Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 30, 2015 to August 17, 2015

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA1537 hisC3076
TA98 hisD3052/R-factor*
TA1535 hisG46 Base-pair
TA100 hisG46/R-factor*
Escherichia coli: tryptophan (Trp+)

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:

S9-mix, rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1,600 and 5,000 µg/plate in triplicates
First experiment (direct plate assay) : 52, 164, 512, 1,600 and 5,000 μg/plate
Second experiment (plate incorporation assay): 17, 52, 164, 512, 1,600 and 5,000 μg/plate

Vehicle / solvent:

Water, saline and DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 & 2-aminoanthracene

Remarks:

Without metabolic activation: TA1535: sodium azide (SA), TA1537: ICR-191, TA1537: 2-nitrofluorene (NF), TA98: 2-nitrofluorene (NF), TA100: methylmethanesulfonate (MMS), WP2uvrA: 4-nitroquinoline N-oxide, With metabolic activation: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Salmonella typhimurium (all strains used) were obtained from Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]. Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) 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 a test. All incubations were carried out in a controlled environment at a temperature of 37.0±1.0°C (actual range 35.8–38.5°C).

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In second mutation experiment, with and without S9 mix

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:

cytotoxicity

Remarks:

in both mutation experiments, with and without S9 mix

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:

cytotoxicity

Remarks:

in second mutation experiment, with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in second mutation experiment without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A range finding study was conducted with the strains TA100 and the WP2uvrA in absence and presence of S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1,600 and 5,000 μg/plate were tested in triplicate.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

First experiment: Direct plate assay
Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1,600 and 5,000 μg/plate.
Precipitate: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period. Toxicity: No reduction of the bacterial background lawn was observed.
Mutagenicity: No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
Second experiment: Pre-incubation assay
A pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 5,000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period.
Toxicity: In tester strain WP2uvrA, a reduction of the bacterial background lawn was only observed at the highest tested concentration in the absence of S9-mix, also the bacterial background lawn was absent. In the Salmonella typhimurium strains no reduction of the bacterial background lawn was observed.
Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Conclusions:

No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay.No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay.

Executive summary:

An in vitro bacterial reverse mutation assay was performed to evaluate the potential of test substance to cause gene mutation according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. A total of two mutation experiments were conducted. Based on the results of the dose range finding test, a first mutation assay, a direct plate assay, was conducted with and without S9-mix in Salmonella typhimurium strains TA1535, TA1537 and TA98 at 52, 164, 512, 1,600 and 5,000 μg/plate. In the second mutation experiment, the substance was tested up to concentrations of 5,000 μg/plate in the S. typhimurium testerstrains TA1535, TA1537, TA98, TA100 and in Escherichia coli strain WP2uvrA in a pre-incubation assay. Toxicity was observed in WP2uvrA (absence of S9-mix only) and in TA1535, TA1537 and TA100 in the absence and presence of S9-mix. The negative and strain-specific positive control values were within the laboratory historical control data ranges, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. No mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay (Verspeek-Rip CM, 2015a).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 07, 2015 to October 21, 2015

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix, rat liver S9-mix induced by phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding study: 5.4, 17, 52, 164 and 512 μg test substance/mL culture medium with and without S9-mix.
First cytogenetic assay (3 h exposure time, 24 h fixation time): without S9-mix : 52, 164 and 512 μg/mL culture medium and with S9-mix : 52, 87, 164 and 512 μg/mL culture medium
Second Cytogenetic assay in the absence of S9-mix: 52, 164 and 512 μg/mL culture medium (24 and 48 h exposure time, 24 h and 48 h fixation time).

Vehicle / solvent:

- Vehicle used: The vehicle for the test substance was culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

a. without metabolic activation: Mitomycin C b: with metabolicactivation: Cyclophosphamide

Details on test system and experimental conditions:

Test system:
Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 y of age).

Cell culture:
Blood samples:

Blood samples were collected by venipuncture 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.
Culture medium:

Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life technologies), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.

Lymphocyte cultures:
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

Environmental conditions:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 - 90%), containing 5.0±0.5% CO2 in air in the dark at 37.0±1.0°C (actual range 35.5 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Dose range finding test:
In order to select the appropriate dose levels for the chromosome aberration test, cytotoxicity data were obtained in a dose range finding test. The test substance was tested in the absence and in the presence of 1.8% (v/v) S9-fraction. 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 48 h and thereafter exposed to selected doses of the test substance for 3, 24 and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.

The highest tested concentration was determined by the solubility of the test substance in the culture medium. After 3 h exposure to the test substance in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time). Cytotoxicity of the test substance in the lymphocyte cultures was determined using the mitotic index. 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.

Cytogenetic assay:
The test substance was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.

First cytogenetic assay:
Lymphocytes were cultured for 48±2 h and thereafter exposed in duplicate to selected doses of the test substance for 3 h in the absence and presence of S9-mix. After 3 h 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 resuspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay. Based on the solubility of the test substance in the culture medium appropriate dose levels were selected for the second cytogenetic assay. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary. The follow up experiment was performed with the following modifications of experimental conditions.

