N,N-bis(2-hydroxyethyl)dodecanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

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, 0.3, 1.0, 3.0, 10.0, 33.0, 66.0, 100.0, 333.0 µg/plate
- with metabolic activation: 0.0, 1.0, 3.0, 10.0, 33.0, 100.0, 166.0, 333.0, 1,000.0 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:

-S9 : 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:

-S9 : 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

Remarks:

-S9 : for strain 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

Remarks:

+S9 : all strains

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.

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 66 µg/plate without metabolic activation; at 1000 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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 66 µg/plate without metabolic activation; at 1000 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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 66 µg/plate without metabolic activation; at 1,000 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:

cytotoxicity

Remarks:

at ≥ 66 µg/plate without metabolic activation; at 1,000 µg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

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.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C12 DEA, according to a design based on OECD Guidance 471, 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 0.3 to 333 µg/plate (without S9-mix) and 1 to 1000 µg/plate (with S9-mix). Cytotoxicity was observed at ≥66 µg/plate without metabolic activation and at 1000 µg/plate with metabolic activation. 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, 1999).

Reference 2

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:

4.99, 15.00, 49.90 and 100.00 µg/mL (without S9);
1.50, 4.99, 15.00 and 30.00 µg/mL (with S9)

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 1 and 5 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 50 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 1: Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells (Loveday KS et al (1990))

Compound	Concentration (µg/mL)	Total Cells Scored	Number of Aberrations	Aberrations/Cell	Cells with Aberrations (%)
Without S9
Solvent control		200	7	0.04	2.5
Mitomycin-C^a	1	200	33	0.17	15.5
	5	50	53	1.06	58
Lauric acid diethanolamine condensate	4.99	200	9	0.05	4
	15	200	3	0.02	1.5
	49.9	200	1	0.01	0.5
					P=0.969^b
With S9
Solvent control		200	10	0.05	4
Cyclophosphamide 50^a	50	50	119	2.3	72
Lauric acid diethanolamine condensate	1.5	200	398.5	597	3.5
	4.99	200	395.01	590.02	5
	15	200	9	0.05	3.5
	30	0
					P=0.494

a = Positive control

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

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, C12 DEA, in a chromosomal aberration assay using Chinese Hamster Ovary (CHO) cells. The concentrations tested were 4.99, 15.00, 49.90 and 100.00 µg/mL without S9 and 1.50, 4.99, 15.00 and 30.00 µg/mL with S9. Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. A single flask per concentration was used and two hundred first-division metaphase cells were scored at each concentration level. 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-mutagenic (NTP, 1999).

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

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 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

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: Trial 1: 2.49, 4.97, 14.90, 49.70 μg/mL; Trial 2: 5.0, 10.0, 15.0, 20.0, 30.0 μg/mL.
- With S9 mix: Trial 1: 2.50, 8.35, 25.0, 83.50 μg/mL; Trial 2: 10.0, 15.0, 20.0, 30.0 μg/mL.

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:

mitomycin C

Remarks:

-S9: at concentrations of 0.002 and 0.010 μg/mL in trials 1 and 2

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:

cyclophosphamide

Remarks:

+S9 : at concentrations of 0.050 and 2.50 μg/mL in trial 1 and 0.75 and 2.50 μg/mL in trial 2

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. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium containing the test substance was removed and replaced with fresh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with 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 and 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-divisionmetaphase 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).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

(Test substance induced sister chromatid exchanges both in the presence and absence of S9).

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 shown to induce sister chromatid exchanges both in the presence and absence of S9.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C12 DEA, in a sister chromatid exchanges assay using Chinese Hamster Ovary (CHO) cells. The concentration studied were 2.49, 4.97, 14.90, 49.70 μg/mL in Trial 1 and 5.0, 10.0, 15.0, 20.0, 30.0 μg/mL in Trial 2 (without S9); 2.50, 8.35, 25.0, 83.50 μg/mL in Trial 1 and 10.0, 15.0, 20.0, 30.0 μg/mL in Trial 2 (with S9). Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. A single flask per concentration was used and fifty second-division metaphase cells were scored at each dose level. Under the study conditions, the test substance was shown to induce sister chromatid exchanges both in the presence and absence of S9 (NTP, 1999).

Reference 4

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:

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

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: 2.5, 5, 10, 20, 30, 40 and 50 μg/mL
With metabolic activation: 5, 10, 20, 30, 40, 50 and 60 μg/mL

Trial 2:
Without metabolic activation: 5, 10, 20 and 30 μg/mL
With metabolic activation: 5, 10, 20, 30 and 40 μg/mL

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

-S9 : 5 μg/mL in both trials 1 and 2

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:

+S9 : 2.5 μg/mL in both trials 1 and 2

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). Both responses would have to be significant (P≤0.05) for test material to be considered positive, i.e., capable of inducing TFT resistance. A single significant response would lead to a call of “questionable,” and the absence of both a trend and peak response results 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.

