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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative (non-mutagenic), In vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2017

Negative (non-clastogenic), In vitro chromosome aberration test with human lymphocytes (with and without S-9 activation), OECD TG 473, 2017

Negative (non-mutagenic), In vitro Mammalian Cell Gene Mutation Test, OECD TG 476, 2018

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Remarks:
The study was conducted under GLP following the most up to date OECD guideline
Adequacy of study:
key study
Study period:
06 June 2017 - 13 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in THF (tetrahydrofuran) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilutions prepared in tetrahydrofuran by mixing on a vortex mixer on the day of each experiment. Formulated concentrations purity correction was not required.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of stock solution = 200 mg/mL.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 micro g/plate in triplicate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: the test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but fully soluble in tetrahydrofuran at 200 mg/mL.
Untreated negative controls:
yes
Remarks:
untreated control
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation; in suspension (main test)
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: presence of bacterial lawn
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a
Rationale for test conditions:
The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out‑of‑historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation: none
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the range-finding test (plate incorporation method) the test item did not cause any visible reduction in the growth of the bacterial background lawns of all of the tester strains, at all dose levels in the absence and presence of S9-mix. Therefore, the maximum recommended dose level of 5000 µg/plate was selected for main experiment.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: Yes (2009 - 2010 data)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: presence of bacterial lawn
- Other observations when applicable: no

Table 1:        Test Results; Experiment 1 – With and Without Metabolic Activation (Plate Incorporation)

Concentration (µg/plate)

 

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

Solvent Control

(THF)

79

81

22

18

29

35

19

20

14

12

1.5 µg

75

75

18

23

24

37

13

24

13

16

5 µg

76

87

14

20

23

31

15

23

16

14

15 µg

78

92

22

21

25

30

13

23

16

15

50 µg

91

85

19

23

34

32

19

21

17

16

150 µg

76

89

18

21

27

33

18

14

18

15

500 µg

73

94P

21P

23P

28P

36P

19P

18P

17P

14P

1500 µg

86

83P

26P

22P

25P

30P

21P

22P

15P

14P

5000 µg

66

62P

16P

20P

22P

26P

16P

19P

10P

9P

Positive controls

Name

ENNG

2AA

ENNG

2AA

ENNG

2AA

4NQO

BP

9AA

2AA

Dose Level

3 µg

1 µg

5 µg

2 µg

2 µg

10 µg

0.2 µg

5 µg

80 µg

2 µg

No. of Revertants

430

1797

313

217

742

153

170

144

397

422

ENNG - N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO - 4-Nitroquinoline-1-oxide

9AA - 9-Aminoacridine

2AA - 2-Aminoanthracene

BP -  Benzo(a)pyrene

P  -  Test item precipitate

Table 2:       Test Results; Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Concentration (µg/plate)

 

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

S9-Mix

(-)

S9-Mix

(+)

Solvent Control

(THF)

76

72

10

17

16

24

13

25

10

9

15 µg

66

67

10

21

17

19

17

23

8

8

50 µg

66

65

10

14

14

19

15

17

8

12

150 µg

62

72

9

13

18

17

14

17

12

8

500 µg

61P

72P

10P

18P

18P

18P

15P

28P

10P

7P

1500 µg

69P

69P

9P

20P

14P

20P

14P

27P

10P

9P

5000 µg

63P

75P

11P

21P

18P

21P

12P

19P

9P

8P

Positive controls

Name

ENNG

2AA

ENNG

2AA

ENNG

2AA

4NQO

BP

9AA

2AA

Dose Level

 

3 µg

1 µg

5 µg

2 µg

2 µg

10 µg

0.2 µg

5 µg

80 µg

2 µg

No. of Revertants

623

977

1434

169

534

120

262

165

225

292

ENNG - N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO - 4-Nitroquinoline-1-oxide

9AA - 9-Aminoacridine

2AA - 2-Aminoanthracene

BP -  Benzo(a)pyrene

P  -  Test item precipitate

Conclusions:
It was concluded that the test itme was non-mutagenic under the conditions of the test.
Executive summary:

OECD 471 (2011) - In a reverse gene mutation assay in bacteria strains: TA100, TA1535, TA98 and TA1537 of S. Typhimurium and WP2uvrA of E. Coli were exposed to

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

in tetrahydrofuran at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate with and without metabolic activation in plate incorporation and pre-incubation methods test system.

