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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames), OECD 471: negative
RA-S CAS 112-84-5, Chromosome aberration in mammalian cells in vitro, OECD 473: negative
RA-S CAS 112-84-5, Mammalian cell gene mutation test in vitro (Mouse Lymphoma), OECD 476: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions. Incomplete strain selection, but adequate at the time of testing.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Incomplete strain selection: TA-102 or equivalent is missing
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
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:
S9-mix prepared from Aroclor 1254 rats
Test concentrations with justification for top dose:
Dose range finding test: 5000, 500, 50, 5 µg/plate
Mutation tests: 5000, 1500, 500, 150, 50 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation)

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: 3, the procedures were repeated in a second mutation assay at a later date using the same dose levels

DETERMINATION OF CYTOTOXICITY
- Method: substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn

Colonies were counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed


Evaluation criteria:
- If treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9-mix, it will be considered to show evidence of mutagenic activity in this test system.
- If treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it will be considered to show no evidence of mutagenic activity in this test system
Statistics:
No statistical analysis performed
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
maximum guideline concentration of 5 mg/plate tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
maximum guideline concentration of 5 mg/plate tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
maximum guideline concentration of 5 mg/plate tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
maximum guideline concentration of 5 mg/plate tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
maximum guideline concentration of 5 mg/plate tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The revertant colony counts for Amides, C16-C18 (even numbered) obtained in the preliminary toxicity test revealed that Amides, C16-C18 (even numbered) was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests.


No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with Amides, C16-C18 (even numbered) at any dose level, either in the presence or absence of S9-mix. It is concluded that Amides, C16-C18 (even numbered) shows no evidence of mutagenic activity when tested in this bacterial system.

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-conducted study performed under current GLP with complete test material characterization available. Reliability was changed from "1" to "2" according to the ECHA guidance document "Practical guide 6: How to report read-across and categories".
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
None
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9, phenobarbital/ß-naphthoflavone induced
Test concentrations with justification for top dose:
4.9 to 1250 µg/ml; additional information below.
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9-mix: 1000 µg/ml (Experiment I); 900 µg/ml (Experiment II)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix: 1.4 µg/ml (Experiment I + II)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 14 h; 18 h treatment: 18 h

SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Large stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany) were stored in liquid nitrogen in the cell bank of Harlan CCR, which allows the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with Neomycin (5 µg/mL) and Amphotericin B (2.5 µg/mL). The cells were sub-cultured twice a week. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
The highest concentration used in the cytogenetic experiments was chosen considering the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible.
With respect to the solubility of the test item, 1250.00 µg/mL of Erucamide (approx. 3.7 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 4.9 and 1250.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item was observed at 312.5 µg/mL and above in the absence of S9 mix and at 625.0 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Dose selection of Experiment II was influenced by test item precipitation obtained in Experiment I and observations made during the experimental performance of Experiment II. Therefore, 450.0 µg/mL (without S9 mix) and 300.0 µg/mL (with S9 mix) were chosen as top treatment concentrations in Experiment II.
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium were added.
Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 14 hours.

Colcemid was added to the culture medium (0.2 µg/mL) 15.5 hours after the start of the treatment. The cells were treated, 2.5 hours later, on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" [5]) using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
The evaluation of cytotoxicity indicated by reduced cell numbers was made after the preparation of the cultures on spread slides. The cell numbers were determined microscopically by counting 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.
Statistics:
Statistical significance was confirmed by Fisher’s exact test (p < 0.05); however, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, classification with regard to historical data and biological relevance is discussed and/or a confirmatory experiment is performed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
noted at 300 µg/ml after 18 hour incubation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation noted at 300 µg/ml after 4 hour incubation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In both experiments, in the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see Table). The aberration rates of the cells after treatment with the test item (0.0 - 3.8 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls.
In both experiments, either EMS (900.0 or 1000.0 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations

Summary of results of the chromosome aberration study with Erucamide

Exp.

Preparation

Test item

Cell numbers

Mitotic indices

Aberrant cells

 

 

interval

concentration

in %

in %

in %

 

 

 

in µg/mL

of control

of control

incl. gaps*

excl. gaps*

with exchanges

 

 

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

100.0

100.0

3.0

1.5

0.5

 

 

 

Positive control2

n.t.