Second cytogenetic assay:

Lymphocytes were cultured for 48±2 h and thereafter exposed in duplicate to selected doses of test substance for 24 h and 48 h in the absence of S9-mix. The cells were not rinsed after exposure but were fixed immediately after 24 and 48 h (24 and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome preparation:

During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid (Merck) fixative (3:1 v/v).

Preparation of slides:
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the WIL Research Europe study identification number and group number. 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. The dry slides were automatically embedded in a 1:10 mixture of xylene and mounted with a coverslip in an automated cover slipper.

Mitotic index/dose selection for scoring of the cytogenetic assay:
The mitotic index of each culture was determined by counting the number of metaphases from at least 1,000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay. The test substance was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analysed was determined by the solubility in the culture medium.

Evaluation criteria:

Acceptability of the assay
A chromosome aberration 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 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). GraphPad PRISM version 4.03 will be used for statistical analysis of the data.

Data evaluation and statistical procedures
- A test substance is 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) Any of the results are outside the 95% control limits of the historical control data range.
- A test substance is 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) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Statistical analysis carried out as per Fisher’s exact test, one-sided, p < 0.05 using GraphPad PRISM version 4.03.
In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p <0.05) should performed to test whether there is a significant trend in the induction.

Key result

Species / strain:

hepatocytes: cultured peripheral human lymphocytes

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:

Dose range finding test
At a concentration of 512 μg/mL the test substance precipitated in the culture medium. Therefore, a concentration of 512 μg/mL was used as the highest concentration of the test substance.

First cytogenetic assay:
Both in the absence and presence of S9-mix, test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Second cytogenetic assay:
The test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. The test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.


COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, 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.

Conclusions:

The test substance was not clastogenic in human lymphocytes.

Executive summary:

Anin vitro mammalian chromosome aberration test with the test substance was conducted in cultured peripheral human lymphocytes according to OECD 473 and EU method B.10, in compliance with GLP. In a first cytogenetic assay, the substance was evaluated up to 512 μg/mL for a 3 h exposure with a 24 h fixation time with and without 1.8% (v/v) S9-fraction. The substance precipitated in the culture medium at this dose level. In a second cytogenetic assay, the substance was also evaluated up to 512 μg/mL for a 24 h continuous exposure with a 24 h fixation time and for a 48 h continuous exposure with a 48 h fixation time in the absence of S9-mix. 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. Positive control substances, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations with and without S9-mix, in either of the two independently performed experiments. There was no effect on the number of polyploid cells and cells with endoreduplicated chromosomes with and without S9-mix. Under the study conditions, the test substance was not clastogenic in human lymphocytes (Buskens CAF, 2015 ).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 30, 2015 to August 17, 2015

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Guideline:

other: “International Workshop on Genotoxicity Tests Workgroup” (the IWGT)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase (TK Locus)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: exposure medium (R5) (RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies, Bleiswijk, The Netherlands).
- Properly maintained: yes
- Periodically checked for mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix, rat liver S9-mix induced by combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding study: test substance concentration range of 17 to 1,600 μg/mL
First mutagenicity test:
Without S9-mix: 0.55, 1.7, 5.4, 17, 52, 164, 200, 300, 400, 500 and 600 μg/mL exposure medium.
With S9-mix: 1.7, 5.4, 17, 52, 164, 200, 300, 400, 500, 600 and 700 μg/mL exposure medium.
Second Mutagenicity test: 5.4, 17, 52, 89, 164, 200, 200, 250, 300, 350, 400 and 450 μg/mL exposure medium in absence S9-mix

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

Without metabolic activation (-S9-mix): Methyl methanesulfonate (MMS) and with metabolic activation (+S9-mix): Cyclophosphamide (CP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in exposure medium

DURATION
- Exposure duration: 3 h and 24 h
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 11-12 d

SELECTION AGENT (mutation assays): trifluorothymidine (TFT-selection).

DETERMINATION OF CYTOTOXICITY
cloning efficiency, relative total growth

Evaluation criteria:

Acceptability of the assay
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 h treatment) and 32-180 (24 h treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). Furthermore, the positive control should have 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 at least 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

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the exposure medium at concentrations of 512 μg/mL and above. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 1,600 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with the test substance concentration range of 17 to 1,600 μg/mL in the absence of S9-mix with 3 and 24 h treatment periods and in the presence of S9-mix with a 3 h treatment period.
In the absence of S9-mix, the relative suspension growth was 14% at the concentration of 512 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the concentration of 1,600 μg/mL. In the presence of S9-mix, the relative suspension growth was 36% at the concentration of 512 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the concentration of 1,600 μg/mL. In the absence of S9-mix, the relative suspension growth was 58% at the concentration of 164 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the concentration of 512 μg/mL and above after 24 h treatment.

Mutation experiment:
Initially a mutation experiment was performed with a 3 h treatment period in the absence and presence of S9-mix. However since due to a technical error, no mutation frequency measurement was performed, this experiment was rejected and no data was reported.