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range.

 

There was no evidence of induced mutant colonies over background in the test item treated colonies.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 May 2018 - 07 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
31 March 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in THF (tetrahydrofuran) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilutions prepared in tetrahydrofuran by mixing on a vortex mixer on the day of each experiment. Formulated concentrations purity correction was not required.
- Preliminary purification step (if any): No
-
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: 18-35 year old non-smoking volunteers (human)
- Suitability of cells: previously screened for suitability
- Cell cycle length, doubling time or proliferation index: doubling time = approximately 16 hours.
- Sex, age and number of blood donors if applicable: 18-35 year old non-smokers- prelim test (female, 22 y/o), 4-h main test (male, 27 y/o) and 24-h main test (male, 27 y/o)
- Whether whole blood or separated lymphocytes were used if applicable: The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Number of passages if applicable: 1
- Methods for maintenance in cell culture if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % fetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 16 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagles Minimal Essential Media
- Properly maintained: n/a - supplied by GIBCO BRL
- Periodically checked for Mycoplasma contamination: n/a - supplied by GIBCO BRL
- Periodically checked for karyotype stability: n/a - supplied by GIBCO BRL
- Periodically 'cleansed' against high spontaneous background: n/a - supplied by GIBCO BRL
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4(20)-hour without S9: 0, 1.25, 2.5, 5, 10, 20, 40, 80 µg/mL
4(20)-hour with S9 (2%): 0, 1.25, 2.5, 5, 10, 20, 40, 80 µg/mL
24-hour without S9: 0, 1.25, 2.5, 5, 10, 20, 40, 80 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The test item was insoluble in MEM at 50 mg/mL, in dimethyl sulphoxide and acetone at 250 and 500 mg/mL but was soluble in THF at 500 mg/mL.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 0.75 mL blood

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h (short-term) or 24 h (continuous)
- Expression time (cells in growth medium): 20 h (short-term) and 0 h (continuous), respectively.
- Selection time (if incubation with a selection agent): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours and 2.5 hours in the Cell Growth Inhibition Test and Main Test, respectively, before the required harvest time.
- Fixation time (start of exposure up to fixation or harvest of cells): After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/mL)

STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

DETERMINATION OF CYTOTOXICITY
- Method: Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
- Any supplementary information relevant to cytotoxicity: no

OTHER EXAMINATIONS:
- Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.
- Determination of endoreplication: If the chromosomes were arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell was scored as endoreduplicated (E).
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): no

- OTHER:

Gaps (g)
Gaps are small areas of the chromosome that are unstained. The chromatids remain aligned as normal and the gap does not extend along the chromatid for a distance greater than the width of a chromatid. If the gap occurs on one chromatid only it is a chromatid gap (g). If a gap appears in both chromatids at the same position it is a chromosome gap (G).

Chromatid Breaks (ctb)
Chromatid breaks (ct) vary in appearance. The chromatid may remain aligned but show a gap which is too large to classify as a gap. Alternatively, the chromatid may be broken so that the broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid fragment (f) should be scored if the chromosome of origin cannot be identified. Very small fragments are scored as minutes (m).

Chromosome Breaks (csb)
Chromosome breaks (CS) are breaks in both chromatids of the chromosome. A fragment with two chromatids is formed and may be displaced by varying degrees. Breaks are distinguished from gaps by the size of the unstained region. A chromosome break is scored if the fragment is associated with a chromosome from which it was probably derived. However, fragments are often seen in isolation and are then scored as chromosome fragments (F). Very small fragments are scored as minutes (M).

Exchanges (cte and cse)
Exchanges are formed by faulty rejoining of broken chromosomes and may be of the chromosome or chromatid type. Chromatid exchanges (c/c,r) have numerous different forms but are generally not further classified. Where multiple exchanges have occurred each exchange point is counted as one chromatid exchange. Chromosome exchanges generally appear as either a dicentric (D) or a ring (R) form, either of which can be associated with a fragment, which if possible should be scored as part of the exchange.

Multiple Aberrations
If many aberrations are present in one metaphase, the exact details may not be scorable. This is particularly the case when chromosome pulverisation occurs. If the number of aberrations is 10 or more then the cell is classified as X.
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
• All the positive control chemicals induced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not affected
- Effects of osmolality: not affected
- Evaporation from medium: not affected
- Water solubility: not affected
- Precipitation: precipitation present in prelim test at and above 39.06 µg/mL. Main test concentrations were tested at and below this level.
- Definition of acceptable cells for analysis:
- Other confounding effects: hemolysis (cytotoxic response) - see below.