104.9

22.0

21.0S

14.0

 

 

 

78.1

88.8

99.6

2.5

2.5

0.5

 

 

 

156.3

77.3

104.4

2.5

2.5

1.0

 

 

 

312.5P##

84.8

76.4

4.0

3.8

1.3

 

 

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

100.0

100.0

3.0

2.5

0.0

 

 

 

Positive control3#

n.t.

40.3

42.0

40.0S

13.0

 

 

 

150.0

67.3

97.8

3.0

1.5

0.0

 

 

 

300.0

51.9

57.5

2.5

2.5

0.0

 

 

 

450.0

60.9

68.4

1.0

1.0

0.5

 

 

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

100.0

100.0

1.5

1.5

0.5

 

 

 

Positive control4

n.t.

52.3

9.5

8.5S

2.5

 

 

 

156.3

96.3

118.2

3.5

2.5

0.0

 

 

 

312.5

82.9

107.7

0.5

0.5

0.0

 

 

 

625.0P

90.5

78.2

2.5

1.5

0.0

 

II

18 hrs

Solvent control1

100

100

0.5

0.5

0.0

 

 

 

Positive control4

n.t.

90.4

13.5

12.0S

5.0

 

 

 

75.0

60.0

102.9

0.0

0.0

0.0

 

 

 

150.0

84.2

106.4

1.5

0.5

0.0

 

 

 

300.0P

67.8

116.4

2.5

1.0

0.5

 

*     Inclusive cells carrying exchanges

#     Evaluation of 50 metaphases per culture

##    Evaluation of 200 metaphases per culture

n.t. Not tested

P     Precipitation occurred at the end of treatment

S     Aberration frequency statistically significant higher than corresponding control values

1     Acetone 0.5 % (v/v)

2           EMS    1000.0 µg/mL;3      EMS    900.0 µg/mL;4    CPA  1.4 µg/mL

Conclusions:
Interpretation of results:
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item Erucamide did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line), when tested up to precipitating or cytotoxic concentrations.
Executive summary:

The test item Erucamide, suspended in acetone, was assessed for its potential to induce structural chromosome aberrations inV79cells of the Chinese hamsterin vitroin two independent experiments. In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated. The highest applied concentration (1250.0 µg/mL; approx. 3.7 mM) was chosen with regard to the solubility properties of the test item in an appropriate solvent and with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In Experiment I in the absence and presence of S9 mix no clear cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred. In Experiment II in the absence of S9 mix clear cytotoxicity was observed at 300.0 and 450.0 µg/mL. Test item precipitation occurred in the presence of S9 mix at the highest evaluated concentration and the cell number was reduced to 60 % and 67.8 % of control after treatment with 75.0 and 300.0 µg/mL, respectively.

No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. Under the experimental conditions reported, the test item did not induce structural chromosome aberrations inV79cells (Chinese hamster cell line)in vitro. Therefore, erucamide is considered to be non-clastogenic in this chromosome aberration test in the presence and absence of metabolic activation when tested up to precipitating or cytotoxic concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well-conducted GLP study with certificate, performed according to accepted guidelines. Complete test material characterization is available. No known or suspected deviations. Reliability was changed from "1" to "2" according to the ECHA guidance document "Practical guide 6: How to report read-across and categories".
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
L5178Y cell line (cell bank of Harlan CCR)
- Absence of Mycoplasma contamination:
yes
- Methods for maintenance in cell culture: Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment
- Cell cycle length, doubling time or proliferation index: 10 - 12 h
- Modal number of chromosomes:
40 ± 2
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium (GIBCO, invitrogen) supplemented with 15% horse serum (HS, GIBCO, invitrogen) (3% HS during 4 hour treatment), 1% of 100 U/100 µg/mL Penicillin / Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75% Amphotericin used as antifungal. The cell cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Rat liver S9, phenobarbital / ß-naphthoflavone induced
- source of S9: Harlan Laboratories GmbH, Borchen, Germany
- method of preparation of S9 mix: The S9 was prepared from 8 - 12 weeks old male Wistar HsdCpb:WU rats, weight approx. 220 - 320 g, induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and β-Naphthoflavone p.o., each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a 150 mM KCl solution (1+3) followed by centrifugation at 9000 g.

