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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 05, 2018 to February 26, 2018
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- No correction for purity required
- The test material is a UVCB
Target gene:
- Salmonella typhimurium
- Tryptophan locus of Escherichia coli strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
The preliminary toxicity assay conducted at dose levels of 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate in water The maximum dose of 5000 µg per plate was achieved using a concentration of 5.00 mg/mL and a 1000 µL plating aliquot. The test article in water formed clear solutions from 0.00667 to 0.333 mg/mL and workable suspensions from 0.667 to 5.00 mg/mL. No toxicity was observed. Precipitate was observed beginning at 667 µg per plate in the absence of S9 activation and beginning at 1000 µg per plate in the presence of S9 activation. Dose responsive increases in revertant counts were observed with tester strain TA98 in the presence of S9 activation.

Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.
Vehicle / solvent:
Water was the vehicle of choice based on compatibility with the target cells. The test article formed workable suspensions in water at concentrations of approximately 10 to 25 mg/mL in the solubility test conducted.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.
Evaluation criteria:
The following criteria must be met for the mutagenicity assay to be considered valid:
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls. The mean revertants per plate must be within the following ranges (inclusive).

95% Control Limits (99% Upper Limit)
TA98 TA100 TA1535 TA1537 WP2 uvrA
-S9 6-26 (31) 66-114 (126) 3-23 (28) 1-13 (16) 9-41 (49)
+S9 9-37 (44) 68-128 (143) 3-23 (28) 3-15 (18) 12-44 (52)
With Study Director justification, values including the 99% control limit and above are acceptable.


To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0 fold increase in the number of revertants over the mean value of the respective vehicle control and exceed the corresponding acceptable vehicle control range cited above.
A minimum of three non toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).
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:
not 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:
not 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:
not 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:
not 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:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.
Remarks on result:
other:
Remarks:
Precipitate was observed beginning at 1500 µg per plate with all conditions

Historical Negative and Positive Control Values

2015

Revertants per plate

Strain

Control

Activation

None

Rat Liver

Mean

SD

Min

Max

95% CL

Mean

SD

Min

Max

95% CL

TA98
(2015)

Neg

16

5

6

43

6-26

23

7

5

53

9-37

Pos

190

191

42

2468

 

329

176

51

1786

 

TA100
(2015)

Neg

90

12

62

233

66-114

98

15

63

157

68-128

Pos

697

172

239

1767

 

671

284

138

2692

 

TA1535
(2015)

Neg

13

5

2

35

3-23

13

5

3

33

3-23

Pos

624

196

50

2509

 

137

110

24

1060

 

TA1537
(2015)

Neg

7

3

1

20

1-13

9

3

2

23

3-15

Pos

392

292

24

2887

 

73

53

19

574

 

WP2uvrA
(2015)

Neg

25

8

7

73

9-41

28

8

10

96

12-44

Pos

336

112

89

1026

 

352

117

78

1409

 

SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Conclusions:
Under the study conditions, results indicate the target substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.
Executive summary:

A study was conducted to determine the mutagenic potential pf the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system according to OECD Guideline 471. Water was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 667 µg per plate in the absence of S9 activation and beginning at 1000 µg per plate in the presence of S9 activation. Dose responsive increases in revertant counts were observed with tester strain TA98 in the presence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Under the study conditions, results indicate the target substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification.
Reason / purpose for cross-reference:
read-across source
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:
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium (Invitrogen Corporation), 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.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Dose range finding study:
- At 3 h exposure time: 3, 10, 33, 100 and 333 µg/mL culture medium with and without S9-mix.
- At 24 and 48 h continuous exposure time: 3, 10, 33, 100, 333 and 1000 µg/mL culture medium without S9-mix


Experiment 1 (First cytogenetic assay):
Without and with S9-mix: 33, 100 and 200 µg/mL culture medium (3 h exposure time, 24 h fixation time)