RANGE-FINDING/SCREENING STUDIES:

The dose range for the Cell Growth Inhibition Test was 4.88 to 1250 µg/mL. The maximum dose was the maximum recommended dose level.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 39.06 μg/mL in all exposure groups with and without metabolic activation.

No hemolysis was observed in all exposure groups with or without metabolic activation.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells with plenty of scorable chromosomes present up to 156.25 µg/mL in all three exposure groups. Above this dose level, in all three exposure groups, there were either insufficient numbers of metaphases or no scorable metaphases present.

The selection of the maximum dose level was based on lowest precipitating dose level (40 µg/mL) for all three exposure groups used in the Main Experiment.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: n/a

Table 1:       Main Experiment: 4(20)-Hour Without Metabolic Activation (S9)

Treatment period

(h)

 

S9 mix

 

Conc.

µg/mL

 

Replicates

Number & Percentage of Cells Showing Structural Chromosome Aberration (%)

 

 

 

 

 

 

g

Cell growth index

 

Number & Percentage of Cells Showing Numerical Aberration (%)

Observed

ctb

cte

csb

cse

Other

Total

Mitotic Index (%)

Observed

polyploids

others

Total

 

 

 

 

 

 

 

4

 

-

Negative Control (THF)

0

A

150

0

0

0

0

0

0

1

5.40

150

0

0

0

B

150

1

0

0

0

0

1

0

5.50

150

0

0

0

Total

 %

300

(100)

1

(0.3)

0

(0)

0

(0)

0

(0)

0

1

(0.3)

1

(0.3)

 

 

(100)

300

 

0

0

0

(0)

 

-

10

A

150

0

0

0

0

0

0

0

5.80

150

0

0

0

B

150

2

0

0

0

0

2

0

4.30

150

0

0

0

Total

 %

300

(100)

2

(0.7)

0

(0)

0

(0)

0

(0)

0

2

(0.7)

0

(0.0)

 

 

(93)

300

 

0

0

0

(0)

 

-

20

A

150

1

0

0

0

0

1

0

5.60

150

0

0

0

B

150

1

0

0

0

0

1

0

7.00

150

0

0

0

Total

 %

300

(100)

2

(0.7)

0

(0)

0

(0)

0

(0)

0

2

(0.7)

0

(0)

 

 

(116)

300

(100)

0

0

0

(0)

 

-

40P

A

150

0

0

0

0

0

0

2

5.95

150

0

0

0

B

150

0

0

0

0

0

0

2

5.95

150

0

0

0

Total

 %

300

(100)

0

(0)

0

(0)

0

(0)

0

(0)

0

0

(0)

4

(1.3)

 

 

(109)

300

(100)

0

0

0

(0)

 

-

Positive Control (MMC)

0.2

A

123a

10

1

4

0

0

15

0

4.10

123

0

0

0

B

120a

7

9

1

0

0

15

0

2.40

120

0

0

0

Total

 %

243

(100)

17

(7.0)

10

(4.1)

5

(2.1)

0

(0)

0

(0)

30***

(12.3)

0

 

(0)

 

 

(60)

243

0

0

0

(0)

MMC = Mitomycin C

***  = p < 0.001

a     = Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed.

Table 2:       Main Experiment: 4(20)-Hour With Metabolic Activation (+S9)

Treatment period

(h)

 

S9 mix

 

Conc.

µg/mL

 

Replicates

Number & Percentage of Cells Showing Structural Chromosome Aberration (%)

 

 

 

 

 

 

g

Cell growth index

 

Number & Percentage of Cells Showing Numerical Aberration (%)

Observed

ctb

cte

csb

cse

Other

Total

Mitotic Index (%)

Observed

polyploids

others

Total

 

 

 

 

 

 

 

4

 

+

Negative Control (THF)

0

A

150

0

0

0

0

0

0

1

6.15

150

0

0

0

B

150

0

0

0

0

0

0

5

11.50

150

0

0

0

Total

 %

300

(100)

0

(0)

0

(0)

0

(0)

0

(0)

0

(0)

0

(0)

6

(2.0)

 

(100)

300

0

0

0

(0)

 

