Docosanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:
Gene mutation in bacteria (Reverse Mutation Test, OECD 471): negative, based on read-across from CAS 112-84-5
Cytogenicity in mammalian cells (Chromosomal Aberration, OECD 473): negative, based on read-across from CAS 112-84-5
Gene mutation in mammalian cells (Mouse Lymphoma Assay, OECD 476): negative, based on read-across from CAS 112-84-5

Link to relevant study records

Reference

Reference 1

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:

Experiment I: 4.9 to 1250 µg/mL (±S9)
Experiment II: 2.3 to 450 µg/mL (-S9) and 4.7 to 300 µg/mL (+S9)

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 activation: 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 activation: 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 5E+05 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 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:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9 mix: noted at 300 µg/ml after 18 hour incubation in Experiment II

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. 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 control^1	100.0	100.0	3.0	1.5	0.5
			Positive control^2	n.t.	104.9	22.0	21.0^S	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
			312.5^P##	84.8	76.4	4.0	3.8	1.3
		Exposure period 18 hrs without S9 mix
II	18 hrs		Solvent control^1	100.0	100.0	3.0	2.5	0.0
			Positive control^3#	n.t.	40.3	42.0	40.0^S	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 control^1	100.0	100.0	1.5	1.5	0.5
			Positive control^4	n.t.	52.3	9.5	8.5^S	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
			625.0^P	90.5	78.2	2.5	1.5	0.0
II	18 hrs		Solvent control^1	100	100	0.5	0.5	0.0
			Positive control^4	n.t.	90.4	13.5	12.0^S	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
			300.0^P	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:

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 inV79 cells of the Chinese hamster in vitro in 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 in V79 cells (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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

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 done according to accepted protocol. No COA for the batch tested was provided; however, sponsor has provided a letter confirming that the test article is representative of a substance of which the exact composition is available. The recommended strains TA 102 or E. coli WP2 were not tested. Only one positive control substance was tested in the presence of S9 mix. Deviations in accordance with guideline at time of study conduction.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

the recommended strains TA 102 or E. coli WP2 were not tested; only one positive control substance was tested in the presence of S9 mix; deviations in accordance with guideline at time of study conduction

GLP compliance:

yes (incl. QA statement)

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

Additional strain / cell type characteristics:

other: uvrB defective, defective lipopolysaccharide barrier on the cell wall (rfa)

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB defective, defective lipopolysaccharide barrier on the cell wall (rfa)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S-9, Aroclor-induced

Test concentrations with justification for top dose:

Pre-experiment:
- 5, 50, 500, 5000 µg/plate
Main experiment:
- 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Remarks:

-S9: ENNG (3 µg/plate TA 100; 5 µg/plate TA 1535), 2-NF (1 µg/plate: TA 98; 2 µg/plate TA 1538), 9-AA (80 µg/plate TA 1537); +S9: 2-AA (0.5 µg/plate TA 1538 and TA 98; 1 µg/plate: TA 100; 2 µg/plate: TA 1535 and TA 1537)

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Remarks:

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine; 2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
A cytotoxicity pre-experiment was carried out with the tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 to determine the nontoxic concentrations for the main genotoxicity experiments.
Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity (relative total growth) and precipitate by using an automatic colony counter.

Evaluation criteria:

A test system is considered as mutagenic if
- there is a clear and dose-related increase in the number of revertants and/or a
- biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

Statistics:

The mean and standard deviation of the number of revertants per plate were calculated and are reported.

