Amides, C18-unsatd., N,N-bis(hydroxyethyl)

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 mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

The genotoxic potential of the test substance was determined by the induction of gene mutations in L5178Y cells in a mouse lymphoma mutagenicity test both with and without metabolic activation as per the Myhr et al., (1985) protocol. 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:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

No data

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Supplemented Fischer’s medium (growth media) maintained at 37°C; normal cycling time was approximately 10 hours. For cloning the horse serum content was increased and noble agar was added.
- Properly maintained: Yes
- Periodically "cleansed" against high spontaneous background: Yes, by exposing to selective media containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days

Additional strain / cell type characteristics:

not applicable

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:

Without metabolic activation: Trial 1: 0, 1.25, 2.5, 5 and 7.5 nL/ mL; Trial 2: 0, 2, 3, 4, 6, 8 and 12 nL/ mL; Trial 3: 0, 3, 4, 6, 8, 12, 15 and 20 nL/ mL
With metabolic activation: Trial 1: 0, 2.5, 5, 7.5, 10 and 15 nL/ mL; Trial 2: 0, 2.5, 5, 7.5, 10, 15 and 20 nL/ mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Untreated negative controls:

yes

Remarks:

(solvent control)

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

-S9 : at 5 μg/mL concentration

Untreated negative controls:

yes

Remarks:

(solvent control)

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

+S9: at 2.5 μg/mL concentration

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: Triplicate (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 as per Caspary et al., (1988). Data was evaluated statistically for trend and peak responses.

Positive response: When there are significant differences for two responses i.e., capable of inducing TFT resistance.
Equivocal response: When a single significant response is observed.
Negative response: When there is absence of both a trend and a peak response.

Statistics:

All data was evaluated statistically for trend and peak responses as per Caspary et al., (1988).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation: The highest doses of 7.5 nL/mL in trial 1 and ≥ 12 nL/mL in trial 3; With metabolic activation: 15 and 20 nL/mL in trial 1 and 2 respectively

Additional information on results:

No induction of TFT resistance was noted in L5178Y mouse lymphoma cells treated with ODEA in the presence or absence of S9 metabolic activation.

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Conclusions:

Under the study conditions, no increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test substance, with or without metabolic activation.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C18-unsatd. DEA, in a mouse lymphoma assay. The cells were exposed for 4 h to concentrations of 1.25, 2.5, 5 and 7.5 nL/ mL without metabolic activation and 2.5, 5, 7.5, 10 and 15 nL/ mL with metabolic activation in Trial 1. Cells were treated with test substance for 4 h at 52, 3, 4, 6, 8 and 12 nL/ mL without metabolic activation and 2.5, 5, 7.5, 10, 15 and 20 nL/ mL with metabolic activation in Trial 2. Cells were treated with test substance for 4 h at 3, 4, 6, 8, 12, 15 and 20 nL/ mL without metabolic activation in Trial 3. 6 X106 cells in triplicate cultures medium were exposed to the test substance (either in the presence or absence of metabolic activation), positive control and solvent control for 4 h. After a 48 h expression period, cells were plated for selection of TFT-resistant cells and cloning efficiency. The plates were scored after an incubation period of 10 to 12 d at 37±1°C in 5% CO2. Under the conditions of the study, no increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test substance, with or without S9 (NTP, 1999).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key 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:

Only four bacterial strains used

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Suspensions of bacterial cells are exposed to the test substance by the plate incorporation method in the presence and in the absence of an exogenous metabolic activation system. In this method, the suspensions are mixed with an overlay agar and plated immediately onto the minimal medium and incubated for two days at 37˚C, The results are 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

Target gene:

No data

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, 0.1, 0.3, 1, 3.3 and 10 µg/plate
With metabolic activation: 0, 3.3, 10, 33, 100 and 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

Remarks:

(for strains TA100 and TA1535)

Positive control substance:

sodium azide

Remarks:

absence of metabolic activation

Untreated negative controls:

yes

Remarks:

(solvent control)

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol)

True negative controls:

not specified

Positive controls:

yes

Remarks:

(for strain TA 98)

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

absence of metabolic activation

Untreated negative controls:

yes

Remarks:

(solvent control)

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol)

True negative controls:

not specified

Positive controls:

yes

Remarks:

(for strain TA 97)

Positive control substance:

9-aminoacridine

Remarks:

absence of metabolic activation

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 mutagenicity.
- Negative response: When no increase in revertant colonies is observed following chemical treatment.
- There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.