+

10

A

150

1

0

0

0

0

1

6

5.05

150

0

0

0

B

150

0

0

0

0

0

0

3

6.35

150

0

0

0

Total

 %

300

(100)

1

(0.3)

0

(0)

0

(0)

0

(0)

0

(0)

1

(0.3)

9

(3.0)

 

(65)

300

0

0

0

(0)

 

+

20

A

150

0

0

0

0

0

0

1

7.05

151

1

0

1

B

150

2

0

0

0

0

2

0

8.25

150

0

0

0

Total

 %

300

(100)

2

(0.7)

0

(0)

0

(0)

0

(0)

0

(0)

2

(0.7)

1

(0.3)

 

(87)

301

1

0

1

(0.3)

 

+

40P

A

150

1

0

0

0

0

1

0

9.00

150

0

0

0

B

150

1

0

1

0

0

2

4

9.85

150

0

0

0

Total

 %

300

(100)

2

(0.7)

0

(0)

1

(0.3)

0

(0)

0

(0)

3

(1.0)

4

(1.3)

 

(107)

300

0

0

0

(0)

 

+

Positive Control (MMC)

0.2

A

150

7

3

1

0

0

10

14

4.95

150

0

0

0

B

70a

11

3

1

0

0

14

6

1.65

70

0

0

0

Total

 %

220

(100)

18

(8.2)

6

(2.7)

2

(0.9)

0

(0)

0

(0)

24***

(10.9)

20

(9.1)

(37)

220

0

0

0

(0)

 MMC = Mitomycin C

***  = p < 0.001

a     = Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed.

Table 3:       Main Experiment 24-Hour Exposure Without Metabolic Activation (-S9)

Treatment period

(h)

 

S9 mix

 

Conc.

µg/mL

 

Replicates

Number & Percentage of Cells Showing Structural Chromosome Aberration (%)

 

 

 

 

 

 

g

Cell growth index

 

Number & Percentage of Cells Showing Numerical Aberration (%)

Observed

ctb

cte

csb

cse

Other

Total

Mitotic Index (%)

Observed

polyploids

others

Total

 

 

 

 

 

 

 

 

 

 

 

 

 

24

 

-

Negative Control (THF)

0

A

150

0

0

0

0

0

0

4

4.90

150

0

0

0

B

150

0

0

0

0

0

0

1

6.05

150

0

0

0

Total

 %

300

(100)

0

(0)

0

(0)

0

(0)

0

(0)

0

(0)

0

(0)

5

(1.7)

 

(100)

300

(100)

0

0

0

(0)

 

-

10

A

150

2

0

0

0

0

2

1

3.20

150

0

0

0

B

150

0

0

0

0

0

0

3

4.70

150

0

0

0

Total

 %

300

(100)

2

(0.7)

0

(0)

0

(0)

0

(0)

0

(0)

2

(0.7)

4

(1.3)

 

(72)

300

(100)

0

0

0

(0)

 

-

20

A

150

0

0

0

0

0

0

2

3.00

150

0

0

0

B

150

3

0

0

0

0

3

1

4.65

150

0

0

0

Total

 %

300

(100)

3

(1.0)

0

(0)

0

(0)

0

(0)

0

(0)

3

(1.0)

3

(1.0)

 

(70)

300

(100)

0

0

0

(0)

 

-

40P

A

150

0

0

0

0

0

0

1

3.90

150

0

0

0

B

150

0

0

1

0

0

1

1

3.10

150

0

0

0

Total

 %

300

(100)

0

(0)

0

(0)

1

(0.3)

0

(0)

0

(0)

1

(0.3)

2

(0.7)

 

(64)

300

(100)

0

0

0

(0)

 

-

Positive Control (MMC)

0.2

A

150

2

8

2

0

0

10

10

1.65

150

0

0

0

B

150

7

3

0

0

0

9

5

3.00

150

0

0

0

Total

 %

300

(100)

9

(3.0)

11

(3.7)

2

(0.7)

0

(0)

0

(0)

19***

(6.3)

15

(5.0)

 

(42)

300

(100)

0

0

0

(0)

MMC = Mitomycin C

***  = p < 0.001

a     = Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed.

Conclusions:
The test item was considered to be non-clastogenic to human lymphocytes in vitro under the conditions of the study.
Executive summary:

OECD 473 (2017) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

at concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, 80 µg/mL for 4 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, 80 µg/mL.