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

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

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

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Table 1: Revertant colony numbers - Experiment I

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
–	Solvent	13 ± 2	14 ±1	10 ± 2.5	30 ± 5.6	84 ± 6
–	0	12 ± 4.9	15 ± 2.5	8 ± 2	29 ± 1.7	79 ± 3.8
–	50	15 ± 4	17 ± 1.7	9 ± 2	27 ± 3.5	87 ± 11.4
–	150	16 ± 3.6	16 ± 1.7	10 ± 0.6	26 ± 1	96 ± 9.1
–	500 P	14 ± 2.5	12 ± 1	11 ± 2.9	28 ± 0.7	90 ± 8.1
–	1500 P	13 ± 2.5	14 ± 2.5	8 ± 0.6	25 ± 2.1	88 ± 2.6
–	5000 P	8 ± 1.5	15 ±1.5	6 ± 2.1	22 ± 2.1	80 ± 6
Positive controls, –S9	Name	ENNG	9AA	NF	NF	ENNG
	Concentrations (μg/plate)	5	80	2	1	3
	Mean No. of colonies/plate (average of 3 ± SD)	137 ± 12.6	797 ± 38.7	50 ± 6	76 ± 5.8	356 ± 14.6
+	Solvent	13 ± 2.1	10 ± 0.6	11 ± 0.6	22 ± 3.6	86 ± 3.6
+	0	11 ± 3.2	10 ± 2	8 ± 1	21 ± 1.2	78 ± 4.6
+	50	16 ± 2.3	10 ± 1.5	13 ± 2.5	25 ± 1.5	95 ± 9.5
+	150	15 ± 2.1	12 ± 1	10 ± 4	23 ± 2.8	90 ± 4.4
+	500 P	11 ± 4.9	12 ± 1.5	10 ± 2.1	20 ± 2.5	84 ± 6
+	1500 P	12 ± 1.5	10 ± 1.5	7 ± 1	20 ± 1.5	83 ± 4.6
+	5000 P	10 ± 1.5	6 ± 2.6	9 ± 2.5	16 ± 2.6	76 ± 4
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	0.5	0.5	1
	Mean No. of colonies/plate (average of 3 ± SD)	99 ± 22.1	95 ± 5.7	122 ± 24.4	116 ± 11.9	369 ± 41.6

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

NF = 2-nitrofluorene

P = Precipitate

Table 2: Revertant colony numbers - Experiment II

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		TA 1535	TA 1537	TA 1538	TA 98	TA 100
–	Solvent	13± 0.6	12± 0.6	12± 1	24±3.2	107± 10.7
–	0	12± 3.1	11± 1.2	11± 1.2	20± 1	98± 5
–	50	13± 1.2	11± 1.7	11± 2.3	20± 2.6	92± 8.1
–	150	11± 1.5	14± 2	9± 2.6	24± 2	99± 14.4
–	500 P	10± 2.6	10± 1.5	11± 1	24± 3.8	105± 4.6
–	1500 P	11± 1.5	9± 0.6	11± 2	15± 4	89± 11
–	5000 P	10± 2.9	9± 2.1	8± 1.2	22± 1.5	75± 9.8
Positive controls, –S9	Name	ENNG	9AA	NF	NF	ENNG
	Concentrations (μg/plate)	5	80	2	1	3
	Mean No. of colonies/plate (average of 3 ± SD)	714 ± 143.7	1190 ± 105.8	62 ± 19.7	103 ± 3.5	512 ± 22.9
+	Solvent	15± 2.5	16± 5.8	11± 2	23± 2	112± 2.5
+	0	11± 1	13± 2.5	12± 0.6	20± 1.5	100± 5.6
+	50	16± 3.2	11± 2.1	13± 3.6	24± 3	119± 7
+	150	10± 1.5	14± 1.2	11± 1.2	26± 3.1	108± 21.1
+	500 P	9± 4	10± 4.6	10± 2	22± 3.5	107± 16.3
+	1500 P	14± 3.2	13± 1.5	14± 0.6	23± 3.8	98± 4
+	5000 P	12± 1.5	11± 4	8± 1.7	20± 2.9	94± 13.5
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	0.5	0.5	1
	Mean No. of colonies/plate (average of 3 ± SD)	88± 9.8	71± 4.2	106± 16.6	141± 5.9	363± 56.1

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

NF = 2-nitrofluorene

P = Precipitate

Conclusions:

When tested at dose levels up to 5000 µg/plate in tetrahydrofuran, erucamide was not mutagenic in this bacterial test system.