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, C12 DEA, in a mouse lymphoma assay. The cells were exposed for 4 h to concentrations of 2.5, 5, 10, 20, 30, 40 and 50 μg/mL without metabolic activation and 5, 10, 20, 30, 40, 50 and 60 μg/mL with metabolic activation in Trial 1. Cells were treated with test substance for 4 h at 5, 10, 20, 30, 40 μg/mL with metabolic activation and 5, 10, 20, 30 without metabolic activation in Trial 2. 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. 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 test substance was found to be non-mutagenic (NTP, 1999).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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 test substance was applied dermally to mice for 14 weeks. Peripheral blood samples were obtained from male and female mice at termination, and smears were immediately prepared and slides were scanned to determine the frequency of micronuclei.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 7 wk
- Housing: Housed individually in Polycarbonate cages
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet, ad libitum
- Water : Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 21.1-23.9°C
- Humidity : 42- 57%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: 1992-01-15 To: 1992-04-17

Route of administration:

dermal

Vehicle:

- Vehicle(s)/solvent(s) used: Ethanol
- Concentration of test material in vehicle:
- Lot/batch no. (if required): 91D22U
- Purity: Purity of the bulk ethanol ranged from 97% to 103% relative to the reference standard

Duration of treatment / exposure:

14 wk

Frequency of treatment:

5 exposures/wk

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Corresponding to 20 mg/mL in ethanol

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Corresponding to 40 mg/mL in ethanol

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Corresponding to 80 mg/mL in ethanol

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Corresponding to 160 mg/mL in ethanol

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Corresponding to 320 mg/mL in ethanol

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Tissues - Peripheral blood.
Cells - Normochromatic erythrocytes.
Slides were scanned to determine the frequency of micronuclei in 2000 Normochromatic erythrocytes (NCEs) in each of five animals per dose group.

Details of tissue and slide preparation:

A detailed discussion of this assay is presented by MacGregor et al, 1990.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Blood samples collected at the end of 14 wk study period

DETAILS OF SLIDE PREPARATION: Peripheral blood samples smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded.

Evaluation criteria:

An individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dose group is less than or equal to 0.025 divided by the number of dose groups.

Statistics:

The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran- Armitage trend test, followed by pairwise comparisons between each dosed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

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 frequency of micronuclei in peripheral blood cells of mice.

Executive summary:

A study was conducted to evaluate the in vivo genetic toxicity of the test substance, C12 DEA, in a peripheral blood micronucleus assay. The substance was applied dermally to mice for 14 weeks with a frequency of 5 exposures/week at doses of 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. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. No increase in the frequency of micronucleated normochromatic erythrocytes was observed in the test at any dose level. Under the study conditions, the test substance did not increase the frequency of micronuclei in peripheral blood cells of mice (NTP, 1999).

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, C12 DEA, according to a design based on OECD Guidance 471, 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 0.3 to 333 µg/plate (without S9-mix) and 1 to 1000 µg/plate (with S9-mix). Cytotoxicity was observed at ≥66 µg/plate without metabolic activation and at 1000 µg/plate with metabolic activation. 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, 1999).

Chromosomal aberration assay 

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C12 DEA, in a chromosomal aberration assay using Chinese Hamster Ovary (CHO) cells. The concentrations tested were 4.99, 15.00, 49.90 and 100.00 µg/mL without S9 and 1.50, 4.99, 15.00 and 30.00 µg/mL with S9. Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. A single flask per concentration was used and two hundred first-division metaphase cells were scored at each concentration level. 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-mutagenic (NTP, 1999).

Sister chromatid exchange assay

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C12 DEA, in a sister chromatid exchanges assay using Chinese Hamster Ovary (CHO) cells. The concentration studied were 2.49, 4.97, 14.90, 49.70 μg/mL in Trial 1 and 5.0, 10.0, 15.0, 20.0, 30.0 μg/mL in Trial 2 (without S9); 2.50, 8.35, 25.0, 83.50 μg/mL in Trial 1 and 10.0, 15.0, 20.0, 30.0 μg/mL in Trial 2 (with S9). Concurrent solvent and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)) were also included. A single flask per concentration was used and fifty second-division metaphase cells were scored at each dose level. Under the study conditions, the test substance was shown to induce sister chromatid exchanges both in the presence and absence of S9 (NTP, 1999).

Mouse lymphoma assay

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C12 DEA, in a mouse lymphoma assay. The cells were exposed for 4 h to concentrations of 2.5, 5, 10, 20, 30, 40 and 50 μg/mL without metabolic activation and 5, 10, 20, 30, 40, 50 and 60 μg/mL with metabolic activation in Trial 1. Cells were treated with test substance for 4 h at 5, 10, 20, 30, 40 μg/mL with metabolic activation and 5, 10, 20, 30 without metabolic activation in Trial 2. 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. 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 test substance was found to be non-mutagenic (NTP, 1999).

In vivo 

Mouse micronucleus assay 

A study was conducted to evaluate the in vivo genetic toxicity of the test substance, C12 DEA, in a peripheral blood micronucleus assay. The substance was applied dermally to mice for 14 weeks with a frequency of 5 exposures/week at doses of 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. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. No increase in the frequency of micronucleated normochromatic erythrocytes was observed in the test at any dose level. Under the study conditions, the test substance did not increase the frequency of micronuclei in peripheral blood cells of mice (NTP, 1999).

Justification for classification or non-classification

C12 DEA was predominantly negative in short-term in vitro and in vivo genotoxicity tests. Although one in vitro cytogenicity study reported an increase in the incidence of sister chromatid exchange in CHO cells treated with the test substance, both in the presence and absence of metabolic activation, the number of chromosomal aberrations was not increased in CHO cells exposed to similar concentrations of the test substance with or without S9. Furthermore, no increase in the frequency of micronucleated normochromatic erythrocytes was observed in an in vivo micronucleus assay in mice. Based on the overall weight of evidence, C12 DEA is not expected to have any genotoxic potential. Therefore, no classification is required for genotoxicity according to CLP (EC 1272/2008) criteria.