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was tested up to precipitating concentrations of 40 µg/mL. In the range-finding test, concentrations above 39.06 μg/mL in all three exposure groups precipitate of the test item was observed in the parallel blood-free cultures, indicating that maximum exposure has been reached.  Positive controls induced the appropriate response.

There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 September 2017 - 22 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidleines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in THF at 500 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To aid dosing, the formulation was maintained at approximately 40 °C. Due to the sensitivity of V79 cells to THF, the formulations were prepared at four times the concentration required in culture and dosed in 100 µL aliquots.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Stock prepared at 500 mg/mL
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): No, applied as a liquid.

OTHER SPECIFICS: n/a
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was prepared by mixing S9 with buffer NADP (5 mM), G­6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary & Mutagenicity Test.
Test concentrations with justification for top dose:
Prelim Test - 0.63, 1.25, 2.5, 5, 10, 20, 40, 60 and 80 μg/mL (limited by precipitation and toxicity observed in concurrent Chromosome Aberration Test).
Main Test (4-hour without S9) - 0*, 1.25*, 2.5*, 5*, 10*, 15* and 20* µg/mL (based on results of prelim).
Main Test (4-hour with S9 (2%) - 0*, 2.5*, 5*, 10* and 15* µg/mL (based on results of prelim).

* = Concentrations plated out for cloning efficiency and mutant frequency.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)

- Justification for choice of solvent/vehicle: The test item was insoluble in MEM at 50 mg/mL, in dimethyl sulphoxide and acetone at 250 and 500 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
THF (tetrahydrofuran)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: Dimethyl benzanthracene (DMBA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-incubation with test item
- Cell density at seeding (if applicable): For mutation expression = 2,000,000 cells per 225 cm2 flask; for cloning efficiency = 200 cells per 25 cm2 flask.

DURATION
- Preincubation period: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items.
- Exposure duration: Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

NUMBER OF REPLICATIONS: 1 for growth and mutation expression and 3 for cloning efficiency.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: n/a

OTHER EXAMINATIONS:
- Determination of polyploidy: n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterizse whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

- OTHER: n/a




Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.

When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Comparisons made between the appropriate vehicle control value and each individual concentration, using Student’s t-test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect
- Effects of osmolality: No effect
- Evaporation from medium: Not observed
- Water solubility: Formulated in acetone, therefore no effect
- Precipitation: Not plated when observed
- Definition of acceptable cells for analysis: Reported and within specification. Fresh cultures used for each test.
- Other confounding effects: None.

Table1:         Preliminary Cytotoxicity Test Results

                         4-hour -S9

4-hour +S9

Dose (µg/mL)

Count

Mean

% CE

% Control

Dose (µg/mL)

Count

Mean

% CE

% Control

0

168

190

192

183.3

91.7

100

0

190

193

188

190.3

95.2

100

0.63

168

160

158

162.0

81.0

88

0.63

163

177

147

162.3

81.2

85

1.25

152

159

139

150.0

75.0

82

1.25

144

190

167

167.0

83.5

88

2.5

149

170

170

163.0

81.5

89

2.5

181

181

191

184.3

92.2

97

5

138

171

148

152.3

76.2

83

5

161

191

168

173.3

86.7

91

10

205

201

171

192.3

96.2

105

10

179

171

202

184.0

92.0

97

20 p

171

187

167

175.0

87.5

95

20 p

141

131

139

137.0

68.5

72

40 p

130

123

151

134.7

67.3

73

40 p

Discarded: excessive precipitate

60 p

Discarded: excessive precipitate

60 p

Discarded: excessive precipitate

80 p

Discarded: excessive precipitate

80 p

Discarded: excessive precipitate

CE= Cloning Efficiency

P = Precipiate

Table 2:       Main Experiment – 4-Hour Exposure without Metabolic Activation (S9)

Day 0 Viability

 

Day 7 Viability

 

Day 7 Mutant

 

Dose (µg/mL)

Replication

Colonies/flask (200 cells plated/flask)

 

% CE

 

% Control

 

Mean % Control

 

Colonies/flask (200 cells plated/flask)

 

% CE

 

% Control

 

Mean % Control

 

Colonies/flask (2x105cells plated/flask)

 

MF

 

FS 10-6

 

SD

Group MFS 10-6

 

