Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro

Ames test

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C16-18 and C18-unsatd. DEA, according to EU Method B.13/14, in compliance with GLP. In a test bacterial strains salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation with rat liver S9 mix were tested with the test concentration 5,000 µg/plate. Under the study conditions, the test substance was considered to be non-mutagenic (Schoberl, 1991).

Also,after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP),an in vitro genotoxicity study according to OECD Guideline 471 is planned with C16-18 and C18-unsatd. DEA in order to further support the read across approach proposed for the FAA category members.

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-mutagenic (NTP, 2001).

Also,after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP),an in vitro genotoxicity study according to OECD Guideline 473 is planned with C16-18 and C18-unsatd. DEA 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 read across substance, C8-18 and C18-unsatd. DEA, in a mouse lymphoma assay using cell line L5178Y. The cells were exposed for 4 h to concentrations of 1.25, 2.5, 5, 6, 10 and 12 nL/mL without metabolic activation and 1.25, 2.5, 5, 10 and 15 nL/mL with metabolic activation in Trial 1. Cells were treated with test substance for 4 h at 4, 5, 6, 8, 10 and 12 nL/mL with metabolic activation and 4, 5, 6, 8, 10 and 12 nL/mLwithout metabolic activation in Trial 2. Cells were treated with test substance for 4 h at 6, 8, 10, 15 and 20 nL/mL with metabolic activation and 1.5, 3, 6, 8, 10 and 12 nL/mL without metabolic activation in Trial 3. Cells were treated with test substance for 4 h at 5, 10, 15, 20, 30 and 40 nL/mL with metabolic activation in Trial 4. 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. No increase in mutant L5178Y mouse lymphoma cell colonies was observed after exposure to test substance, with or without S9. A single positive response noted at 8 nL/mL in the second trial conducted without S9 was not reproducible and the test results overall were considered to be negative. Under the study conditions, the test substance was found to be non-mutagenic (NTP, 2001).

Also,after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP),an in vitro genotoxicity study according to OECD Guideline 476 is planned with C16-18 and C18-unsatd. DEA in order to further support the read across approach proposed for the FAA category members.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From March 13, 1991 to April 23, 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to the section 13 for details on the category justification.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Luminal-induced rat liver microsomes
Test concentrations with justification for top dose:
5,000 µg/plate
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
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 considered to be non-mutagenic.
Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C16-18 and C18-unsatd. DEA (95.9% active) using EU Method B.13/14, in compliance with GLP. In a test bacterial strains salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation with rat liver S9 mix were tested with the test concentration 5,000 µg/plate. Under the study conditions, the test substance was considered to be non-mutagenic (Schoberl, 1991).

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:
guideline study with acceptable restrictions
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
Reason / purpose for cross-reference:
read-across: supporting information
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-mutagenic (NTP, 2001).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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
Reason / purpose for cross-reference:
read-across: supporting information
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: 1.25, 2.5, 5, 6, 10 and 12 nL/mL
With metabolic activation: 1.25, 2.5, 5, 10 and 15 nL/mL

Trial 2:
Without metabolic activation: 4, 5, 6, 8, 10 and 12 nL/mL
With metabolic activation: 4, 5, 6, 8, 10 and 12 nL/mL

Trial 3:
Without metabolic activation: 1.5, 3, 6, 8, 10 and 12 nL/mL
With metabolic activation: 6, 8, 10, 15 and 20 nL/mL

Trial 4:
With metabolic activation: 5, 10, 15, 20, 30 and 40 nL/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
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
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:
Mutant fraction (MF) (frequency) is a ratio of the mutant count to the cloning efficiency, divided by 3 (to arrive at MF/106 cells treated).

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). ll data were evaluated statistically for trend and peak responses. 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 mutant L5178Y mouse lymphoma cell colonies was observed after exposure to coconut oil acid diethanolamine condensate, with or without S9. A single positive response noted at 8 nL/mL in the second trial conducted without S9 was not reproducible and the test results overall were considered to be negative.

For detailed tables 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 mouse lymphoma assay using cell line L5178Y. The cells were exposed for 4 h to concentrations of 1.25, 2.5, 5, 6, 10 and 12 nL/mL without metabolic activation and 1.25, 2.5, 5, 10 and 15 nL/mL with metabolic activation in Trial 1. Cells were treated with read across substance for 4 h at 4, 5, 6, 8, 10 and 12 nL/mL with metabolic activation and 4, 5, 6, 8, 10 and 12 nL/mLwithout metabolic activation in Trial 2. Cells were treated with read across substance for 4 h at 6, 8, 10, 15 and 20 nL/mL with metabolic activation and 1.5, 3, 6, 8, 10 and 12 nL/mL without metabolic activation in Trial 3. Cells were treated with read across substance for 4 h at 5, 10, 15, 20, 30 and 40 nL/mL with metabolic activation in Trial 4. 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. No increase in mutant L5178Y mouse lymphoma cell colonies was observed after exposure to test substance, with or without S9. A single positive response noted at 8 nL/mL in the second trial conducted without S9 was not reproducible and the test results overall were considered to be negative. Under the study conditions, the test substance was found to be non-mutagenic (NTP, 2001). Based on the results of the read across study, the test substance is also expected to have similar mutagencity potential in vitro.

