipitate of the test material was detected in the Preliminary Range Finding Test.

INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
- In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at the concentration of 0.5 μg/plate. The mutation factor value was 1.91. Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain with metabolic activation. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration with metabolic activation. The mutation factor values were 1.80. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Test in some other cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value and were within the historical control range, they were considered as reflecting the biological variability of the test.
- Inhibitory, cytotoxic effects of the test material (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the Initial Mutation Test at 1581 and 500 μg/plate concentrations in Salmonella typhimurium TA98, TA100, TA1537 strains with and without metabolic activation, in Salmonella typhimurium TA1535 strain without metabolic activation; at 1581 μg/plate concentration in Salmonella typhimurium TA1535 strain with metabolic activation and at 5 000 and 1 581 μg/plate concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation.
- Similar but stronger inhibitory, cytotoxic effects of the test material were observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains at 1581, 500 and 158.1 μg/plate concentrations without metabolic activation and at 1 581 and 500 μg/plate concentrations with metabolic activation; in Escherichia coli WP2 uvrA strain without metabolic activation at 5 000, 1 581 and 500 μg/plate concentrations and at 5 000 and 1 581 μg/plate concentrations with metabolic activation.
- Slight precipitate was observed in the Confirmatory Mutation Test in Salmonella typhimurium strains with metabolic activation at the concentration of 1 581 and in Escherichia coli WP2 uvrA strain with metabolic activation at 5 000 and 1 581 μg/plate concentrations.

VALIDITY OF THE TESTS
- Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were in good correlation with the historical control data. At least five analysable concentrations were presented in all strains of the main tests.
- The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.
Executive summary:

The test material was tested for potential mutagenic activity in accordance with the standardised guidelines OECD 471, EU Method B13/14 and OPPTS 870.5100, under GLP conditions using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test material was dissolved in DMSO. Concentrations of 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test and Confirmatory Mutation Test for Salmonella typhimurium strains were 1 581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate and for Escherichia coli WP2 uvrA strain were 5 000, 1 581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.

Inhibitory, cytotoxic effects of the test material were observed in the Initial Mutation Test at 1 581 and 500 μg/plate concentrations in Salmonella typhimurium TA98, TA100, TA1537 strains with and without metabolic activation, in Salmonella typhimurium TA1535 strain without metabolic activation; at 1581 μg/plate concentration in Salmonella typhimurium TA1535 strain with metabolic activation and at 5 000 and 1581 μg/plate concentrations in Escherichia coli WP2 uvrA strain with and without metabolic activation.

Similar but stronger inhibitory, cytotoxic effects of the test material were observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains at 1 581, 500 and 158.1 μg/plate concentrations without metabolic activation and at 1 581 and 500 μg/plate concentrations with metabolic activation; in Escherichia coli WP2 uvrA strain without metabolic activation at 5 000, 1 581 and 500 μg/plate concentrations and at 5 000 and 1 581 μg/plate concentrations with metabolic activation.

Slight precipitate was observed in the Confirmatory Mutation Test in all tested strains with metabolic activation at the concentrations of 5 000 and/or 1 581 μg/plate.

The mean values of revertant colonies of the solvent control plates were in good correlation with the historical control data, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material had no mutagenic activity in the applied bacterium tester strains.

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