0

A

170

179

196

90.8

100

100

179

140

178

82.2

100

100

1

3

1

1

1

1

2

1

0

2

6.5

7.9

0.86

7

B

170

167

180

86.2

100

177

197

177

91.8

100

1

3

0

1

1

1

0

2

2

2

6.5

7.1

1.25

A

192

196

186

95.7

105.3

103

141

164

160

77.5

94.3

93

4

3

2

2

1

3

0

0

1

2

9

11.6

1.13

11

B

170

181

169

86.7

100.6

167

163

177

84.5

92.0

1

0

3

2

2

3

2

1

1

1

8

9.5

2.5

A

170

179

181

88.3

97.2

99

153

175

148

79.3

96.6

93

1

3

2

2

2

3

2

2

0

2

9.5

12.0

1.17

12

B

170

181

169

86.3

100.6

173

151

173

82.8

90.2

2

3

1

0

2

4

0

4

1

2

9.5

11.5

5

A

169

186

122

79.5

87.5

93

175

180

200

92.5

112.6

106

0

3

1

0

1

1

1

1

1

1

5

5.4

1.02

7

B

176

149

180

84.2

97.7

166

205

180

91.8

100.0

0

3

2

0

2

2

1

2

3

0

7.5

8.2

10

A

177

179

169

87.5

96.3

103

130

161

168

76.5

93.1

97

1

0

3

0

2

1

1

2

2

0

6

7.8

1.36

9

B

186

167

209

93.7

108.7

167

184

201

92.0

100.2

3

0

3

5

1

1

1

1

0

3

9

9.8

15

A

153

203

157

85.5

94.1

88

136

163

126

70.8

86.2

97

0

3

1

1

0

3

1

0

1

0

5

7.1

1.52

6

B

136

141

144

70.2

81.4

181

202

213

99.3

108.2

0

0

1

0

0

0

6

1

2

0

5

5.0

20

A

140

140

122

67.0

73.8

69

170

154

174

83.0

101.0

92

0

0

0

0

1

1

0

0

2

2

3

3.6

0.69

3

B

122

97

113

55.3

64.2

150

136

176

77.0

83.8

0

0

1

1

0

1

0

1

1

0

2.5

3.2

EMS 500

A

152

177

168

82.8

91.2

89

149

145

154

74.7

90.9

87

32

26

31

32

40

3

28

26

33

28

153.5

205.6

4.85

190

B

141

162

147

75.0

87.0

170

121

169

76.7

83.5

28

38

28

26

22

28

27

25

27

19

134

174.8

EMS 700

A

138

129

126

65.5

72.1

63

145

143

138

71.0

86.4

91

52

57

43

45

46

39

34

41

40

33

215

302.8

7.48

276

B

117

82

82

46.8

54.4

173

186

172

88.5

96.4

34

39

38

38

60

45

49

49

40

50

221

249.7

 EMS = Ethyl methane sulphonate


CE = Cloning efficiency

MF = Mutant frequency


MFS = Mutant frequency per survivor SD = Standard deviation

p = Precipitate

Table 3:       Main Experiment – 4-Hour Exposure with Metabolic Activation (S9)

Day 0 Viability

 

Day 7 Viability

 

Day 7 Mutant

 

Dose (µg/mL)

Replication

Colonies/flask (200 cells plated/flask)

 

% CE

 

% Control

 

Mean % Control

 

Colonies/flask (200 cells plated/flask)

 

% CE

 

% Control

 

Mean % Control

 

Colonies/flask (2x105cells plated/flask)

 

MF

 

FS 10-6

 

SD

Group MFS 10-6

 

0

A

142

135

155

72.0

100

100

 184

 189

 183

 92.7

 100

100

3

0

2

5

3

3

1

1

5

3

13

14.0

1.50

11

B

149

148

179

79.3

100

 158

 184

 194

 89.3

 100

1

3

0

2

1

1

1

3

0

1

6.5

7.3

1.25

A

137

140

150

71.2

98.8

95

Discarded: surplus to requirements

 

Discarded: surplus to requirements

 

 