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

Genetic toxicity in vivo

Description of key information

In vivo

Mouse micronucleus assay

A study was conducted to evaluate the in vivo genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to the method recommended in the First Addendum to OECD Guideline 474 and Method No. 431, Annex V of EEC Directive 79/831. The experimental animals were 70 NMRI mice, divided into 5 groups. Of the 5 groups, 3 were test groups, 1 negative control group and 1 positive control group. The test groups were treated with 15000 mg test substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively, after treatment. From bone marrow smears, micronucleus counts were made per 1000 polychromatic erythrocytes. The test substance did not induce an increase in the frequency of micronucleated normochromatic erythrocytes in peripheral blood samples from both male and female mice. Under the study conditions, the test substance did not induce an increase in the frequency of micronuclei in bone marrow cells of the mouse and was therefore considered to be non-mutagenic (Kallesen, 1985).

A study was conducted to evaluate the in vivo genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, in a mouse peripheral blood micronucleus assay. The test substance (in ethanol) was applied dermally for 14 weeks with the frequency of 5 exposures/week 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. 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. Significant increases in the frequencies of micronucleated normochromatic erythrocytes (NCEs) were seen in peripheral blood of both male and female mice at the end of 14 weeks. Statistical analysis of the data showed positive trends for both data sets as well as significantly elevated micronucleus frequencies at the highest dose tested (800 mg/kg bw) (NTP, 2001).

A study was conducted to evaluate the in vivo genetic toxicity of the read across 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).

A study was conducted to evaluate the in vivo genetic toxicity of the read across 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).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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 section 13 of IUCLID for details on the category justification.
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 (PJ 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 80 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 assess the in vivo genetic toxicity of of the read across 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:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
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:
guideline study with acceptable restrictions
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
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 read across 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:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting 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
Justification for type of information:
Refer to the section 13 for details on the category justification.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
The micronucleus assay was carried out at the end of the 14-wk dermal toxicity study. Test substance is applied dermally to mice for 14 wk. Peripheral blood samples were obtained from male and female mice, 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:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 16 to 17 d

ENVIRONMENTAL CONDITIONS
- Temperature : 20.6 -22.8°C
- Humidity : 41-58%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Feb. 12, 1992 To: May 15, 1992
Route of administration:
dermal
Vehicle:
Ethanol (Purity: 95% (Gas chromatography with a flame ionization detector))



Details on exposure:
Test material formulation applied daily on shaved skin. No further details available.
Duration of treatment / exposure:
14 wk (The micronucleus assay was carried out at the end of the 14-wk dermal toxicity study)
Frequency of treatment:
5 exposures/wk
Post exposure period:
Not applicable
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Correspodning to 20 mg/mL in ethanol
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Correspodning to 40 mg/mL in ethanol
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Correspodning to 80 mg/mL in ethanol
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Correspodning to 160 mg/mL in ethanol
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
Correspodning to 320 mg/mL in ethanol
No. of animals per sex per dose:
10/sex/dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Not applicable
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 the assay is presented by MacGregor et al, 1990.

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

DETAILS OF SLIDE PREPARATION: 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.





Evaluation criteria:
Reproducible and significant increase in the number of micronucleated NCEs in the test group over that of the control group.
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 dose 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. 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.
Key result
Sex:
male/female
Genotoxicity:
positive
Toxicity:
yes
Remarks:
For details, refer to section 'details on results' of 14 wk dermal toxicity study in mice
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
At the end of 14 wk, significant increases in the frequencies of micronucleated NCEs were seen in peripheral blood of both male and female mice. Statistical analysis of the data showed positive trends for both data sets as well as significantly elevated micronucleus frequencies at the highest dose tested (800 mg/kg) in male and female mice.