Statistics:

Not reported

Key result

Species / strain:

other: S. typhimurium TA97, TA98, TA100 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at ≥3.3 µg/plate without metabolic activation; at 200 µg/plate with metabolic activation) .

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

None

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Conclusions:

Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without S9 metabolic activation.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C18-unsatd. DEA, in a bacterial mutation assay, 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 five concentration levels for each bacterial strain, in triplicate, both with and without metabolic activation (S9 mix; Aroclor induced rat and hamster liver homogenate metabolising system). The concentration range was 0.1 to 10 µg/plate (-S9 -mix) and 3.3 to 200 µg/plate (+S9 -mix). Cytotoxicity was observed at ≥3.3 µg/plate without metabolic activation and at 200 µg/plate with metabolic activation. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any concentration tested, either with or without metabolic activation. The vehicle (ethanol) control 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 the S9-mix. Under the conditions of the study, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without S9 metabolic activation (NTP, 1999).

Reference 3

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 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 the section 13 for details on the category justification.

Reason / purpose for cross-reference:

reference to other study

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, 50 µg/mL (-/+ S9)

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9: 0.0625 and 0.25 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:

+S9: 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 coconut oil acid diethanolamine condensate for 10 h; Colcemid was added and incubation continued for 2 h. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the test with S9, cells were treated with coconut oil acid diethanolamine condensate and S9 for 2 h, after which the treatment medium was removed and the cells were incubated for 11 h in fresh medium, with Colcemid present for the final 2 h. Cells were harvested in the same manner as for the treatment without S9.
- Harvest time: 12 h (without S9); 13 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.

Evaluation criteria:

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).

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. Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.

Statistics:

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, in a chromosomal aberrations assay using Chinese Hamster Ovary (CHO) cells. The concentrations tested were 16, 30 and 50 µg/mL with and without metabolic activation. 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). All slides were scored blind and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Chromosomal aberration data were presented as percentage of cells with aberrations. Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose. The test substance did not induce an increase in the number of chromosomal aberrations in cultured Chinese hamster ovary cells after incubation, with or without S9. Under the study conditions, the test substance was found to be non-genotoxic (NTP, 2001).

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

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, 50 µg/mL (-/+ S9)

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9: 0.0625 and 0.25 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:

+S9: 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 coconut oil acid diethanolamine condensate for 10 h; Colcemid was added and incubation continued for 2 h. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the test with S9, cells were treated with coconut oil acid diethanolamine condensate and S9 for 2 h, after which the treatment medium was removed and the cells were incubated for 11 h in fresh medium, with Colcemid present for the final 2 h. Cells were harvested in the same manner as for the treatment without S9.
- Harvest time: 12 h (without S9); 13 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.

Evaluation criteria:

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).

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. Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.

Statistics:

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C8-18 and C18-unsatd. DEA, in a chromosomal aberrations assay using Chinese Hamster Ovary (CHO) cells. The concentrations tested were 16, 30 and 50 µg/mL with and without metabolic activation. 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). All slides were scored blind and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Chromosomal aberration data were presented as percentage of cells with aberrations. Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose. The test substance did not induce an increase in the number of chromosomal aberrations in cultured Chinese hamster ovary cells after incubation, with or without S9. Under the study conditions, the test substance was found to be non-genotoxic (NTP, 2001).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A study was conducted to evaluate the potential of the test material to induce micronuclei in B6C3F1 mice. After completion of a 13-wk dermal treatment period, the study animals were sacrificed and the peripheral blood samples were collected and blood smears were processed as per the standard NTP protocol. Thereafter, the slides from different test groups and control were evaluated for the induction of micronucleus.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

dermal

Vehicle:

No data

Details on exposure:

No data

Duration of treatment / exposure:

13 wk

Frequency of treatment:

Assumed to be daily

Post exposure period:

Not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose were evaluated for micronuclei induction

Control animals:

yes

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

Tissue: Peripheral blood
Cell: Normochromatic erythrocytes

Evaluation criteria:

No data

Statistics:

No data

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Conclusions:

Under the study conditions, the test substance did not increase the frequencies of micronucleated normochromatic erythrocytes (NCEs) in peripheral blood of both male and female mice at the end of 13 weeks.