First mutagenicity test:
In the absence of S9-mix, the dose levels of 0.55 to 52 μg/mL showed no cytotoxicity. Therefore, the dose levels of 5.4 and 17 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 200 and 300 μg/mL showed similar cytotoxicity. Therefore, the dose level of 200 μg/mL was not regarded relevant for mutation frequency measurement. In the presence of S9-mix, the dose levels of 400 to 700 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the absence of S9-mix, the relative total growth of the highest test substance concentration was 8% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was 6% 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 test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test: Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 5.4, 17, 52, 89, 164, 200, 200, 250, 300, 350, 400 and 450 μg/mL exposure medium.

Evaluation of toxicity
The dose levels of 5.4 to 89 μg/mL showed no cytotoxicity. Therefore, the dose level of 5.4 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 400 to 450 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: 17, 52, 89, 164, 200, 250, 300 and 350 μg/mL exposure medium. The relative total growth of the highest tested dose was 18% 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 substance. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Conclusions:

The test substance was not mutagenic in the in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

Executive summary:

An in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells was conducted according to OECD Guideline 476 and EU Method B.10, in compliance with GLP. The objective of this test was to evaluate the mutagenic activity of the substance by evaluating 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 activation system (S9 -mix). Two independent experiments were performed. In the first, the substance was tested up to concentrations of 600 and 300 μg/mL in the absence and presence of S9-mix, respectively. In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency. This result was confirmed in a repeat experiment with modification in the duration of treatment. In the presence of S9-mix, the test substance also did not induce a significant increase in the mutation frequency. Mutation frequencies of the positive control substances were within the acceptability criteria of this assay. The spontaneous mutation frequencies in the solvent-treated control cultures were within the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Hence under the study conditions, the test substance was not mutagenic in the in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (Verspeek-Rip CM, 2015b).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Bacterial reverse mutation assay

An in vitro bacterial reverse mutation assay was performed to evaluate the potential of test substance to cause gene mutation according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. A total of two mutation experiments were conducted. Based on the results of the dose range finding test, a first mutation assay, a direct plate assay, was conducted with and without S9-mix in Salmonella typhimurium tester strains TA1535, TA1537 and TA98 at 52, 164, 512, 1,600 and 5,000 μg/plate. In the second mutation experiment, the substance was tested up to concentrations of 5,000 μg/plate in the S. typhimurium strains TA1535, TA1537, TA98, TA100 and in Escherichia coli strain WP2uvrA in a pre-incubation assay. Toxicity was observed in WP2uvrA (absence of S9-mix only) and in TA1535, TA1537 and TA100 in the absence and presence of S9-mix. The negative and strain-specific positive control values were within the laboratory historical control data ranges, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. No mutagenic effect of the test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay (Verspeek-Rip CM, 2015a).

In vitro chromosomal aberration assay

An in vitro mammalian chromosomal aberration assay with the test substance was conducted in cultured peripheral human lymphocytes according to OECD 473 and EU method B.10, in compliance with GLP. In a first cytogenetic assay, the substance was evaluated up to 512 μg/mL for a 3 h exposure with a 24 h fixation time with and without 1.8% (v/v) S9-fraction. The substance precipitated in the culture medium at this dose level. In a second cytogenetic assay, the substance was also evaluated up to 512 μg/mL for a 24 h continuous exposure with a 24 h fixation time and for a 48 h continuous exposure with a 48 h fixation time in the absence of S9-mix. 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. Positive control substances, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations with and without S9-mix, in either of the two independently performed experiments. There was no effect on the number of polyploid cells and cells with endoreduplicated chromosomes with and without S9-mix. Under the study conditions, the test substance was not clastogenic in human lymphocytes (Buskens CAF, 2015 ).

In vitro mammalian cell gene mutation assay

An in vitro mammalian cell gene mutation assay with L5178Y mouse lymphoma cells was conducted according to OECD Guideline 476 and EU Method B.10, in compliance with GLP. The objective of this test was to evaluate the mutagenic activity of the substance by evaluating 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 activation system (S9 -mix). Two independent experiments were performed. In the first, the substance was tested up to concentrations of 600 and 300 μg/mL in the absence and presence of S9-mix, respectively. In the absence of S9-mix, the test substance did not induce a significant increase in the mutation frequency. This result was confirmed in a repeat experiment with modification in the duration of treatment. In the presence of S9-mix, the test substance also did not induce a significant increase in the mutation frequency. Mutation frequencies of the positive control substances were within the acceptability criteria of this assay. The spontaneous mutation frequencies in the solvent-treated control cultures were within the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Hence under the study conditions, the test substance was not mutagenic in the in vitro mammalian cell gene mutation assay with L5178Y mouse lymphoma cells (Verspeek-Rip CM, 2015b).

Justification for classification or non-classification

Based on the results of in vitro genotoxicity studies, the substance does not warrant classification according to EU CLP (EC 1272/2008) criteria.