Amides, C12-18 and C18-unsatd., N,N-bis(hydroxyethyl)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 26, 1996 to June 2, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI2004/0994)

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

Without metabolic activation: 0, 1.5, 5, 15, 50, 150 and 500 µg/plate
With metabolic activation: 0, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

3 µg/plate for TA100 and 5 µg/plate for TA1535

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine, 5 µg/plate for TA1538

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA1538 and TA98

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 0.5 µg/plate for TA1538 and TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation).

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
The cytotoxicity was rated as follows:
S=sparse background lawn
T=toxic
V=very thin background lawn
C=contaminated
P=precipitate
X=plate unscorable

Evaluation criteria:

The test item was considered positive if there is a dose related increase in the number of revertants or a biologically relevant increase for at least onetest concentration.
The test item was considered negative if the number of induced revertants is less than that of the spontaneous revertants by two fold at each dose level.

Statistics:

No data

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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:

valid

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:

valid

Positive controls validity:

valid

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

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test material exhibited toxicity initially at and above 150 µg/plate to the strain of Salmonella used (TA100).

Conclusions:

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

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C8-18 and C18-unsatd. DEA, according to OECD Guideline 471 and Method B.14, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance using the Ames plate incorporation method at up to seven dose levels for each bacterial strain, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 500 µg/plate (-S9 mix) and 1.5 to 1500 µg/plate (+S9 mix) in the first experiment. The experiment was repeated on a separate day using a similar dose range to experiment 1, fresh cultures of the bacterial strains and fresh test substance formulations. Extra doses were incorporated into each experiment to allow for test substance toxicity. The vehicle (ethanol) 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. The test substance caused a visible reduction in the growth of bacterial lawn to all of the strains of Salmonella tested both with and without metabolic activation. The substance was, therefore, tested up to its toxic limit. 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. Under the study conditions, the test substance was found to be non-mutagenic (Thompson, 1996).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 6, 2014 to March 7, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Range finding test in strain TA100 and E.Coli WP2 uvrA with and without metabolic activation: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330; 5000 µg/L

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

Experiment 2: Tester trains TA1535, TA1537, TA98, TA100: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 µg/L. Tester strain E.Coli WP2 uvrA: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330 and 5000 µg/L

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Negative solvent / vehicle controls:

yes

Remarks:

Saline

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

TA1535; concentration/plate 5µg

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: ICR-191

Remarks:

TA1537; concentration/plate 2.5µg

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

TA98; concentration/plate 10µg

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

TA100; concentration/plate 650µg

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2 uvrA; concentration/plate 15µg

Positive controls:

yes

Remarks:

with metabolic activation and solvent DMSO

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535 2.5µg S9-mix 5 and 10%; TA1537 2.5µg S9-mix 5%; TA1537 5µg S9-mix 10%; TA98 1µg S9-mix 5 and10%; TA100 1µg S9-mix 5% ; TA100 2µg S9-mix 10% ; W2P uvrA 15µg S9-mix 5 and 10%

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
NUMBER OF REPLICATIONS: Triplicates
DETERMINATION OF CYTOTOXICITY: Reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a)The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain
b)The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean
c)The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extendto 5 mg/plate.

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is not greater than three (3) times the concurrent vehicle control.
b)The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is greater than three (3) times the concurrent vehicle control.
b)In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

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

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Dose range finding/Experiment 1

The test substance was tested in the tester strains TA100 and WP2 uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 3, 10, 33, 100, 333 and 1000 μg/plate.The results are shown in Table 3 and Table 4 of the study report.  

Precipitate

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

Toxicity

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

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

 

Strain	Without S9-mix		With S9-mix
Dose        Bacterial                Revertant                (μg/plate)  background lawn   colonies			Dose       Bacterial                Revertant (µg/plate) background lawn  colonies
TA1535		333          extreme          microcolonies 1000          extreme         microcolonies	1000        extreme         microcolonies
TA1537		100          normal               moderate  333          extreme         microcolonies 1000          extreme        microcolonies	1000          extreme         microcolonies
TA98		333          moderate          extreme 1000          extreme        microcolonies	1000          extreme         microcolonies
TA100		100-333     moderate          extreme 1000-5000  extreme         microcolonies	333          moderate          extreme 1000-5000  extreme         microcolonies
WP2 uvrA		5000          normal               -^1	5000          normal             slight

-¹  No reduction in the number of revertant colonies

Experiment 2

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

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

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

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

Precipitate

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

Toxicity

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

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

 

Strain	Without S9-mix		With S9-mix
Dose         Bacterial              Revertant              (μg/plate)  background lawn  colonies			Dose         Bacterial                   Revertant (µg/plate)  background lawn    colonies
TA1535		333      extreme           microcolonies 1000     extreme           microcolonies	1000          extreme              microcolonies
TA1537		333      absent             complete 1000     absent             complete	333          slight                 -^1 1000          extreme              microcolonies
TA98		333      slight                 extreme 1000     extreme           microcolonies	1000          moderate            extreme
TA100		100      normal             moderate  333      extreme           microcolonies 1000     extreme           microcolonies	1000          extreme              microcolonies
WP2 uvrA		5000     normal                     -^2	5000           normal                  -^2

-¹  No reduction in the number of revertant colonies

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

Conclusions:

