(Z)-N-[2-(2-hydroxyethoxy)ethyl]-9-octadecenamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 10, 2017 to June 17, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Stability at higher temperatures: yes, maximum temperature: 30°C
light yellow paste

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells: cells from healthy, adult, non smoking humans were used as recommended in international guidelines (e.g. OECD).
- Average Generation Time: 13.7 h (dose-range finding), 15.8 h (first cytogenetic assay), 13.4 (second cytogenic assay) and 14.2 h (additional second cytogenetic assay)
- Sex, age and number of blood donors: approx. 22 years of age
- Whole blood treated with heparin was used


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix for all exposures: 78, 156, 313, 625, 1250 and 2500 µg/mL
The highest tested concentration was determined by the solubility of the test substance in the culture medium.

First cytogenetic test:
Without S9 (3h exposure time, 24h fixation time): 25, 100, 200, 225, 250, 275 and 300 µg/mL (25, 200 and 250 µg/mL were selected for scoring)
With S9 (3h exposure time, 24h fixation time): 25, 100, 200, 225, 250, 275, 300 and 350 µg/mL (25, 250 and 300 µg/mL were selected for scoring)
The test substance precipitated in the culture medium from 250 µg/mL and upwards.

Second cytogenetic test:
Without S9 (24 h exposure time, 24 h fixation time and 48 h exposure time, 48 h fixation time): 10, 50, 100, 125, 150, 175, 200 and 250 µg/mL (no cytotoxic dose levels could be selected for scoring)
In the 24 h exposure time, the test substance precipitated in the culture medium at 250 µg/mL

Additional second cytogenetic test:
Without S9 (24 h exposure time, 24 h fixation time): 50, 100, 150, 180, 200, 220 and 240 µg/mL (50, 100 and 180 µg/mL and 150, 175 and 200 µg/mL were selected for scoring in the 24 h exposure and 48 h exposure times, respectively)

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test substance was dissolved in dimethyl sulfoxide of spectroscopic quality.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24, 48 h (without S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

ENVIRONMENTAL CONDITIONS:
- Humidity: 38-91%
- Temperature: 34.5-37.1 °C

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96%
(v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: In the first assay, the test substance was difficult to dissolve in aqueous solutions, the highest concentration analysed was determined by the solubility in the culture medium.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

EVALUATION CRITERIA:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

ACCEPTABILITY 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 substance induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).

Statistics:

Fisher's exact test, one-sided, was used to compare the incidence of abberant cells cells with the concurrent negative control.
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second assay the highest concentration (250 μg/ml) was too toxic for scoring (83%)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

RANGE-FINDING/SCREENING STUDIES:
- The test substance precipitated in the test medium at and above a dose level of 313 µg/mL. At and above this concentration, no metaphases were observed and cell lysis occurred.

- Precipitation in the main tests: In the first cytogenetic test, the test substamce precipitated in the culture medium from 250 µg/mL and upwards. In the second cytogenetic test, in the 24 h exposure time, the test substance precipitated in the culture medium at 250 µg/mL. No precipitation was observed in the additional second test.

DOSE LEVELS SELECTED FOR SCORING:
- First cytogenetic test:
Without S9: 25, 200 and 250 µg/mL
With S9: 25, 250 and 300 µg/mL

- Second cytogenetic test:
At the 24 h exposure time, 24 h fixation time, no cytotoxic dose levels could be selected for scoring since at the concentration of 200 μg/ml not enough cytotoxicity was observed (44%), whereas the next higher concentration of 250 μg/ml was too toxic for scoring (83%). Therefore, an additional second cytogenetic assay was performed with a 24 h exposure time and 24 h fixation time only.

- Additional second cytogenetic test:
24 hour exposure time, 24 hour fixation time: 50, 100 and 180 µg/mL
48 hour exposure time, 48 hour fixation time: 150, 175 and 200 µg/mL


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses (see attached illustration for details).

Conclusions:

Under the study conditions, it is concluded that the test substance is not clastogenic in human lymphocytes.

