Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of Alkaterge E was investigated in a bacterial reverse mutation assay (Ames test) according to OECD 471, in the in vitro micronucleus assay (OECD 487) and in an in vitro mammalian cell mutation assay

according to OECD 476. Alkaterge E showed no evidence of a mutagenic or clastogenic potential, with and without metabolic activation, in these in vitro test systems.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: Alkaterge E
Chemical name (IUPAC, synonym or trade name): 4-Ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol
CAS number: 68140-98-7
Molecular formula: C23H43NO2
Molecular weight: 365.60
Appearance: Brown viscous liquid
Batch: D598F47BC1
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 05 April 2020 (retest date)

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat S9, obtained from Trinova Biochem GmbH, Giessen (Germany) is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

In the first experiment, Alkaterge E was tested up to concentrations of 38 µg/mL in the absence and presence S9-mix. In the absence of S9-mix, relative total growth (RTG) was reduced to 7% and 36% at the concentrations of 25 and 38 µg/mL, respectively. In the presence of S9-mix, no toxicity was observed at the dose level of 38 µg/mL. Alkaterge E precipitated in the culture medium at the dose level of 38 µg/mL. In the second experiment, Alkaterge E was tested up to concentrations of 25 µg/mL in the absence of S9-mix.

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test System L5178Y/TK+/--3.7.2C mouse lymphoma cells. Source American Type Culture Collection, (ATCC, Manassas, USA) (2001)

Stock cultures of these cells are stored in liquid nitrogen (-196°C). The cultures are checked for mycoplasma contamination. Cell density will be preferably kept below 1 x 106 cells/mL.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Alkaterge E precipitated in the exposure medium at concentrations of 38 μg/mL and above. Alkaterge E was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 150 μg/mL.

Dose-range Finding Test
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of from 4.7 to 150 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and with a range of from 4.3 to 136 µg/mL in the presence of S9-mix with a 3 hour treatment period. In the absence of S9-mix, the relative suspension growth was 16% at the test item concentration of 38 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 75 μg/mL and above. In the presence of S9-mix, the relative suspension growth was 5% at the test item concentration of 136 μg/mL compared to the relative suspension growth of the solvent control. Due to a technical error, the dose level of 150 µg/mL was not used for cytotoxicity determination. The relative suspension growth was 36% at the test item concentration of 19 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at the test item concentrations of 38 μg/mL and above.

First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without S9-mix: 0.074, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.5, 15, 19, 25 and 38 μg/mL exposure medium.
With S9-mix: 0.074, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, 9.5, 19 and 38 μg/mL exposure medium.

Evaluation of toxicity
In the absence of S9-mix, the dose levels of 0.074 to 15 μg/mL showed no cytotoxicity or showed an inconsistent RSG (0.074 µg/mL). Therefore, the dose levels of 0.074, 0.3, 1.2 and 9.5 µg/mL were not regarded relevant for mutation frequency measurement. In the presence of S9-mix, no cytotoxicity was observed. Therefore, the dose levels of 0.074 and 0.15 µg/mL were not regarded relevant for mutation frequency measurement. The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.15, 0.6, 2.4, 4.8, 15, 19, 25 and 38 μg/mL exposure medium.
With S9-mix: 0.3, 0.6, 1.2, 2.4, 4.8, 9.5, 19 and 38 μg/mL exposure medium.

In the absence of S9-mix, the relative total growth was 7% and 36% at the concentrations of 25 and 38 µg/mL, respectively.In the presence of S9-mix, no toxicity was observed up to and including the highest tested dose level.

Evaluation of the mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with Alkaterge E either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Alkaterge E treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second Mutagenicity Test
To obtain more information about the possible mutagenicity of Alkaterge E, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. Based on the results of the dose-range finding test, the following dose levels were selected for mutagenicity testing: 0.63, 1.25, 2.5, 5, 10, 15, 20, 25, 30 and 40 µg/mL exposure medium. Further investigation showed that at a concentration of 30 µg/mL Alkaterge E already precipitated in the exposure medium.

Evaluation of toxicity
The dose levels of 30 and 40 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 0.63, 1.25, 2.5, 5, 10, 15, 20 and 25 µg/mL exposure medium. The relative total growth of the highest test item was 14% compared to the total growth of the solvent controls.

Evaluation of mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item.

Conclusions:

Alkaterge E is not mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of Alkaterge E by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  The study procedures described in this report were based on OECD guideline No. 490. Alkaterge E was a brown viscous liquid. The vehicle of the test item was ethanol. The concentrations analyzed for the dose formulation samples at intermediate and high concentration level were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). For the dose formulation samples at low concentration level the mean accuracy were slightly above the target concentration (i.e. 118% of target). A small response at the retention time of the test item was observed in the chromatograms of the vehicle group, however,this response was only 0.025-12% of the Group Low Samples. Dose formulation samples were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

In the first experiment, Alkaterge E was tested up to concentrations of 38 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. In the absence of S9-mix, relative total growth (RTG) was reduced to 7% and 36% at the concentrations of 25 and 38 µg/mL, respectively. In the presence of S9-mix, no toxicity was observed at the dose level of 38 µg/mL. Alkaterge E precipitated in the culture medium at the dose level of 38 µg/mL. In the second experiment, Alkaterge E was tested up to concentrations of 25 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 14%. The mean mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.  It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Alkaterge E did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, Alkaterge E did not induce a significant increase in the mutation frequency.