B

144

132

162

73.0

92.0

Discarded: surplus to requirements

Discarded: surplus to requirements

2.5

A

116

134

128

63.0

87.5

92

139

149

145

72.2

77.9

91

0

2

1

2

2

0

1

2

0

3

6.5

9.0

1.09

8

B

149

148

160

76.2

96.0

195

190

175

93.3

104.5

0

2

3

3

0

0

2

2

1

1

7

7.5

5

A

148

157

159

77.3

107.4

98

181

194

172

91.2

98.4

96

3

4

2

1

4

4

1

2

2

6

14.5

15.9

1.73

11

B

146

134

140

70.0

88.2

173

160

164

82.8

92.7

1

1

2

1

1

1

0

1

0

2

5

6.0

10

A

126

129

133

64.7

89.8

91

158

184

167

84.8

91.5

93

0

3

2

1

4

4

1

2

4

2

11.5

13.5

1.17

12

B

139

169

133

73.5

92.6

168

176

166

85.0

95.1

4

2

1

2

1

2

2

1

2

2

9.5

11.2

15

A

153

138

155

74.3

103.2

99

165

166

154

80.8

87.2

94

2

2

1

3

1

2

2

2

2

2

9.5

11.8

0.88

10

B

160

149

140

74.8

94.3

166

190

181

89.5

100.2

0

1

0

2

2

2

2

3

0

1

6.5

7.3

20

A

142

142

148

72.0

100

96

Discarded: excessive precipitation

 

Discarded: excessive precipitation

 

 

B

157

120

162

73.2

92.2

Discarded: excessive precipitation

Discarded: excessive precipitation

DMBA 1

A

106

111

97

52.3

72.7

72

177

148

151

79.3

85.6

81

57

51

49

48

53

50

53

33

51

59

252

317.6

6.76

350

B

109

121

111

56.8

71.6

125

137

151

68.8

77.1

48

50

56

60

52

50

58

38

54

60

263

382.1

DMBA 2

A

110

101

116

54.5

75.7

73

131

143

148

70.3

75.9

77

76

81

79

77

91

77

84

94

82

87

414

588.6

12.97

511

   B  120 96  114  55.0  69.3     137 142  136  69.2  77.4    66  59  68  60  52  56  55  57  61  66  300  433.7  12.97  

DMBA= Dimethyl benzanthracene


CE = Cloning efficiency

MF = Mutant frequency


MFS = Mutant frequency per survivor

SD = Standard deviation

p = Precipitate

Conclusions:
The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Executive summary:

OECD 476 (2018) - In a mammalian cell gene mutation assay (HPRT locus), Chinese hamster (V79) cells cultured  were exposed in vitro  to the test item,

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

at concentrations under GLP conditions to assess its mutagenicity potential. Following a preliminary cytotoxicity test at up to 80 µg/mL concentration, a definitive test was performed at concentrations of 0, 1.25, 2.5, 5, 10, 15, 20 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively. The vehicle control values were all considered to be within the historical control range with an average absolute cloning efficiency above 50 % and the positive controls all gave marked increases in mutant frequency within the historical control range, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system.

The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test. Both exposure groups met the requirements of the OECD 476 guideline, with one precipitating dose level.

The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 471 (2011) - In a reverse gene mutation assay in bacteria strains: TA100, TA1535, TA98 and TA1537 of S. Typhimurium and WP2uvrA of E. Coli were exposed to

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

in tetrahydrofuran at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation in plate incorporation and pre-incubation methods test system.

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in the test item treated colonies.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

OECD 473 (2017) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

at concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, 80µg/mL for 4 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested without metabolic activation at test item concentrations of 0, 1.25, 2.5, 5, 10, 20, 40, 80 µg/mL.

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was tested up to precipitating concentrations of 40 µg/mL. In the range-finding test, at concentrations above 39.06 μg/mL in all three exposure groups precipitate of the test item was observed in the parallel blood-free cultures, indicating that maximum exposure has been reached.  Positive controls induced the appropriate response.

There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.

OECD 476 (2018) - In a mammalian cell gene mutation assay (HPRT locus), Chinese hamster (V79) cells cultured were exposed in vitro to the test item,

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

at different concentrations under GLP conditions to assess its mutagenicity potential. Following a preliminary cytotoxicity test at up to 80 µg/mL concentration, a definitive test was performed at concentrations of 0, 1.25, 2.5, 5, 10, 15, 20 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix), respectively. The vehicle control values were all considered to be within the historical control range with an average absolute cloning efficiency above 50 % and the positive controls all gave marked increases in mutant frequency within the historical control range, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolism activation system.

The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test. Both exposure groups met the requirements of the OECD 476 guideline, with one precipitating dose level. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Justification for classification or non-classification

The substance did not meet the classification criteria in accordance with Regulation (EC) No 1272/2008 (CLP).