Executive summary:

A study was conducted to determine the potential of the test substance to induce structural chromosome aberrations in cultured human lymphocytes according to OECD Guideline 473, in compliance with GLP. Two independent experiments were performed. In the first cytogenetic assay, the test substance was tested up to 250 μg/mL and 300 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix, respectively. The test substance precipitated in the culture medium at these dose levels. In the second cytogenetic assay, test substance was tested up to 180 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 200 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The negative control used in the test system was the vehicle for the test substance DMSO. Mitomycin C and Cyclophosphamide served as positive controls. The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two experiments. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. Tests conducted with the positive control chemicals, confirmed that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it is concluded that the test substance is not clastogenic in human lymphocytes (Buskens, 2017).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 13, 2017 to April 16, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B

Deviations:

no

Principles of method if other than guideline:

In the second mutation experiment, S9-mix contained per 10 ml: 15.2 mg NADP and 30 mg glucose-6-phosphate (G-6-P).
Evaluation: Due to a calculation error not the correct composition of the S9-mix was prepared. According to OECD guideline 471 the amount of S9-fraction should be in the range of 5 to 30% in the metabolic activation mixture. Furthermore, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Therefore, this deviation in the composition of the S9-mix had no effect on the results of the study.

GLP compliance:

yes

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

Metabolic activation:

with and without

Metabolic activation system:

5% (v/v) and 10% (v/v) rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

In the dose-range finding test, the test substance was tested up to concentrations of 5000 μg/plate.
First test: concentration range of 17 to 5000 μg/plate (TA1535, TA1537 and TA98).
Second test: concentration range of 154 to 2800 μg/plate (TA1535, TA1537, TA98, TA100 and WP2uvrA).

Vehicle / solvent:

The test substance was dissolved in dimethyl sulfoxide. No correction was made for the purity/composition of the test substance. A solubility test was performed based on visual assessment. No analysis of the formulated test substance was conducted for this study with respect to either test substance concentration, homogeneity or of the test substance in the vehicle. Preparations were visually homogenous prior to use and all preparations were used within 3 hours after adding the vehicle to the test substance.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria
Source: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]

The characteristics of the different Salmonella typhimurium strains were as follows:
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

CELL CULTURE

Preparation of bacterial cultures:
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for testing.

Agar plates:
Agar plates (ø 9 cm) containing 25 ml glucose agar medium were used. Glucose agar medium contained per liter: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan.

Top agar:
Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions:
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.4 - 39.0°C). The temperature was continuously monitored throughout the experiment.

METABOLIC ACTIVATION SYSTEM
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

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 MgCl2 solution; 1 ml 0.33 M KCl 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.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation 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 vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated attest labolatroy.
b) 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.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

Critical Computerized Systems:
REES Centron (Temperature Data collection), version: SQL 2.0,
Ames study Manager (Revertant Colony Count Collection), version: 1.23.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

FIRST MUTATION EXPERIMENT:

Precipitate:
Precipitation of the test substance on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and at 1600 and 5000 μg/plate at the end of the incubation period.

Toxicity:
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
In strains TA1535 and TA1537, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed in the absence of S9-mix. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test substance. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.

Mutagenicity:
No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

SECOND MUTATION EXPERIMENT:

Precipitate:
Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 1568 and 2800 μg/plate. At the end of the incubation period the test substance was tested up to or beyond a precipitating dose level.

Toxicity:
In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In strains TA1535, TA1537 (absence of S9-mix) and TA100 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.

Mutagenicity:
In tester strain TA1537, the test substance induced an up to 6-fold increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix. However, these increases were only observed in the second experiment and related to a very low mean solvent control value. Furthermore, the highest number of revertants was not higher than 12 and within our historical control data range. Therefore, these increases are considered to be not biologically relevant. In the other test strains, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Remarks on result:

other: Experiment I and II

Conclusions:

Under the study conditions, it is concluded that the test substance is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay.

Executive summary:

A study was conducted to determine the potential of the test substance and/or its metabolites to induce reverse mutations at the histidine locus in several strains of S. typhimurium and at the tryptophan locus of E. coli according to OECD Guideline 471 and EC Guideline No. 440/2008. Part B. The test substance was examined using five strains of S. typhimurium: TA98, TA100, TA1535, and TA1537, and strain WP2uvrA of E. coli in the presence or absence of an exogenous mammalian metabolic activation system (S9). The test substance was dissolved in dimethyl sulfoxide. Based on the results of the dose-range finding test, the test substance was tested in the first mutation assay at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 154 to 2800 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it is concluded that the test substance is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay (Gijsbrechts, 2017).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 14, 2017 to April 24, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical concurrent solvent control database, except in the first experiment in the absence of S9-mix in which the mutation frequency of one of the solvent control cultures was not within the range of the acceptability criteria.
Since the mutation frequency was just below the lower limit of the acceptability criteria range and the mutation frequency of the other solvent control culture was within the acceptability criteria range, this deviation in the mutation frequency had no effect on the validity of the results of the first mutation experiment.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- No correction for purity required.
- Stable at higher temperatures: yes, maximum temperature: 30°C
- Solubility and stability in vehicle: not indicated

