Amides, vegetable-oil, N,N-bis(hydroxyethyl)

Genetic toxicity: in vitro

Administrative data

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was well documented, meets generally accepted scientific principles, acceptable for assessment; compliant with GLP.

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was well documented, meets generally accepted scientific principles, acceptable for assessment; compliant with GLP.

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

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, 0.1, 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

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

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

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

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.
- There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.

Statistics:

not reported

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

Conclusions:

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

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance 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 (Irwin, 1999).

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was well documented, meets generally accepted scientific principles, acceptable for assessment; compliant with GLP.

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

The genotoxic potential of the test substance was determined by the induction of sister chromatid exchanges in Chinese Hamster Ovary Cells both with and without metabolic activation.

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

no data

Additional strain / cell type characteristics:

not applicable

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:

- Without S9 mix: 0.5, 1.6, 5.0 and 16 μg/mL.
- With S9 mix: Trial 1: 0.5, 5 and 16 μg/mL; Trial 2: 5, 10, 16 and 30 μg/mL.

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

- Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substituted DNA.
- Details for SCE test without S9: CHO cells were incubated for 26 hours with the test substance in supplemented McCoy’s 5A medium. Bromodeoxyur idine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium containing the test substance was removed and replaced with fre sh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained wi th Hoechst 33258 and Giemsa.
- Details on SCE test with S9: Cells were incubated with the test substance, serum-free medium, and S9 for 2 hours. The medium was then removed an d replaced with medium containing serum and BrdU and no test substance. Incubation proceeded for an additional 26 hours, with Colcemid (spindle inhibitor) present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9.
- Number of replications: A single flask per dose was used, and tests yielding equivocal or positive results were repeated.
- Scoring and number of cells evaluated: All slides were scored blind and those from a single test were read by the same person. Fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level.

Evaluation criteria:

- Statistically conservative positive response: When an SCE frequency was 20% above the concurrent solvent control value at two or more doses.
- Weak response: When an increase of 20% or greater is observed at any single dose.
- Equivocal response: A statistically significant (P<0.005) absence of any responses reaching 20% above background.

Statistics:

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points (i.e., by the linear regression trend test versus log of the dose).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(the test substance did not induce sister chromatid exchanges both in the presence and absence of S9).

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, the read across test substance did not induce significant sister chromatid exchanges in the Chines hamster overy cells both in the presence and absence of S9.

Executive summary:

A NTP study was conducted to determine the genotoxic potential of 'amides, C8-18 (even-numbered) and C18-unsatd., N,N-bis(hydroxyethyl)' (also known ascoconut oil acid diethanolamine condensate)by the induction of frequencies of sister chromatid exchanges in Chinese Hamster Ovary cells.

The doses studied were 0.5, 1.6, 5.0 and 16 μg/mL without metabolic activation (-S9); 0.5, 5 and 16 μg/mL in trail-1 and 5, 10, 16 and 30 μg/mL in trial-2 with metabolic activation (+S9). Concurrent solvent and positive controls (i.e., mitomycin-C (without S9 mix) and cyclophosphamide (with S9 mix)) were also included. A single flask per dose was used and fifty second-division metaphase cells were scored at each dose level.

The test susbtance did not induce any significant increase in the frequencies of sister chromatid exchanges. The solvent control and the positive controls induced exchanges within the normal range indicating the test conditions to be valid. 

Under the test conditions, the read acorss test substance did not induce significant sister chromatid exchanges in the Chines hamster ovary cells both in the presence and absence of S9.

Cross-reference 4

Reason / purpose for cross-reference:

read-across source

Reference

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:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was well documented, meets generally accepted scientific principles, acceptable for assessment; compliant with GLP.

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

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:

yes

Type of assay:

other: 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 applicable

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 (with and without S9 mix)

Vehicle / solvent:

ethanol

Untreated negative controls:

no

Details on test system and experimental conditions:

Detailed protocol of this study has been presented by (Galloway et.al, (1987).