Experiment 2 (Second cytogenetic assay):
- Without S9-mix: 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (24 h exposure time, 24 h fixation time)
10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (48 h exposure time, 48 h fixation time)
- With S9-mix: 50, 100 and 200 µg/mL culture medium (3 h exposure time, 48 h fixation time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 10 µg/mL 3 h exposure period (24 h fixation time)
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.1 µg/mL (48 h exposure), 0.2 (24 h exposure) and 0.5 µg/mL (3 h exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3h (Experiment 2 with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 48 h (Experiment 2 with S9 mix)


SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µ g/mL medium)
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: Two


NUMBER OF CELLS EVALUATED: 1000 cells


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

Evaluation criteria:
Evaluation criteria
A test substance was considered clastogenic if:
a) A dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations
b) A significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations in the absence of a clear dose-response relationship

A test substance was considered non-clastogenic if:
a) None of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
Statistics
One sided, Chi-square test to calculate dose-related statistically significant increase in the number of cells with chromosome aberrations
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: At the 24 and 48 h exposure time, test material was tested beyond the limit of solubility to obtain adequate toxicity data. - Precipitate of the test material was seen at 333 µg /mL


COMPARISON WITH HISTORICAL CONTROL DATA: Yes, test data were within the laboratory historical control data range


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Increased number of polyploid cells in the absence of S9-mix in a dose dependent manner in the first cytogenetic assay indicating potential to inhibit mitotic processes and to induce numerical chromosome aberrations.

The doses selected for scoring of chromosome aberrations:

Without S9-mix: 50, 100 and 150 µg/mL culture medium (24 h exposure time, 24 h fixation time).

50, 100 and 125 µg/mL culture medium (48 h exposure time, 48 h fixation time)

With S9-mix:50, 100 and 200µg/mLculture medium (3 h exposure time, 48 h fixation time)

Conclusions:
Under the study conditions, test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro.
Executive summary:

A study was conducted to evaluate the ability of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473 and EU Method B. 10, in compliance with GLP. Peripheral human lymphocytes were treated with the test substance (experiment 1: 33, 100 and 200 µg/mL without and with S9-mix; experiment 2: 10 – 300 µg/mL without S9-mix, 50, 100 and 200 µg/mL with S9-mix) for either 3, 24 or 48 h. The frequency of cells with aberrations in the vehicle control group was within the historical control data range. Both of the positive control substances induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence of metabolic activation, in either independent repeat experiment. No effects on the number of polyploid cells were observed both in the absence and presence of S9-mix. The substance did not disturb the mitotic processes, cell cycle progression and did not induce numerical chromosome aberrations. Under the study conditions, test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip, 2009).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification.
Reason / purpose for cross-reference:
read-across source
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
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) 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: For 3 h exposure cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5-medium) and for 24 h exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat inactivated horse serum (R10-medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First mutagenicity test:
Without S9-mix: 0.3, 1, 3, 10, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL exposure medium
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 30, 50, 100, 150, 200, 250, 300 and 350 µg/mL exposure medium
Second mutagenicity test:
Without S9-mix: 0.3,1,3,10,15,20,22.5,25,27.5,30,32.5,35,40,45 and 50 µg/mL exposure medium
With 12% (v/v) S9-mix: 0.3, 1, 3, 10, 30, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL exposure medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation: 15 and 5 µg/mL for a 3 and 24 h treatment period
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3h (Experiment 2 with S9 mix)
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 d after the treatment period. During this culture period at least 4 x 106 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CE day 2) and the mutation frequency (MF).


SELECTION AGENT (mutation assays): Trifluorothymidine 0.5 mg/mL


NUMBER OF REPLICATIONS: Two


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more then MF(controls) + 126 in a dose-dependent manner; or
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 independently repeated experiment.
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:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
No data
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 33 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 µg/mL and above. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 333 µg/mL.


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 and within the acceptability criteria of this assay.