Under the conditions of the study, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A study was conducted to evaluate the in vitro genotoxic toxicity of the test substance, C8-18 and C18-unsatd. DEA, according to OECD Guideline 471, in compliance with GLP. In the dose range finding study, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and upwards in the absence of metabolic activation (S9-mix) and at dose levels of 333 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, toxicity was only observed at the dose level of 5000 μg/plate in the presence of S9-mix. Based on the results of the dose range finding study, the substance was tested in the first mutation assay at a concentration range of 3 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains. In the second mutation assay, the substance was tested up to concentrations of 1000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA100, TA1535, TA1537 and TA98 and up to concentrations of 5000 µg/plate in tester strain WP2 uvrA. Toxicity was observed in all tester strains, except in tester strain WP2 uvrA. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of the study, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Verspeek-Rip, 2014).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12-03-1991 to 23-04-1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Target gene:

Not reported

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:

5000 µg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Details on test system and experimental conditions:

Not reported

Evaluation criteria:

Not reported

Statistics:

Not reported

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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:

valid

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

not specified

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C8-18 and C18-unsatd. DEA, according to EU Method B 13/14, in compliance with GLP. The test concentration was 5000 µg/plate. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay (Schöberl, 1991).

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the section 13 for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

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

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix

Test concentrations with justification for top dose:

16, 30, 50 µg/mL (-/+ S9)

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9: 0.0625 and 0.25 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(Ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9: 2.5 and 7.5 µg/mL

Details on test system and experimental conditions:

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

DURATION
- Exposure duration: In the test without S9, cells were incubated in McCoy’s 5A medium with coconut oil acid diethanolamine condensate for 10 h; Colcemid was added and incubation continued for 2 h. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the test with S9, cells were treated with coconut oil acid diethanolamine condensate and S9 for 2 h, after which the treatment medium was removed and the cells were incubated for 11 h in fresh medium, with Colcemid present for the final 2 h. Cells were harvested in the same manner as for the treatment without S9.
- Harvest time: 12 h (without S9); 13 h (with S9)

NUMBER OF REPLICATIONS: A single flask per dose was used, and tests yielding equivocal or positive results were repeated.
NUMBER OF CELLS EVALUATED: Two hundred first-division metaphase cells were scored at each dose level.

Evaluation criteria:

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverised cells, despiralised chromosomes, and cells containing 10 or more aberrations).

Statistical analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P ≤0.05) difference for one dose point and a significant trend (P ≤0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicates the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call. Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.

Statistics:

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 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 (Irwin, 2001).

Reference 5

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:

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

Remarks:

KL2 due to RA

Justification for type of information:

Refer to the section 13 for details on the category justification.

Reason / purpose for cross-reference:

read-across source

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: 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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/mL without 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 (Irwin, 2001). Based on the results of the read across study, the test substance is also expected to have similar mutagenicity potential in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

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:

read-across source

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 (90% active) 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 (Irwin, 1999).

Reference 2

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:

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:

read-across source

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 (Irwin, 2001). Based on the results of the read across study, the test substance is also expected to have similar clastogenic potential in vivo MNT.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: 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:

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:

read-across source

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

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 (purity not specified) 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) (Irwin, 2001).

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

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

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:

read-across source

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 (48 -56% active) 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

In vitro

In vitro gene mutation in bacteria

A study was conducted to evaluate the in vitro genotoxic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to OECD Guideline 471, in compliance with GLP. In the dose range finding study, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and upwards in the absence of metabolic activation (S9-mix) and at dose levels of 333 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, toxicity was only observed at the dose level of 5000 μg/plate in the presence of S9-mix. Based on the results of the dose range finding study, the substance was tested in the first mutation assay at a concentration range of 3 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains. In the second mutation assay, the substance was tested up to concentrations of 1000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA100, TA1535, TA1537 and TA98 and up to concentrations of 5000 µg/plate in tester strain WP2 uvrA. Toxicity was observed in all tester strains, except in tester strain WP2 uvrA. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of the study, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays (Verspeek-Rip, 2014).

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to OECD Guideline 471 and Method B.14, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance using the Ames plate incorporation method at up to seven dose levels for each bacterial strain, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 500 µg/plate (-S9 mix) and 1.5 to 1500 µg/plate (+S9 mix) in the first experiment. The experiment was repeated on a separate day using a similar dose range to experiment 1, fresh cultures of the bacterial strains and fresh test substance formulations. Extra doses were incorporated into each experiment to allow for test substance toxicity. The vehicle (ethanol) 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. The test substance caused a visible reduction in the growth of bacterial lawn to all of the strains of Salmonella tested both with and without metabolic activation. The substance was, therefore, tested up to its toxic limit. 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. Under the study conditions, the test substance was found to be non-mutagenic (Thompson, 1996).

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to EU Method B 13/14, in compliance with GLP. The test concentration was 5000 µg/plate. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay (Schöberl, 1991).

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 (Irwin, 2001).

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 (Irwin, 2001).

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 test 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) (Irwin, 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 (Irwin, 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 (Irwin, 2001).

Overall evaluation

The test read across substance was negative in vitro 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 (Irwin, 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.

Justification for classification or non-classification

The test read across substance was negative in vitro 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 (Irwin, 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 required for this endpoint according to CLP (EC 1272/2008) criteria.