Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the absence of genotoxicity in available in vitro read across studies, the test substance, C10-12 and C18-unsatd. DEA, is not considered to be mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
February 6, 2014 to March 7, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 as RA study
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Range finding test in strain TA100 and E.Coli WP2 uvrA with and without metabolic activation: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330; 5000 ug/L

Experiment 1: Tester trains TA1535, TA1537, TA98 with and without metabolic activation: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 ug/L.

Experiment 2: Tester trains TA1535, TA1537, TA98, TA100: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 ug/L. Tester strain E.Coli WP2 uvrA: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330 and 5000 ug/L
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Saline
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA1535; concentration/plate 5µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: ICR-191
Remarks:
TA1537; concentration/plate 2.5µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
TA98; concentration/plate 10µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
TA100; concentration/plate 650µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2 uvrA; concentration/plate 15µg
Positive controls:
yes
Remarks:
with metabolic activation and solvent DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1535 2.5µg S9-mix 5 and 10%; TA1537 2.5µg S9-mix 5%; TA1537 5µg S9-mix 10%; TA98 1µg S9-mix 5 and10%; TA100 1µg S9-mix 5% ; TA100 2µg S9-mix 10% ; W2P uvrA 15µg S9-mix 5 and 10%
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
NUMBER OF REPLICATIONS: Triplicates
DETERMINATION OF CYTOTOXICITY: Reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
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 negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain

b)The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean c)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 extendto 5 mg/plate.

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:

a)The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is not greater than three (3) times the concurrent vehicle control.

b)The negative response should be reproducible in at least one independently repeated experiment.


A test substance is considered positive (mutagenic) in the test if:

a)The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is greater than three (3) times the concurrent vehicle 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 independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.


Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no mutagenic potential

Dose range finding/Experiment 1

The test substance was tested in the tester strains TA100 and WP2 uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. 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: 3, 10, 33, 100, 333 and 1000 μg/plate.The results are shown in Table 3 and Table 4 of the study report.  

Precipitate

Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

Toxicity

To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. The reduction of the bacterial background lawn and the reduction in the number of revertants is presented in Table1.

Table 1: Toxicity of the test substance in the dose range finding test/first mutation experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

 

Strain

Without S9-mix

With S9-mix

                Dose        Bacterial                Revertant

               (μg/plate)  background lawn   colonies

Dose       Bacterial                Revertant

(µg/plate) background lawn  colonies

TA1535

 333          extreme          microcolonies

1000          extreme         microcolonies

 1000        extreme         microcolonies

TA1537

 100          normal               moderate

 333          extreme         microcolonies

1000          extreme        microcolonies

1000          extreme         microcolonies

TA98

 333          moderate          extreme

1000          extreme        microcolonies

1000          extreme         microcolonies

TA100

100-333     moderate          extreme

1000-5000  extreme         microcolonies

 333          moderate          extreme

1000-5000  extreme         microcolonies

WP2 uvrA

5000          normal               -1

5000          normal             slight     

-1  No reduction in the number of revertant colonies

Experiment 2

To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the following dose range was selected for the second mutation assay:

TA1535, TA1537, TA98, TA100: Without and with S9-mix: 3, 10, 33, 100, 333 and 1000 μg/plate

WP2 uvrA: Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

The results are shown in Table 5 of the study report.

Precipitate

Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period.

Toxicity

The reduction of the bacterial background lawn and the reduction in the number of revertants is presented inTable 2.

Table 2: Toxicity of the test substance in the second mutation experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

 