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


ADDITIONAL INFORMATION ON CYTOTOXICITY: First mutagenicity test: Evaluation of toxicity: In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 74% 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 reduced by 77% compared to the total growth of the solvent controls.
Second mutagenicity test: In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 95% 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 reduced by 72% compared to the total growth of the solvent controls.

The growth rate over the two-day expression period for cultures treated with DMSO was between 13 and 23 (3 h treatment) and 37 and 38 (24 h treatment). Mutation frequencies in cultures treated with positive control chemicals were increased by 8.2 and 16-fold for MMS in the absence of S9-mix, and by 10- and 13-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Conclusions:
Under the study conditions, the test substance was not mutagenic in the TK mutation test system both with and without metabolic activation.

Executive summary:

A study was conducted to evaluate the mutagenic potential of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), in L5178Y mouse lymphoma cells according to OECD Guideline 476 and EU method B.17, in compliance with GLP. The test was performed in two independent experiments with L5178Y mouse lymphoma cells, in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 60 and 200 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. Test material was tested up to cytotoxic levels of 74 and 77% in the absence and presence of S9-mix, respectively. In the second experiment, test material was tested up to concentrations of 45 and 225 µg/mL in the absence and presence of 12% (v/v) S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 72% (presence of S9-mix), but failed to induce a significant increase in the frequency of mutations. The spontaneous mutation frequencies in the solvent-treated control cultures were within historical control data range and therefore within the acceptability criteria of the assay. Mutation frequencies in positive control cultures were elevated 8.2- and 16-fold for MMS (absence of S9-mix), and 10- and 13-fold for CP (presence of S9-mix). Negative results were confirmed in an independent repeat experiment with extended exposures. Under the study conditions, the test substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip, 2009).

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 mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system according to OECD Guideline 471. Water was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 667 µg per plate in the absence of S9 activation and beginning at 1000 µg per plate in the presence of S9 activation. Dose responsive increases in revertant counts were observed with tester strain TA98 in the presence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Under the study conditions, results indicate the target substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system (Pant, 2018).

Chromosome aberration assay

A study was conducted to evaluate the ability of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473 and EU Method B. 10, in compliance with GLP. Peripheral human lymphocytes were treated with the test substance (experiment 1: 33, 100 and 200 µg/mL without and with S9-mix; experiment 2: 10 – 300 µg/mL without S9-mix, 50, 100 and 200 µg/mL with S9-mix) for either 3, 24 or 48 h. The frequency of cells with aberrations in the vehicle control group was within the historical control data range. Both of the positive control substances induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence of metabolic activation, in either independent repeat experiment. No effects on the number of polyploid cells were observed both in the absence and presence of S9-mix. The substance did not disturb the mitotic processes, cell cycle progression and did not induce numerical chromosome aberrations. Under the study conditions, test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip, 2009).

Mouse lymphoma assay

A study was conducted to evaluate the mutagenic potential of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), in L5178Y mouse lymphoma cells according to OECD Guideline 476 and EU method B.17, in compliance with GLP. The test was performed in two independent experiments with L5178Y mouse lymphoma cells, in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 60 and 200 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. Test material was tested up to cytotoxic levels of 74 and 77% in the absence and presence of S9-mix, respectively. In the second experiment, test material was tested up to concentrations of 45 and 225 µg/mL in the absence and presence of 12% (v/v) S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 72% (presence of S9-mix), but failed to induce a significant increase in the frequency of mutations. The spontaneous mutation frequencies in the solvent-treated control cultures were within historical control data range and therefore within the acceptability criteria of the assay. Mutation frequencies in positive control cultures were elevated 8.2- and 16-fold for MMS (absence of S9-mix), and 10- and 13-fold for CP (presence of S9-mix). Negative results were confirmed in an independent repeat experiment with extended exposures. Under the study conditions, the test substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip, 2009).

Justification for classification or non-classification

Based on the results of in vitro testing on the substance itself or the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), no classification for genetic toxicity is proposed according to CLP (EC 1272/2008) criteria.