In conclusion, Alkaterge E is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Alkaterge E
Chemical name (IUPAC, synonym or trade name): 4-Ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol
CAS number: 68140-98-7
Molecular formula: C23H43NO2
Molecular weight: 365.60
Appearance: Brown viscous liquid
Batch: D598F47BC1
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 05 April 2020 (retest date)

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human

Metabolic activation:

with and without

Test concentrations with justification for top dose:

In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. Alkaterge E was tested in the absence and presence of S9-mix. Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of Alkaterge E for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time. The highest tested concentration was determined by the solubility of Alkaterge E in the culture medium. Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was determined by the solubility or showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

First Cytogenetic Assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Alkaterge E for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasin B (5 µg/mL) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay. A repeat of the first cytogenetic assay was performed because the vehicle was changed, no scoring was performed.

Second Cytogenetic Assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix. Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Alkaterge E with cytochalasin B (5 µg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

Rationale for test conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.

Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2017) are presented below:
Dose-range finding study: age 26, AGT = 14.2 h
First cytogenetic assay: age 23, AGT = 14.2 h
Second cytogenetic assay:age 28, AGT = 13.4 h
Cytogenetic assay 2A: age 32, AGT = 14.1 h

Evaluation criteria:

An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a). The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b). The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c). The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a). At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b). The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c). Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a). None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b). There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c). All results are inside the 95% control limits of the negative historical control data range.

The Chi-square test showed that there were statistically significant differences between one or more of the test item groups and the vehicle control group. A Cochran Armitage trend test (p < 0.05) was performed to test whether there was a significant trend in the induction.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Dose-range finding test

At a concentration of 250 µg/mL Alkaterge E precipitated in the culture medium. The vehicle used was dimethyl sulfoxide. In the dose-range finding test blood cultures were treated with 7.8, 15.6, 31.3, 62.5, 125 and 250 µg Alkaterge E/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

First cytogenetic assay

At a concentration of 78 µg/mL Alkaterge E precipitated in the culture medium in the first cytogenetic assay. The vehicle used was ethanol. Based on the cytotoxicity observed in the dose range finding test and on the results of precipitation in the culture medium, the following dose levels were selected for the first cytogenetic assay with ethanol as the vehicle: 19.5, 39 and 78 µg/mL culture medium, with and without S9-mix (3 hours exposure time, 27 hours harvest time). All dose levels were selected for scoring of micronuclei. In the absence of S9-mix, Alkaterge E did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronu

In the presence of S9-mix, Alkaterge E induced a statistically significant increase in the number of binucleated with micronuclei at the lowest concentration tested. This increase was observed at the lowest dose level, only. Although a statistical significant trend was observed all results are well within the 95% control limits of the distribution of the historical negative control database, therefore this increase was not biologically relevant.

Second cytogenetic assay

Alkaterge E, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay. Without S9-mix:  5, 25, 50, 60, 70 and 80 µg/mL culture medium  (24 hours exposure time, 24 hours harvest time). The test item precipitated in the culture medium at concentrations of 50 µg/mL and above. The following dose levels were selected for the scoring of micronuclei: 5, 25, 50 and 60 µg/mL culture medium  (24 hours exposure time, 24 hours harvest time). Alkaterge E did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

Conclusions:

Alkaterge E is negative in the in vitro micronucleus test in cultured human lymphocytes.

Executive summary:

The objective of this study was to evaluate Alkaterge E for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of Alkaterge E was tested in two independent experiments. The study procedures described in this report are in compliance with the most recent OECD guideline No. 487. Alkaterge E was a brown viscous liquid. The v hicle of the test item was dimethyl sulfoxide (dose range finding) or ethanol (cytogenetic assays). The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

In the first cytogenetic assay, Alkaterge E was tested up to 78 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Alkaterge E precipitated in the culture medium at this dose level. In the second cytogenetic assay, Alkaterge E was tested up to 60 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level and the test item precipitated in the culture medium. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in the absence of S9-mix the response of colchicine was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Alkaterge E did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, this test is valid and Alkaterge E is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report. 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2010 - 19 November 2010

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:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2 uvrA in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 or at 5000 μg per plate. Toxicity was observed beginning at concentrations ranging from 150 to 5000 μg per plate with most test conditions. Strain TA1537was retested based on the precipitate/toxicity profile observed. The maximum concentration in the retest and confirmatory mutagenicity assays were 500 μg per plate with all Salmonella tester strains without S9 and 5000 μg per plate with the remaining test conditions. In the retest of the initial toxicity-mutation assay, the concentrations were 1.5, 5.0, 15, 50, 150 and 500 μg per plate with tester strain TA1537 without S9 and 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strain TA1537 with S9. No positive mutagenic response was observed with tester strain TA1537 either with or without S9. Precipitate was observed beginning at 500 μg per plate. Toxicity was observed beginning at 150 or 1500 μg per plate.