Strain

Without S9-mix

With S9-mix

             Dose         Bacterial              Revertant

             (μg/plate)  background lawn  colonies

Dose         Bacterial                   Revertant

(µg/plate)  background lawn    colonies

TA1535

 333      extreme           microcolonies

1000     extreme           microcolonies

1000          extreme              microcolonies

TA1537

 333      absent             complete

1000     absent             complete

 333          slight                 -1

1000          extreme              microcolonies

TA98

 333      slight                 extreme

1000     extreme           microcolonies

1000          moderate            extreme

TA100

 100      normal             moderate

 333      extreme           microcolonies

1000     extreme           microcolonies

1000          extreme              microcolonies

WP2 uvrA

5000     normal                     -2

5000           normal                  -2

-1  No reduction in the number of revertant colonies

-2  Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

Conclusions:
Based on the results of the read across study, the test substance, C10-12 and C18-unsatd. DEA can also be considered to be non-mutagenic in Ames test, with and without metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, C8-18 and C18-unsatd. DEA, in the Salmonella typhimurium reverse mutation assay (strains TA 1535, TA 1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay strain WP2 uvrA according to OECD Guideline 471, in compliance with GLP. In the dose range finding study, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and upwards in the absence of metabolic activation (S9-mix) and at dose levels of 333 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, toxicity was only observed at the dose level of 5000 μg/plate in the presence of S9-mix. Based on the results of the dose range finding study, the substance was tested in the first mutation assay at a concentration range of 3 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains. In the second mutation assay, the substance was tested up to concentrations of 1000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA100, TA1535, TA1537 and TA98 and up to concentrations of 5000 µg/plate in tester strain WP2 uvrA. Toxicity was observed in all tester strains, except in tester strain WP2 uvrA. 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 tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, 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. Under the conditions of the study, the read across substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Verspeek-Rip, 2014). Based on the results of the read across study, the test substance, C10-12 and C18-unsatd. DEA can also be considered to be non-mutagenic in Ames test, with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
reference to other study
Reason / purpose:
read-across source
Principles of method if other than guideline:
Suspensions of bacterial cells were exposed to the test substance by the plate incorporation method in the presence and in the absence of an exogenous metabolic activation system. The suspensions were mixed with an overlay agar and plated immediately onto the minimal medium and incubated for 2 d at 37˚C. The results were interpreted by counting the revertant colonies and comparing to the number of spontaneous revertant colonies on solvent-control plates.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA97, TA98, TA100 and TA1535
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver).
Test concentrations with justification for top dose:
- without metabolic activation:0.0, 0.1, 0.3, 1.0, 3.3, 6.7 µg/plate
- with metabolic activation: 0.0, 3.3, 10.0, 33.0, 100.0, 200 µg/plate
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
absence of metabolic activation (for strains TA100 and TA1535)
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
absence of metabolic activation (for strain TA97)
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-nitro-o- phenylenediamine (for strain TA98)
Remarks:
absence of metabolic activation (For TA98)
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
metabolic activation
Details on test system and experimental conditions:
- Test medium: Top agar supplemented with L-histidine and d-biotin
- Method of application: In agar (plate incorporation)
- Duration of incubation: 2 d at 37 °C
- Number of replicates: Three
Evaluation criteria:
- Positive response: Reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination.
- Equivocal response: An increase in revertants that are not dose related, is not reproducible, or is not of sufficient magnitude to support a determination of m utagenicity.
- Negative response: When no increase in revertant colonies is observed following chemical treatment.
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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:
not specified
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:
Slighlty toxic at ≥ 200 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
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:
not specified
Positive controls validity:
valid
Conclusions:
Under the study conditions, the test substance was not mutagenic.
Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, C8-18 and C18-unsatd. DEA, according to an Ames protocol, in compliance with GLP. Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with the test substance using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of S9 mix (Aroclor-induced rat and hamster liver homogenate-metabolising system). The dose range was 0.1 to 6.7 µg/plate (without S9 -mix) and 0.33 to 200 µg/plate (with S9-mix). Cytotoxicity was observed at ≥ 200 µg/plate with metabolic activation on strain TA100. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. The vehicle control (ethanol) or the negative control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without S9-mix. Under the study conditions, the test substance was not mutagenic (NTP, 2001). Based on the result of the read across substance, C8-18 and C18-unsatd. DEA, the test substance, C10-12 and C18-unsatd. DEA can be considered as not mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
reference to other study
Reason / purpose:
read-across source
Principles of method if other than guideline:
The genotoxic potential of the test substance was determined by the induction of chromosomal aberrations in Chinese Hamster Ovary Cells both with and without metabolic activation.

GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix
Test concentrations with justification for top dose:
16, 30 and 50 µg/mL (without S9);
16, 30 and 50 µg/mL (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: None
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9, at 0.0625 and 0.2500 µg/mL)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9, at 2.5 and 7.5 µg/mL)
Details on test system and experimental conditions:
Detailed protocol of this study has been presented by (Galloway et al (1987)).