Method details: In the test without S9, cells were incubated in McCoy’s 5A medium with the test substance for 8 hours; Colcemid was added and incubation continued for 2 hours. For the test with S9, cells were treated with the test substance and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa.

Harvest time: Without S9: 12 h; with S9: 13 h

Number of replications: A single flask per dose was used, and tests yielding equivocal or positive results were repeated.

Number of cells evauated: Two hundred first-division metaphase cells were scored at each dose level.

SCORING AND SELECTION OF CELLS: 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). Chromosomal aberration data are presented as percentage of cells with aberrations.

Evaluation criteria:

- Weak evidence for a positive response: When there are significant differences for only one dose.
- Positive response: When there are significant differences for two or more doses.
- Equivocal response: When a positive trend is observed in the absence of a statistically significant increase at any one dose.

Statistics:

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

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the conditions of the study, the read across test substance was found to be non-clastogenic in the presence or absence of metabolic activation.

Executive summary:

A study was conducted by NTP to determine the genotoxic potential of the read across test substance,

'amides C8-18 (even-numbered) and C18 -unsatd. N,N-bis(hydroxyethyl)' (in the form ofcoconut oil acid diethanolamine condensate)by the induction of chromosomal aberrations in Chinese Hamster Ovary cells.

The doses studied were16, 30, 50 μg/mL with and without S9 mix. Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. A single flask per dose was used and two hundred first-division metaphase cells were scored at each dose level.

 

The test substance did not induce an significant increase in the number of chromosomal aberrations.The vehicle (ethanol) control or the negative control flasks induced an increase in the number of chromosomal aberrations within the normal range. All the positive control chemicals used in the testinduced a marked increase in the number of chromosomal aberrationsboth with and without the S9 -mix.

 

Under the conditions of the study,the test substancewas found to be non-clastogenic in the presence or absence of metabolic activation.

Cross-reference 5

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was well documented, meets generally accepted scientific principles, acceptable for assessment; compliant with GLP.

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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.

GLP compliance:

yes

Type of assay:

other: 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 (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 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:

Without metabolic activation (-S9): Trial 1: 0, 1.25, 2.5, 5, 6, 10, 12 nL/mL; Trial 2: 0, 4, 5, 6, 8, 10, 12 nL/mL; Trial 3: 0, 1.5, 3, 6, 8, 10, 12, 15 nL/mL
- With metabolic activation (+S9): Trial 1: 0, 1.25, 2.5, 5, 10, 15 nL/mL; Trial 2: 0, 4, 5, 6, 8, 10, 12 nL/mL; Trial 3: 0, 6, 8, 10, 12, 15, 20 nL/mL;
Trial 4: 0, 5, 10, 15, 20, 30, 40, 50 nL/mL

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Details on test system and experimental conditions:

Detailed protocol of this study has been presented 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): 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).

Species / strain:

mouse lymphoma L5178Y cells

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

Additional information on results:

No induction of TFT resistance was noted in L5178Y mouse lymphoma cells treated with coconut oil acid diethanolamine condensate in the presence or absence of S9 metabolic activation.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, the read across test substance did not produce any increase in mutant L5178Y mouse lymphoma cell in the presence and absence of metabolic activation

Executive summary:

A study was conducted by NTP todetermine the genotoxic potential of the read across test substance, 'amides C8-18 (even-numbered) and C18 -unsatd. N,N-bis(hydroxyethyl)' (in the form of coconut oil acid diethanolamine condensate)byinducing the mutations in the L5178Y cell line.

The test substance was tested at the following concentrations :

Without metabolic activation (-S9): Trial 1: 0, 1.25, 2.5, 5, 6, 10, 12 nL/mL; Trial 2: 0, 4, 5, 6, 8, 10, 12 nL/mL; Trial 3: 0, 1.5, 3, 6, 8, 10, 12, 15 nL/mL

With metabolic activation (+S9): Trial 1: 0, 1.25, 2.5, 5, 10, 15 nL/mL; Trial 2: 0, 4, 5, 6, 8, 10, 12 nL/mL; Trial 3: 0, 6, 8, 10, 12, 15, 20 nL/mL; Trial 4: 0, 5, 10, 15, 20, 30, 40, 50 nL/mL.6 X10⁶cells in triplicate cultures were exposed to the test substance (either in the presence or absence of metabolic activation), positive control and solvent control for 4 h.

