[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 14, 2017 to July 14, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD guidelines for testing of chemicals no. 471 (1997) "Bacterial Reverse Mutation Test".

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C16-18 and ethoxylated C16-18 , phosphates (20 moles ethoxylation)

Molecular formula:

C18H39O4P1 (monoester representative, i.e., mono- C18 PSE) C56H115O24P1 (ethoxylated monoester representative, i.e., mono- C16 AE20 PSE) C32H67O4P1 (diester representative, i.e., di- C18 PSE) C112H227O44P1 (ethoxylated diester representative, i.e., di- C16 AE20 PSE)

IUPAC Name:

Alcohols, C16-18 and ethoxylated C16-18 , phosphates (20 moles ethoxylation)

Test material form:

solid

Method

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:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Eight concentrations of the test substance (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method in experiment 1. Experiment 2 was performed using pre-incubation method in the presence and absence of metabolic activation. The test substance concentrations were: Salmonella strains TA100 and TA1535 (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate. Salmonella strain TA1535 (presence of S9): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was initially used in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test substance induced toxicity as weakened bacterial background lawns to the extent where a two of the strains required repeat analysis employing an amended test substance dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test substance was employed in the second mutation test.

Vehicle / solvent:

Identity: Tetrahydrofuran
Batch number (purity): 1707545 (99.9%)

The test substance was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. No correction was required for purity. Tetrahydrofuran is toxic to the bacterial cells at and above 50 uL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 uL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran, Batch no. - 1707545, Purity- 99.9%, Expiry: 02/2019

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

not specified

Details on test system and experimental conditions:

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 μg/plate because of test item precipitation. A number of manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 - 7 to 40
TA100 - 60 to 200
TA1537 - 2 to 30
TA98 - 8 to 60
WP2uvrA - 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative 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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was initially used in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test substance induced toxicity as weakened bacterial background lawns to the extent where a two of the strains required repeat analysis employing an amended test substance dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test substance was employed in the second mutation test. Results from the second mutation test showed that the test substance induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 µg/plate (TA1535), 500 µg/plate (TA100) and 1500 µg/plate (TA98 and TA1537). In the presence S9-mix, weakened bacterial background lawns were initially noted from 500 µg/plate (TA1535) and 1500 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA at any test substance dose level in either the absence or presence of S9-mix or Salmonella strains TA98 and TA1537 dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

A test substance precipitate (particulate in appearance) was noted at and above 1500 mg/plate, this observation did not prevent the scoring of revertant colonies. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.4 times the concurrent vehicle control.

Further statistically significant increases were observed in Experiment 2 (TA100 dosed in the presence of S9 at 1500 µg/plate and TA1535 at 150 and 500 µg/plate in the absence of S9 and 500, 1500 and 5000 µg/plate in the presence of S9). These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional histidine being available to His-bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation.

Executive summary:

A study was conducted to determine mutagenic potential of the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' by Ames test according to OECD 471, in compliance with GLP. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was initially used in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test substance induced toxicity as weakened bacterial background lawns to the extent, where a two of the strains required repeat analysis employing an amended test substance dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test substance was employed in the second mutation test. Results from the second mutation test showed that the test substance induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 µg/plate (TA1535), 500 µg/plate (TA100) and 1500 µg/plate (TA98 and TA1537). In the presence S9-mix, weakened bacterial background lawns were initially noted from 500 µg/plate (TA1535) and 1500 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA at any test substance dose level in either the absence or presence of S9-mix or Salmonella strains TA98 and TA1537 dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test substance precipitate (particulate in appearance) was noted at and above 1500 mg/plate, this observation did not prevent the scoring of revertant colonies. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.4 times the concurrent vehicle control. Further statistically significant increases were observed in Experiment 2 (TA100 dosed in the presence of S9 at 1500 µg/plate and TA1535 at 150 and 500 µg/plate in the absence of S9 and 500, 1500 and 5000 µg/plate in the presence of S9). These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional histidine being available to His-bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017).