DURATION
- Exposure duration: In the test without S9, cells were incubated in McCoy’s 5A medium with lauric acid diethanolamine condensate for 8 h; Colcemid was added and incubation continued for 2 h. For the test with S9, cells were treated with lauric acid diethanolamine condensate and S9 for 2 h, after which the treatment medium was removed and the cells were incubated for 10 h in fresh medium, with Colcemid present for the final 2 h. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa.
- Harvest time: 10 h (without S9); 12 h (with S9)
NUMBER OF REPLICATIONS: A single flask per dose was used, and tests yielding equivocal or positive results were repeated.


NUMBER OF CELLS EVALUATED: Two hundred first-division metaphase cells were scored at each dose level.


OTHER: Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverised cells, despiralised chromosomes, and cells containing 10 or more aberrations).
Evaluation criteria:
No data
Statistics:
Statistical analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P ≤0.05) difference for one dose point and a significant trend (P ≤0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicates the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Additional information on results:
None

Table: Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by test substance a

Compound Concentration (μg/mL) Total Cells Scored Number of Aberrations Aberrations/Cell Cells with Aberrations (%)
`-S9          
Harvest time: 12 hours          
Summary: Negative          
Solvent control   200 2 0.01 1
Mitomycin-Cb 0.0625 200 35 0.18 16.5
  0.25 50 26 0.52 32
Test substance 16 200 3 0.02 1.5
  30 200 3 0.02 1.5
  30 200 5 0.02 2.5
          P=0.134c
`+S9          
Harvest time: 13 hours          
Summary: Negative          
Solvent control   200 2 0.01 1
Cyclophosphamideb 2.5 200 39 0.2 17.5
  7.5 50 24 0.48 38
Test substance 16 200 5 0.03 2.5
  30 200 4 0.02 2
  30 200 4 0.02 2
          P=0.280

a Study was performed at Environmental Health Research and Testing. The detailed protocol is presented by Galloway et al. (1987).

b Positive control

c Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose

Conclusions:
Based on the result of the read across substance, C8-18 and C18-unsatd. DEA, the test substance, C10-12 and C18-unsatd. DEA can be considered to be non-clastogenic in chromosomal aberration assay, with and without metabolic activation.
Executive summary:

An in vitro study was conducted to determine the genotoxic potential of the read across substance, C8-18 and C18-unsatd. DEA, by induction of chromosomal aberrations in Chinese Hamster Ovary (CHO) cells, both in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. The CHO cells were treated at 16, 30 and 50 µg/mL without S9 and 16, 30 and 50 µg/mL with S9 test test concentrations respectively. A single flask per dose was used, and tests yielding equivocal or positive results were repeated. Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). Two hundred first-division metaphase cells were scored at each concentration level and chromosomal aberration data were presented as percentage of cells with aberrations. The test substance did not induce an increase in the number of chromosomal aberrations. Under the study conditions, the test substance was found to be non-clastogenic with and without metabolic activation (NTP, 2001). Based on the result of the read across substance, C8-18 and C18-unsatd. DEA, the test substance, C10-12 and C18-unsatd. DEA can be considered to be non-clastogenic in chromosomal aberration assay, with and without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Cells deficient in thymidine kinase (TK) due to the mutation of TK+/- to TK-/- are resistant to the cytotoxic effects of trifluorothymidine (TFT). Thymidine kinase proficient cells (TK+/-) are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not able to proliferate.

GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Supplemented Fischer’s medium
- Properly maintained: Yes
- Periodically "cleansed" against high spontaneous background: Yes, by exposing to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 d; to medium containing thymidine, hypoxanthine, and glycine for 1 d; and to normal medium for 3 to 5 d
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from the livers of Aroclor 1254-induced male Fischer 344 rats
Test concentrations with justification for top dose:
Trial 1:
Without metabolic activation: 0, 1.25, 2.5, 5, 6, 10 and 12 nL/mL
With metabolic activation: 0, 1.25, 2.5, 5, 10, 15 nL/mL
Trial 2:
Without metabloic actvation: 0, 4, 5, 6, 8, 10 and 12 nL/mL
With metabolic activation: 0,4 5, 6, 8, 10 and 12 nL/mL
Trial 3:
Without metabolic activation: 0, 1.5, 3, 6, 8, 10, 12 and 15 nL/mL
With metabolic activation: 0, 6, 8, 10, 12, 15 and 20 nL/mL
Trial 4:
With metabolic activation: 0, 5, 10, 15, 20, 30, 40 and 50 nL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
at 5 μg/mL in trials 1, 2 and 3 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Ethanol)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methyl cholanthrene
Remarks:
2.5 μg/mL in trial 1, 2, 3 and 4 with metabolic avtivation
Details on test system and experimental conditions:
The experimental protocol is presented in detail by Myhr et al, 1985

METHOD OF APPLICATION: In medium


DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10 to 12 d (at 37 °C in 5 % CO2)


SELECTION AGENT (mutation assays): Yes, Trifluorothymidine (TFT)



NUMBER OF REPLICATIONS: Duplicate (all treatment levels within an experiment, including concurrent positive and solvent controls, were replicated)


NUMBER OF CELLS EVALUATED: 6 × 10(6) cells in 10 mL medium


DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency
Evaluation criteria:
Minimum criteria for accepting an experiment as valid and a detailed description of the statistical analysis and data evaluation are presented by Caspary et al. (1988). All data were evaluated statistically for trend and peak responses. Both responses had to be significant (P#0.05) for test substance to be considered positive, i.e., capable of inducing TFT resistance. A single significant response led to a “questionable” conclusion, and the absence of both a trend and peak response resulted in a “negative” call.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
No increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test material, with or without S9.

Conclusions:
Based on the result of the read across substance, C8-18 and C18-unsatd. DEA, the test substance, C10-12 and C18-unsatd. DEA can be considered to be non-mutagenic mouse lymphoma assay, with and without metabolic activation.
Executive summary:

An in vitro study was conducted to assess the mutagenicity potential of the read across substance, C8-18 and C18-unsatd. DEA, in the mouse lymphoma cell line L5178Y with and without S9 metabolic activation. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated 4 times. In Trial 1, the cells were exposed for 4 h to concentrations of 0, 1.25, 2.5, 5, 6, 10 and 12 nL/mL without metabolic activation and 0, 1.25, 2.5, 5, 10, 15 nL/mL with metabolic activation. In Trial 2, the cells were treated with test substance for 4 h at 0, 4, 5, 6, 8, 10 and 12 nL/mL without metabolic activation and 0,4 5, 6, 8, 10 and 12 nL/mL with metabolic activation. In Trial 3, the cells were exposed for 4 h 0, 1.5, 3, 6, 8, 10, 12 and 15 nL/mL without metabolic activation and 0, 6, 8, 10, 12, 15 and 20 nL/mL with metabolic activation. In Trial 4, the cells were exposed for 4 h 0, 5, 10, 15, 20, 30, 40 and 50 nL/mL with metabolic activation. After the 48 h expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells and cells were plated in nonselective medium and soft agar to determine cloning efficiency. A single positive response noted at 8 nL/mL in the trial 2 conducted without metabolic activation was not reproducible, and the test results overall were considered to be negative. Under the test conditions, no increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test substance, with or without S9. Under the study conditions, the read across substance was found to be non-mutagenic to mouse lymphoma cell line L5178Y (NTP, 2001). Based on the result of the read across substance, C8-18 and C18-unsatd. DEA, the test substance, C10-12 and C18-unsatd. DEA can be considered to be non-mutagenic mouse lymphoma assay, with and without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Study 1: An in vitro study was conducted to determine the mutagenic potential of the read across substance, C8-18 and C18-unsatd. DEA, in the Salmonella typhimurium reverse mutation assay (strains TA 1535, TA 1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay strain WP2 uvrA according to OECD Guideline 471, in compliance with GLP. In the dose range finding study, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and upwards in the absence of metabolic activation (S9-mix) and at dose levels of 333 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, toxicity was only observed at the dose level of 5000 μg/plate in the presence of S9-mix. Based on the results of the dose range finding study, the substance was tested in the first mutation assay at a concentration range of 3 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains. In the second mutation assay, the substance was tested up to concentrations of 1000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA100, TA1535, TA1537 and TA98 and up to concentrations of 5000 µg/plate in tester strain WP2 uvrA. Toxicity was observed in all tester strains, except in tester strain WP2 uvrA. 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 tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, 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. Under the conditions of the study, the read across substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Verspeek-Rip, 2014). Based on the results of the read across study, the test substance, C10-12 and C18-unsatd. DEA can also be considered to be non-mutagenic in Ames test, with and without metabolic activation.