 

After the 48-hours expression period, cells were plated for selection of TFT-resistant cells and for cloning efficiency.The plates were scored after an incubation period of 10 to 12 days at 37±1°C in 5% CO2.

 

Under the test conditions, the read across test substance did not produce any increase in mutant L5178Y mouse lymphoma cell in the presence and absence of metabolic activation.

Cross-reference 6

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was well documented, meets generally accepted scientific principles, acceptable for assessment; compliant with GLP.

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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:

other: 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 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:

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:

ethanol

Untreated negative controls:

yes

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

without metabolic activation system)

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation system)

Positive control substance:

3-methylcholanthrene

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).

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.

Remarks on result:

other: all strains/cell types tested

Conclusions:

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 metabolic activation.

Cross-reference 7

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance, although study was conducted was acceptable.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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:

other: 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 weeks

Frequency of treatment:

Assumed to be daily

Remarks:

Doses / Concentrations:
0, 50, 100, 200, 400 and 800 mg/kg bw
Basis:
nominal conc.

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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the test, structurally similar 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 wk

Executive summary:

A study was conducted to assess the clastogenic potential of structurally similar 'amides, C18(unsatd.), N,N-bis(hydroxyethyl)' (in the form of oleic acid diethanolamine condensate (CAS 93-83-4)).The test substance was applied dermally for 13 wk at 0, 50, 100, 200, 400 and 800 mg/kg bw.Peripheral blood samples were obtained from male and female mice, and smears were immediately prepared and fixed in absolute methanol.

 

Under the conditions of the test, structurally similar 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 wk.

Data source

Reference

Reference Type:

publication

Title:

NTP TR 479

Author:

NTP

Year:

2001

Bibliographic source:

NIH Publication No. 01-3969

Materials and methods

Test guideline

Qualifier:

no guideline followed

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

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 003

Cas Number:

68603-42-9

Method

Species / strain

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 and 6.7 µg/plate
- With metabolic activation: 0.0, 3.3, 10, 33, 100 and 200 µg/plate

Vehicle / solvent:

ethanol

Controls

Untreated negative controls:

yes

Positive controls:

yes

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.
- There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.

Statistics:

not reported

Results and discussion

Test results

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 6.7 µg/plate without metabolic activation in the strain TA 1535; at ≥100 and 200 µg/plate with metabolic activation in the strains TA 97 and TA 100 respectively)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative with metabolic activation

Under the test conditions, the read across 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 by NTP to determine the mutagenic potential of the read across test substance, 'amides, C8 -18 (even-numbered) and C18 -unsatd., N, N-bis(hydroxyethyl)' (in the form ofcoconut oil acid diethanolamine condensate)inSalmonella typhimurium.

Salmonella typhimuriumstrainsTA97, TA98, TA100 and TA1535were treated with the test substance using the Ames plate incorporation method at up to eight dose levels for each bacterial strain, in triplicate, both with and without the addition of S9 mix (i.e., Aroclor induced rat and hamster liver homogenate metabolising system). The dose range was 0.1 to 6.7 µg/plate in the absence of S9 mix and from 3.3 to 200 µg/plate in the presence of S9 mix.

 

Cytotoxicity was observed at 6.7µg/plate without metabolic activation in the strain TA 1535 and at≥100 and200 µg/plate with metabolic activation in the strains TA 97 and 100 respectively.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 (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 test conditions, the read across test substancewas not mutagenic inSalmonella typhimuriumstrain TA97, TA98, TA100, or TA1535, with or without S9 metabolic activation.