Executive summary:

A study was conducted to evaluate the in vivo genetic toxicity of the test substance, C18-unsatd. DEA, in a micronucleus assay. The substance was applied dermally in mice for 13 weeks at 0, 50, 100, 200, 400 and 800 mg/kg bw. Peripheral blood samples were obtained from male and female animals, and smears were immediately prepared and fixed in absolute methanol. Under the study conditions, the test substance did not increase the frequencies of micronucleated normochromatic erythrocytes (NCEs) in peripheral blood of both male and female mice at the end of 13 weeks (NTP, 2001).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Ames test

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C18-unsatd. DEA, in a bacterial mutation assay, 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 five concentration levels for each bacterial strain, in triplicate, both with and without metabolic activation (S9 mix; Aroclor induced rat and hamster liver homogenate metabolising system). The concentration range was 0.1 to 10 µg/plate (-S9 -mix) and 3.3 to 200 µg/plate (+S9 -mix). Cytotoxicity was observed at ≥3.3 µg/plate without metabolic activation and at 200 µg/plate with metabolic activation. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any concentration tested, either with or without metabolic activation. The vehicle (ethanol) control 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 the S9-mix. Under the conditions of the study, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without S9 metabolic activation (NTP, 1999).

Chromosomal aberration assay

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, in a chromosomal aberrations assay using Chinese Hamster Ovary (CHO) cells. The concentrations tested were 16, 30 and 50 µg/mL with and without metabolic activation. 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). All slides were scored blind and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Chromosomal aberration data were presented as percentage of cells with aberrations. Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose. The test substance did not induce an increase in the number of chromosomal aberrations in cultured Chinese hamster ovary cells after incubation, with or without S9. Under the study conditions, the test substance was found to be non-genotoxic (NTP, 2001).

Also, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), in vitro genotoxicity testing according to OECD Guidelines 473 is planned with C16-18 MEA in order to further support the read across approach proposed for the FAA category members.

Mouse lymphoma assay

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C18-unsatd. DEA, in a mouse lymphoma assay. The cells were exposed for 4 h to concentrations of 1.25, 2.5, 5 and 7.5 nL/ mL without metabolic activation and 2.5, 5, 7.5, 10 and 15 nL/ mL with metabolic activation in Trial 1. Cells were treated with test substance for 4 h at 52, 3, 4, 6, 8 and 12 nL/ mL without metabolic activation and 2.5, 5, 7.5, 10, 15 and 20 nL/ mL with metabolic activation in Trial 2. Cells were treated with test substance for 4 h at 3, 4, 6, 8, 12, 15 and 20 nL/ mL without metabolic activation in Trial 3. 6 X106 cells in triplicate cultures medium were exposed to the test substance (either in the presence or absence of metabolic activation), positive control and solvent control for 4 h. After a 48 h expression period, cells were plated for selection of TFT-resistant cells and cloning efficiency. The plates were scored after an incubation period of 10 to 12 d at 37±1°C in 5% CO2. Under the conditions of the study, no increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test substance, with or without S9 (NTP, 1999).

In vivo

Mouse micronucleus assay

A study was conducted to evaluate the in vivo genetic toxicity of the test substance, C18-unsatd. DEA, in a micronucleus assay. The substance was applied dermally in mice for 13 weeks at 0, 50, 100, 200, 400 and 800 mg/kg bw. Peripheral blood samples were obtained from male and female animals, and smears were immediately prepared and fixed in absolute methanol. Under the study conditions, the test substance did not increase the frequencies of micronucleated normochromatic erythrocytes (NCEs) in peripheral blood of both male and female mice at the end of 13 weeks (NTP, 2001).

Justification for classification or non-classification

The absence of mutagenic and clastogenic effects in short-termin vitroandin vivogenetic toxicity tests on C18-unsatd. DEA and the read across substance C8-18 and C18-unsatd. DEA suggests that the substance does not have genotoxic potential. Therefore, no classification for this endpoint is warranted according to CLP (EC 1272/2008) criteria.