Study 2: An in vitro study was conducted to determine the mutagenic potential of the read across substance, C8-18 and C18-unsatd. DEA, according to an Ames protocol, in compliance with GLP. Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with the test substance using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of S9 mix (Aroclor-induced rat and hamster liver homogenate-metabolising system). The doses ranged from 0.1 to 6.7 µg/plate (without S9 -mix) and 0.33 to 200 µg/plate (with S9-mix). Cytotoxicity was observed at ≥ 200 µg/plate with metabolic activation on strain TA100. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. The vehicle control (ethanol) or the negative control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without S9-mix. Under the study conditions, the test substance was not mutagenic (NTP, 2001). Based on the results of the read across study, the test substance, C10-12 and C18-unsatd. DEA can also be considered to be non-mutagenic in Ames test, with and without metabolic activation.

Study 3: An in vitro study was conducted to determine the genotoxic potential of the read across substance, C8-18 and C18-unsatd. DEA, by induction of chromosomal aberrations in Chinese Hamster Ovary (CHO) cells, both in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. The CHO cells were treated at 16, 30 and 50 µg/mL without S9 and 16, 30 and 50 µg/mL with S9 test test concentrations respectively. A single flask per dose was used, and tests yielding equivocal or positive results were repeated. Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). Two hundred first-division metaphase cells were scored at each concentration level and chromosomal aberration data were presented as percentage of cells with aberrations. The test substance did not induce an increase in the number of chromosomal aberrations. Under the study conditions, the test substance was found to be non-clastogenic with and without metabolic activation (NTP, 2001). Based on the result of the read across substance, C8-18 and C18-unsatd. DEA, the test substance, C10-12 and C18-unsatd. DEA can be considered to be non-clastogenic in chromosomal aberration assay, with and without metabolic activation.

 

Study 4: An in vitro study was conducted to assess the mutagenicity potential of the read across substance, C8-18 and C18-unsatd. DEA, in the mouse lymphoma cell line L5178Y with and without S9 metabolic activation. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated 4 times. In Trial 1, the cells were exposed for 4 h to concentrations of 0, 1.25, 2.5, 5, 6, 10 and 12 nL/mL without metabolic activation and 0, 1.25, 2.5, 5, 10, 15 nL/mL with metabolic activation. In Trial 2, the cells were treated with test substance for 4 h at 0, 4, 5, 6, 8, 10 and 12 nL/mL without metabolic activation and 0,4 5, 6, 8, 10 and 12 nL/mL with metabolic activation. In Trial 3, the cells were exposed for 4 h 0, 1.5, 3, 6, 8, 10, 12 and 15 nL/mL without metabolic activation and 0, 6, 8, 10, 12, 15 and 20 nL/mL with metabolic activation. In Trial 4, the cells were exposed for 4 h 0, 5, 10, 15, 20, 30, 40 and 50 nL/mL with metabolic activation. After the 48 h expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells and cells were plated in nonselective medium and soft agar to determine cloning efficiency. A single positive response noted at 8 nL/mL in the trial 2 conducted without metabolic activation was not reproducible, and the test results overall were considered to be negative. Under the test conditions, no increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test substance, with or without S9. Under the study conditions, the read across substance was found to be non-mutagenic to mouse lymphoma cell line L5178Y (NTP, 2001).Based on the result of the read across substance, C8-18 and C18-unsatd. DEA, the test substance, C10-12 and C18-unsatd. DEA can be considered to be non-mutagenic mouse lymphoma assay, with and without metabolic activation.

Justification for classification or non-classification

Based on the absence of genotoxicity in the in vitro read across studies, the test substance, C10-12 and C18-unsatd. DEA, is concluded not to warrant classification for genotoxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).