Target gene:

- Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: not indicated

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose-range finding test (with and without S9; cytotoxicity test): 15.63, 31.25, 62.5, 125, 250 and 500 μg/mL
Since the test substance was poorly soluble in the exposure medium, the highest tested concentration was 500 μg/mL exposure medium.
Experiment 1 (without S9): 2.5, 5, 10, 20, 30, 40, 45 and 50 μg/mL
Experiment 1 (with S9): 25, 50, 60, 70, 80, 90, 100 and 110 μg/mL
Experiment 2 (without S9): 1.25, 2.5, 5, 10, 15, 20, 25 and 30 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: as recommended in OECD test guideline 490

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: below 1 x 10^6 cells/mL

DURATION
- Cleansing period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT: TFT

STAIN: 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For determination of the cloning efficiency the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for cloning efficiency and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFTselection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the cloning efficiency and MF were scored with the naked eye or with the microscope.

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test substance is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the 1st exp.: at and above 55 μg/mL (without S9) and at and above 70 μg/mL (with S9). In the 2nd exp.: at and above 20 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility in medium: the test item precipitated in the exposure medium at concentrations of 250 μg/mL and above. The test item 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 500 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
- After 3 hours of treatment, in the absence of S9-mix, the relative suspension growth was 7% at the test item concentration of 62.5 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test item concentrations of 125 μg/ml and above.
- After 3 hours of treatment, in the presence of S9-mix, the relative suspension growth was 67% at the test item concentration of 62.5 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test item concentrations of 125 μg/ml and above.
- After 24 hours of treatment, the relative suspension growth was 26% at the test item concentration of 31.25 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at the test item concentration of 62.5 μg/ml and above.

FIRST MUTAGENICITY TEST:
- Dose levels which showed no cytotoxicity or were too toxic for further testing, were not used for mutation frequency measurements.
- In the absence of S9-mix, the relative total growth of the highest test item concentration was 16% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth of the highest test item concentration was 20% compared to the total growth of the solvent controls.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the DGA oleamide treated cultures were comparable to the numbers of small and large colonies of the solvent controls. In addition, none of the tested concentrations reached a mutation frequency of MF(controls) + 126, it is a GEF of 192 and 199 per 10^6 survivors in the absence and presence of S9-mix, respectively.

SECOND MUTAGENICITY TEST:
- Dose levels which showed no cytotoxicity or were too toxic for further testing, were not used for mutation frequency measurements.
- The relative total growth of the highest test item was 12% compared to the total growth of the solvent controls.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls. In addition, none of the tested concentrations reached a mutation frequency of MF(controls) + 126, it is a GEF of 250 per 10^6 survivors.

HISTORICAL CONTROL DATA: see table 1 and table 2

ACCEPTABILITY:
- The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database, except in the first experiment in the absence of S9-mix, in which the mutation frequency of one of the solvent control cultures was just below the acceptability criteria of this assay. Since the mutation frequency was just below the lower limit of the range and the mutation frequency of the other solvent control culture (85 survivors per 10^6 survivors) was within the range, this deviation in the mutation frequency had no effect on the results of the study.
- Positive control chemicals both produced significant increases in the mutation frequency and the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

For detailed results, see attached illustration.

Table 1 Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	86	81	87
SD	23	26	28
n	220	202	273
Upper control limit (95% control limits)	135	135	145
Lower control limit (95% control limits)	37	28	28

SD = Standard deviation

n = Number of observations

Table 2 Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	857	688	1710
SD	246	187	815
n	110	102	139
Upper control limit (95% control limits)	1425	1124	4214
Lower control limit (95% control limits)	289	253	-793

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between January 2013 and November 2016. 