The protein concentration of the S9 preparation was 31.7 mg/mL in the pre-experiment, 32.7 mg/mL in experiment I and 35.0 mg/mL in experiment II.

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to give a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4

- concentration of S9 mix in the final culture medium: 5% (v/v)
Test concentrations with justification for top dose:
Preliminary study:
- 9.4, 18.8, 37.5, 75, 150, 300, 600, 1200 µg/mL (with and without metabolic activation)
Experiment I:
- 9.4, 18.8, 37.5, 75, 150, 300 µg/mL (with and without metabolic activation)
Experiment II:
- 4.7, 9.4, 18.8, 37.5, 75, 150 µg/mL (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties and its relative non-toxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 19.5 µg/ml (experiment I), 13.0 µg/ml (experiment II)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9: 3.0 µg/ml (experiment I), 4.5 µg/ml (experiment II)
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1E07 cells / flask (4 h treatment) and 3E06 cells / flask (24 h treatment)
- Test substance added in medium

FOR GENE MUTATION:
- Exposure duration: Experiment I: 4 h exposure with and without S9 mix, Experiment II: 4 h exposure with S9 mix, 24 h exposure without S9 mix
- Expression time: 48 h
- Selection time: 10 – 15 days
- Method used: microwell plates for the mouse lymphoma assay
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of mutant frequency, cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4E03 cells in selective medium with TFT. Viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT).
- Criteria for small (slow growing) and large (fast growing) colonies: absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

SELECTION AGENT: 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, cloning efficiency
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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 applicable
Positive controls validity:
valid
Additional information on results:
Full study data tables and figures are presented in Attachment 1.

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 9.4 µg/mL and 1200 µg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the solubility of the test item in organic solvents and aqueous media.

No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 and 24 h of treatment.

The test medium was checked for precipitation at the end of each treatment period (4 or 24 h) before the test item was removed. Precipitation was observed by the unaided eye at 150 µg/mL and above with and without metabolic activation after 4 and 24 h of treatment.

MAIN STUDY RESULTS
:
Precipitation of the test item visible to the naked eye was noted in Experiment I at 37.5 µg/mL and above with and without metabolic activation. In the second experiment, precipitation occurred at 75.0 µg/mL and above with and without metabolic activation. This precipitation did not interfere with the ability to assess viability or mutation frequency, with at least four analysable concentrations available for each experimental condition, therefore, satisfying the guideline requirement.

No relevant toxic effect indicated by a relative total growth of less than 50 % of survival in both parallel cultures was observed up to the maximum concentration with and without metabolic activation, following 4 and 24 h of treatment.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. An isolated increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control occurred at 9.4 µg/mL in the first culture of the first experiment with metabolic activation. This increase was not considered relevant since it was not reproduced in the parallel culture under identical conditions or at any other, even higher concentration in both cultures. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.

In the study, the range of the solvent controls was 103 - 214 mutant colonies per 10E6 cells; the range of the groups treated with the test item was 102 - 368 mutant colonies per 10E6 cells. In Experiment I, Culture I without metabolic activation and in Experiments I and II, Culture II with metabolic activation the solvent controls exceeded the recommended 50 – 170E06 control range as stated in the acceptability criteria. However, the number of mutant colonies per 10E6 cells in the parallel cultures (138, 148, and 163 mutant colonies / 10E6 cells) was acceptable.

MMS (19.5 µg/mL in Experiment I and 13.0 µg/mL in Experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

HISTORICAL CONTROL DATA
See Attachment 2

No relevant toxic effect indicated by a relative total growth of less than 50 % of survival in both parallel cultures was observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. An isolated increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control occurred at 9.4 µg/mL in the first culture of the first experiment with metabolic activation. This increase was not considered relevant since it was not reproduced in the parallel culture under identical conditions or at any other, even higher concentration in both cultures. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In this study the range of the solvent controls was from 103 up to 214 mutant colonies per 106cells; the range of the groups treated with the test item was from 102 up to 368 mutant colonies per 106cells. In experiment I, culture I without metabolic activation and in experiments I and II, culture II with metabolic activation the solvent controls exceeded the recommended 50 – 170 x 106control range as stated under criteria for acceptability of the assay of this report. However, the number of mutant colonies per 106cells in the parallel cultures (138, 148, and 163 mutant colonie/106cells) was acceptable.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