In the confirmatory mutagenicity assay, the concentrations were 1.5, 5.0, 15, 50, 150 and 500 μg per plate with all Salmonella tester strains without S9 and 15, 50, 150, 500, 1500 and 5000 μg per plate with the remaining test conditions. No positive mutagenic responses were observed with any of the tester strains with S9 or with tester strains TA98, TA100, TA1535 and WP2 uvrA without S9. Precipitate was observed at 500 and 5000 μg per plate. Toxicity was observed at concentrations ranging from 50 to 5000 μg per plate with all Salmonella tester strains. Strain TA1537 without S9 was retested. In the second retest of the confirmatory mutagenicity assay, the concentrations were 1.5, 5.0, 15, 50, 150 and 500 μg per plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

not applicable

Positive controls validity:

valid

Conclusions:

ALKATERGE-E Oxazoline was concluded to be negative in the bacterial reverse mutation assay.

Executive summary:

ALKATERGE-E Oxazoline, was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence or absence of Aroclor-induced rat liver S9. The assay was performed in two phases using the preincubation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article. Dosing formulation samples were adjusted for the initial sponsor-provided purity (81.4%), using a correction factor of 1.23. However, the Certificate of Analysis, provided after experimental completion, reported a purity of 80.0%. All dose levels cited in this report, except for the formulation analysis results, represent the nominal values based on the initially reported purity, and are consequently 98.3% of the intended dose levels based on the purity stated on the Certificate of Analysis. Dimethyl sulfoxide (DMSO) was selected as the solvent based on the solubility of the test article and compatibility with the target cells. After sonication for 10 minutes at 32°C, the test article formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, which is the maximum concentration recommended by test guidelines. This concentration was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The concentrations tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. The test article formed a soluble but cloudy solution in DMSO at 100 mg/mL and soluble and clear solutions from 0.030 to 30 mg/mL. No positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 or WP2 uvrA in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 or at 5000 μg per plate. Toxicity was observed beginning at concentrations ranging from 150 to 5000 μg per plate with most test conditions. Due to unacceptable vehicle control values tester strain TA1537 was not evaluated for mutagenicity but was retested based on the precipitate/toxicity profile observed. Based on the findings of the initial toxicity-mutation assay, the maximum concentrations to be tested in the retest and confirmatory mutagenicity assays were 500 μg per plate with all Salmonella tester strains in the absence of S9 activation and 5000 μg per plate with the remaining test conditions. The maximum concentration of 5000 μg per plate was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The plating aliquot was increased to 100 μL, which decreased the maximum concentration from 100 mg/mL to 50 mg/mL, in order to collect solutions for dose formulation analysis as opposed to suspensions. A 100 μL plating aliquot was used for all subsequent assays.

In the retest of the initial toxicity-mutation assay, the concentrations tested were 1.5, 5.0, 15, 50, 150 and 500 μg per plate with tester strain TA1537 in the absence of S9 activation and 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strain TA1537 in the presence of S9 activation. No positive mutagenic response was observed with tester strain TA1537 in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 μg per plate. Toxicity was observed beginning at 150 or 1500 μg per plate. In the confirmatory mutagenicity assay, the concentrations tested were 1.5, 5.0, 15, 50, 150 and 500 μg per plate with all Salmonella tester strains in the absence of S9 activation and 15, 50, 150, 500, 1500 and 5000 μg per plate with the remaining test conditions. No positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation or with tester strains TA98, TA100, TA1535 and WP2 uvrA in the absence of S9 activation. Precipitate was observed beginning at 500 or at 5000 μg per plate. Toxicity was observed beginning at concentrations ranging from 50 to 5000 μg per plate with all Salmonella tester strains. Due to an unacceptable vehicle control value, tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested based on the precipitate/toxicity profile observed. For the retest of the confirmatory mutagenicity assay, a technical error occurred in deviation from the protocol, in which the titer plates for tester strain TA1537 were not dosed. Therefore, tester strain TA1537 in the absence of S9 activation was not evaluated for mutagenicity but was retested. In the second retest of the confirmatory mutagenicity assay, the concentrations tested were 1.5, 5.0, 15, 50, 150 and 500 μg per plate. No positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation. Precipitate was observed at 500 μg per plate. Toxicity was observed beginning at 50 μg per plate. All criteria for a valid study were met as described in the protocol. The vehicle controls and positive controls in the initial toxicity-mutation, confirmatory mutagenicity and retest assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay except as indicated above.

Under the conditions of this study, ALKATERGE-E Oxazoline was concluded to be negative in the bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.