Conclusions:

Under the study conditions, the test substance is not mutagenic in the mouse lymphoma L5178Y test system.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells according to OECD Guideline 490, in compliance with GLP. 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. In the first experiment, test substance was tested up to concentrations of 50 and 110 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative Total Growth (RTG) was 16 and 20% in the absence and presence of S9-mix, respectively. The test substance did not precipitate in the culture medium. In the second experiment, test substance was tested up to concentrations of 30 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 12%. The test substance did not precipitate in the culture medium. The negative control used in the test system was the vehicle for the test substance DMSO. Methyl methanesulfonate and cyclophosphamide served as positive control chemicals. In the absence of S9-mix, the test substance 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, the test substance did not induce a significant increase in the mutation frequency based on the Global Evaluation Factor (GEF) of 126-E6. The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical concurrent solvent control database, except in the first experiment in the absence of S9-mix in which the mutation frequency of one of the solvent control cultures was not within the range of the acceptability criteria. Since the mutation frequency was just below the lower limit of the acceptability criteria range and the mutation frequency of the other solvent control culture was within the acceptability criteria range, this deviation in the mutation frequency had no effect on the validity of the results of the first mutation experiment. Tests conducted with the positive control chemicals, confirmed that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance is not mutagenic in the mouse lymphoma L5178Y test system (Verspeek-Rip, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

A study was conducted to determine the potential of the test substance and/or its metabolites to induce reverse mutations at the histidine locus in several strains of S. typhimurium and at the tryptophan locus of E. coli according to OECD Guideline 471 and EC Guideline No. 440/2008. Part B. The test substance was examined using five strains of S. typhimurium: TA98, TA100, TA1535, and TA1537, and strain WP2uvrA of E. coli in the presence or absence of an exogenous mammalian metabolic activation system (S9). The test substance was dissolved in dimethyl sulfoxide. Based on the results of the dose-range finding test, the test substance was tested in the first mutation assay at a concentration range of 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 154 to 2800 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it is concluded that the test substance is not mutagenic in the S. typhimurium reverse mutation assay and in the E. coli reverse mutation assay (Gijsbrechts, 2017).

Mammalian cell gene mutation tests:

A study was conducted to investigate the potential of the test substance to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells according to OECD Guideline 490, in compliance with GLP. 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. In the first experiment, test substance was tested up to concentrations of 50 and 110 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Relative Total Growth (RTG) was 16 and 20% in the absence and presence of S9-mix, respectively. The test substance did not precipitate in the culture medium. In the second experiment, test substance was tested up to concentrations of 30 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 12%. The test substance did not precipitate in the culture medium. The negative control used in the test system was the vehicle for the test substance DMSO. Methyl methanesulfonate and cyclophosphamide served as positive control chemicals. In the absence of S9-mix, the test substance 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, the test substance did not induce a significant increase in the mutation frequency based on the Global Evaluation Factor (GEF) of 126-E6. The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical concurrent solvent control database, except in the first experiment in the absence of S9-mix in which the mutation frequency of one of the solvent control cultures was not within the range of the acceptability criteria. Since the mutation frequency was just below the lower limit of the acceptability criteria range and the mutation frequency of the other solvent control culture was within the acceptability criteria range, this deviation in the mutation frequency had no effect on the validity of the results of the first mutation experiment. Tests conducted with the positive control chemicals, confirmed that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance is not mutagenic in the mouse lymphoma L5178Y test system (Verspeek-Rip, 2017).

Mammalian chromosome aberration test:

A study was conducted to determine the potential of the test substance to induce structural chromosome aberrations in cultured human lymphocytes according to OECD Guideline 473, in compliance with GLP. Two independent experiments were performed. In the first cytogenetic assay, the test substance was tested up to 250 μg/mL and 300 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix, respectively. The test substance precipitated in the culture medium at these dose levels. In the second cytogenetic assay, test substance was tested up to 180 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 200 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The negative control used in the test system was the vehicle for the test substance DMSO. Mitomycin C and Cyclophosphamide served as positive controls. The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two experiments. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. Tests conducted with the positive control chemicals, confirmed that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it is concluded that the test substance is not clastogenic in human lymphocytes (Buskens, 2017).

Justification for classification or non-classification

Based on the results of in vitro testing, the test substance does not require classification for mutagenicity according to EU CLP (EC 1272/2008) criteria).