Statistical Analysis

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

p-value

experiment I, culture I without S9 mix

0.927

experiment I, culture II without S9 mix

0.729

experiment I, culture I with S9 mix

0.404

experiment I, culture II with S9 mix

0.144

experiment II, culture I without S9 mix

0.976

experiment II, culture II without S9 mix

0.151

Summary Results

Table 1: Summary of results – Experiment I    

 

 

 

 

 

Dose (µg/mL)

RTG

(%)

Mutant colonies/10E6 cells

RTG

(%)

Mutant colonies/10E6 cells

without metabolic activation

4 hours treatment

Culture 1

Culture 2

SC

-

100

204

100

138

MMS

19.5

38

368

34.8

315

Test item

9.4

185.6

103

92.8

190

Test item

18.8

120.6

127

94.2

188

Test item

37.5 (P)

117.2

155

165.2

118

Test item

75 (P)

126.1

158

102.6

154

Test item

150 (P)

124.9

154

101.8

152

Test item

300 (P)

culture was not continued#

culture was not continued#

 

 

with metabolic activation

4 hours treatment

Culture 1

Culture 2

SC

-

100

148

100

192

CPA

3

36.3

303

20.6

672

CPA

4.5

10.8

833

23.2

578

Test item

9.4

58.5

368

78.4

197

Test item

18.8

69.2

225

96.9

163

Test item

37.5 (P)

97.6

131

134.1

159

Test item

75 (P)

72.8

246

97.7

185

Test item

150 (P)

184.2

116

80.3

235

Test item

300 (P)

culture was not continued#

culture was not continued#

# culture not continued to avoid analysis of too many precipitating concentrations

SC: Solvent control

MMS: Methyl methanesulfonate

CPA: Cyclophosphamide

P: Precipitation of the test material

Table 2: Summary of results – Experiment II

 

 

 

Dose (µg/mL)

RTG

(%)

Mutant colonies/10E6 cells

RTG

(%)

Mutant colonies/10E6 cells

without metabolic activation

24 hours treatment

Culture 1

Culture 2

SC

-

100

103

100

148

MMS

13

26.6

383

26.4

397

Test item

4.7

culture was not continued#

culture was not continued#

Test item

9.4

112.7

183

112.5

131

Test item

18.8

133.8

111

94.8

136

Test item

37.5

56.8

127

64.4

191

Test item

75 (P)

113.5

141

131.9

144

Test item

150 (P)

105.5

130

82.7

196

 

 

with metabolic activation

4 hours treatment

Culture 1

Culture 2

SC

-

100

163

100

214

CPA

3

27

482

48.4

225

CPA

4.5

30.9

569

41.7

423

Test item

4.7

culture was not continued#

culture was not continued#

Test item

9.4

98.5

173

70.1

229

Test item

18.8

105.6

123

59.2

142

Test item

37.5

91.8

176

103.3

102

Test item

75 (P)

122.9

106

70.9

181

Test item

150 (P)

78.4

209

99.6

129

#culture was not continued since a minimum of only four analysable concentrations is required

SC: Solvent control

MMS: Methyl methanesulfonate

CPA: Cyclophosphamide

P: Precipitation of the test material

Conclusions:
Interpretation of results:
negative

Under the conditions of this study, erucamide (CAS 112-84-5) was neither mutagenic nor cytotoxic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo:
Waiving - No in vivo testing required as none of the in vitro genetic toxicity studies were positive for the substance itself (Amides, C16 -C18 (even numbered) or for the structurally related substance CAS 112-84-5.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data on genetic toxicity of Amides, C16-C18 (even numbered) is available from a bacterial reverse mutation assay (Ames test) addressing the mutagenicity of the test material in the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in the absence and presence of metabolic activation (Jones, 1989). As metabolic activation system rat liver S9-mix, prepared from Aroclor 1254-treated rats, was utilised. Although the strain selection is incomplete according to today's standards, as the S. typhimurium strain TA102 or an equivalent is missing, which serve to detect cross-linking agents, it was appropriate at the time of the study. The substance was tested up to the guideline limit concentration of 5 mg/plate. No cytotoxicity was observed up to the highest tested concentration, and no significant increases in revertant numbers occurred, either in absence or in presence of metabolic activation. Therefore, Amides, C16-C18 (even numbered) is not considered as mutagenic in bacteria.