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

Conclusions:
Under the study conditions, significant increases in the frequencies of micronucleated normochromatic erythrocytes (NCEs) were seen in peripheral blood of both male and female mice at the end of 14 weeks.
Executive summary:

A study was conducted to evaluate the in vivo genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, in a mouse peripheral blood micronucleus assay. The test substance (in ethanol) was applied dermally for 14 weeks with the frequency of 5 exposures/week 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. 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. Significant increases in the frequencies of micronucleated normochromatic erythrocytes (NCEs) were seen in peripheral blood of both male and female mice at the end of 14 weeks. Statistical analysis of the data showed positive trends for both data sets as well as significantly elevated micronucleus frequencies at the highest dose tested (800 mg/kg bw) (NTP, 2001).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: EEC Directive 79/831, Annex V, Method No. 431
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeders were purchased from G1. Bomholtgard Ltd., but the mice used were born in the Scantox Laboratories
- Age at study initiation: 6-7 wk
- Weight at study initiation: 25-29 g
- Assigned to test groups randomly: Yes, in 5 groups
- Fasting period before study: No
- Housing: 5 animals/cage, males and females separately in type III Macrolone cages, bedding used was special softwood sawdust "Spanvall Special White " from Spanvall Ltd., DK-4535 Vallekilde
- Diet: Complete rodent diet "Altromin 1314" from Chr. Petersen Ltd., DK-4100 Ringsted, ad libitum
- Water (e.g. ad libitum): Drinking water adjusted to pH 2.5 with hydrochloric acid

ENVIRONMENTAL CONDITIONS
- Temperature: 21±2 °C
- Humidity (%): 55±15%
- Air changes: 10 per hr
- Photoperiod: 12 hrs dark / 12 hrs light


Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dilution of the test material in distilled water

Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Once
Post exposure period:
24, 48 and 72 h
Dose / conc.:
15 000 mg/kg bw/day (nominal)
Remarks:
equivalent to 14.7 mL/kg
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 30 mg/10 mL
Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Preliminary investigations - A few mice were treated orally by various concentrations of the test material diluted with distilled water. Thereby the maximum tolerated dose was estimated at 15 g/kg bw. At this dosage bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE). At a dose exceeding 15 g/kg bw the mortality was too high.

DETAILS OF SLIDE PREPARATION: Immediately after sacrifice, femurs of a mouse were dissected free of muscle, and by a 1 mL syringe with needle the bone marrow was flushed out into 5 mL of fetal calf serum. After thorough shaking, the mixture was centrifuged for 10 min. at about 1000 rpm. Thereafter, smears were made after removal of the supernatant. The specimens were fixed in methanol and stained with May-Grunwald/Giemsa.

METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
Number of normochromatic erythrocytes (NCE) per 1000 erythrocytes
Number of polychromatic erythrocytes (PCE) per 1000 erythrocytes
Number of micronuclei (MN) in 1000 normochromatic erythrocytes
Number of micronuclei (MN) in 1000 polychromatic erythrocytes.

Evaluation criteria:
Increase in the frequency of micronucleated polychromated erythrocytes in treated animals as compared to controls.
Statistics:
The statistical difference was analysed by one-way ANOVA. In the case of PCE (%) the test was performed on the values observed, and for the MN (per thousand) the test was done on computed rank values transformed to normal scores according to Blom's method. (G. Blom Statistical Estimates and Transformed Beta Variables. New York: John Wiley and Sons, Inc., 1958).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Up to 15 g/kg bw
- Clinical signs of toxicity in test animals: Maximum tolerated dose was estimated at 15 g/kg bw. At a dose exceeding 15 g/kg bw, the mortality was too high.
- Evidence of cytotoxicity in tissue analyzed: At 15 g/kg bw, bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant difference as compared to controls.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

For detailed results and bar charts kindly refer to the attached background materials section of the IUCLID.

Conclusions:
Under the study conditions, the test substance did not induce an increase in the frequency of micronuclei in bone marrow cells of the mouse, and was therefore considered to be non-mutagenic.
Executive summary:

A study was conducted to evaluate the in vivo genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to the method recommended in the First Addendum to OECD Guideline 474 and Method No. 431, Annex V of EEC Directive 79/831. The experimental animals were 70 NMRI mice, divided into 5 groups. Of the 5 groups, 3 were test groups, 1 negative control group and 1 positive control group. The test groups were treated with 15000 mg read across substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively, after treatment. From bone marrow smears, micronucleus counts were made per 1000 polychromatic erythrocytes. The read across substance did not induce an increase in the frequency of micronucleated normochromatic erythrocytes in peripheral blood samples from both male and female mice. Under the study conditions, the test substance was considered to be non-mutagenic (Kallesen, 1985).

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

Additional information

Justification for classification or non-classification

The test read across substance was negative in in vitro genotoxicity studies and negative in all in vivo micronucleus tests except one in which significantly elevated micronucleus frequencies was detected at the highest dose (800 mg/kg bw) in male and female mice (NTP, 2001). However, given that all in vitro test results were negative, a similar in vivo study with a high reliability score (Klimisch score 1) was negative at a maximum tolerated oral dose of 15000 mg/kg bw and no effects were seen in in vivo studies conducted with the read across substances C12 DEA and C18-unsatd. DEA, the test substance is overall not considered have any genotoxic potential. Therefore, no classification is warranted for this endpoint according to CLP (EC 1272/2008) criteria.