No further data on genetic toxicity was available for Amides, C16-C18 (even numbered). Therefore, read-across data from the structurally related substance erucamide (CAS 112-84-5) was included as read-across for regulatory purposes. For this substance a chromosomal aberration test and a mouse lymphoma study, both conducted in 2010, are available.

In the in vitro chromosomal aberration test, performed according to OECD Guideline 473, Chinese Hamster V79 cells were treated with erucamide (CAS 112-84-5) in the absence and presence of a metabolic activation system consisting of rat liver S9-mix prepared from phenobarbital / β-naphtoflavone-treated rats (Hall, 2010). Two separate experiments were performed, in the first one, which was originally regarded as preliminary test, the cells were exposed for 4 hours to concentrations ranging from 4.9 to 1250 µg/mL in the absence and presence of metabolic activation, in the second experiment the cells were exposed to 2.3 to 450 µg/mL for 18 hours in the absence and to 4.7 to 300 µg/mL for 4 hours in the presence of S9-mix. Structural chromosomal aberrations were assessed by visual inspection.

In the first experiment precipitation was observed after 4 hours incubation without S9 starting at a concentration of 312.5 µg/mL and with S9 after incubation with 625 µg/mL. Therefore, concentrations were adjusted accordingly in the second experiment. In this experiment cytotoxicity was observed at 300 µg/mL after 18 hours incubation without S9; precipitation was noted at 300 µg/mL after 4 hours incubation with S9. No statistically significant or biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed in any of the experiments. The aberration rates were close to the values of the solvent control values and within the range of the historical control data. No increase in polyploid metaphases was observed. Therefore, the test substance erucamide was considered not to induce structural chromosomal or numerical aberrations under the conditions of the study. Considering structural and physical chemical similarities to erucamide it is justified to assume that Amides, C16-C18 (even numbered) does not induce an increase in chromosomal aberrations in cultured mammalian cells, either.

The in vitro mutagenicity test in mammalian cells with erucamide (CAS 112-84-5) was performed with mouse lymphoma L5187Y cells, according to OECD Guideline 476 in the absence and presence of a metabolic activation system consisting of rat liver S9-mix prepared from phenobarbital / β-naphtoflavone-treated rats (Wollny, 2010). Here, the ability of the test substance to induce mutations in the thymidine kinase gene in cultured mammalian cells was investigated in two independent experiments. In the first one, the preliminary test, the cells were exposed to test substance concentrations of 9.4 to 300 µg/mL for 4 hours with and without metabolic activation; in the second experiment the cells were exposed to 4.7 to 150 µg/mL for 4 hours with and for 24 hours without metabolic activation. There was no cytotoxic effect observed up to the maximum concentration in presence and absence of metabolic activation following exposure for 4 and 24 hours, respectively. No significant and reproducible dose dependent increase of mutation frequency was observed in both experiments, and no significant dose dependent trend of the mutation frequency was determined in any of the experimental groups. Under the conditions of the experiment erucamide did not induce mutations in the thymidine kinase locus in the L5178Y cell line. Therefore, it is reasonable and justified to anticipate that Amides, C16-C18 (even numbered) does not induce gene mutations in cultured mammalian cells in vitro either, due to the structural and physical chemical similarities to erucamide.

As there is reliable read-across data for a structurally related substance available addressing clastogenicity and mutagenicity in cultured mammalian cells, the conduction of corresponding studies for Amides, C16-C18 (even numbered) would not provide new information relevant for assessment. Therefore, no further studies for Amides, C16-C18 (even numbered) are proposed.

According to Regulation (EC) No 1907/2006, Annex IX, 8.4, column 2, testing for genetic toxicity in vivo is not indicated as the available data for the substance itself and a structurally related substance did not demonstrate any genotoxic activity in bacteria or mammalian cells in vitro.


Justification for classification or non-classification

According to the criteria of Regulation (EC) No 1272/2008, Amides, C16-C18 (even numbered) does not have to be classified for genetic toxicity.