Executive summary:

In this in vitro assessment of the mutagenic potential of erucamide, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to the test material diluted in tetrahydrofuran, which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150 and 50 µg/plate.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

No evidence of mutagenic activity was seen at any dose level of erucamide in either mutation test.

It is concluded that, when tested at dose levels up to 5000 µg/plate in tetrahydrofuran, erucamide was not mutagenic in this bacterial test system.

Reference 3

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. Reliability was changed from "1" to "2" according to the ECHA guidance document "Practical guide 6: How to report read-across and categories". Complete test material characterization is available.

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 1E+06 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 50-170E+06 control range in the study report. However, the number of mutant colonies per 106cells in the parallel cultures (138, 148, and 163 mutant colonies/1E+06 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.

Statistical Analysis

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT^â11statistics 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 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

There is no data available on the genetic toxicity of docosanamide (CAS 3061-75-4).

In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance is conducted.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).

In vitro

There exist three reliable studies on genetic toxicity in vitro of the structurally related substance (Z)-Docos-13-enamide (CAS 112-84-5, erucamide), which is used for read across based on the analogue approach.

- Gene mutation in bacteria

The mutagenic potential of (Z)-Docos-13-enamide

(CAS 112-84-5) was assessed in a GLP-compliant gene mutation assay in bacteria (Ames test) performed similar to OECD 471 (Jones, 1990). In two independent experiments, the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to the test material diluted in tentrahydrofuran in the presence and absence of metabolic activation (S9 mix). Since no cytotoxicity was observed in a preliminary range finding study up to 5000 µg/plate, concentrations of 50, 150, 500, 1500 and 5000 µg/plate were used for treatment of bacteria in the main experiments. Likewise, no cytotoxic effects were observed in any tester strains at any concentration in the main experiments. No evidence of mutagenic activity, indicated by similar revertant colonies between treated bacteria and controls, was observed at any concentration in either mutation test in the presence and absence of S9 mix. The concurrent positive and solvent controls demonstrated the expected results, and thus verified the sensitivity of the assay. Based on these results, erucamide was not considered mutagenic in the presence and absence of metabolic activation in the selected strains of S. typhimurium.

- Chromosome aberrations

In the in vitro chromosomal aberration test, performed according to OECD Guideline 473, Chinese Hamster V79 cells were treated with (Z)-Docos-13-enamide (CAS 112-84-5) (Hall, 2010). The treatment of cells was carried out in two independent experiments in the absence and presence of a metabolic activation system (S9 mix). In the first experiment, which was originally regarded as preliminary test, the cells were exposed for 4 h to concentrations ranging from 4.9 to 1250 µg/mL in the absence and presence of metabolic activation. In the second experiment, continuous treatment of cells was performed by exposure to 2.3 to 450 µg/mL for a period of 18 h in the absence and to 4.7 to 300 µg/mL for 4 h in the presence of S9-mix. Structural chromosomal aberrations were assessed by visual inspection. Precipitation was observed in the first experiment after 4 h incubation without S9 mix starting at a concentration of 312.5 µg/mL and with S9 mix 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 h incubation without S9 mix; precipitation was noted at 300 µg/mL after 4 h 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.

- Gene mutation in mammalian cells

The in vitro mutagenicity test in mammalian cells with (Z)-Docos-13-eanmide (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 (Wollny, 2010). 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 experiment, which was performed as preliminary cytotoxicity test, the cells were exposed to concentrations of 9.4 to 300 µg/mL for 4 h with and without metabolic activation (S9 mix). In the second experiment, the cells were treated with concentrations ranging from 4.7 to 150 µg/mL for 4 h with and for 24 h without metabolic activation. No cytotoxic effect was observed up to the maximum concentration in the presence and absence of metabolic activation following exposure for 4 and 24 h, respectively. No significant and reproducible dose dependent increase of mutation frequency was noted 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.

In vivo

No study investigating the genetic toxicity in vivo of docosanamide (CAS 3061-75-4) is available. However, there exist reliable data on genetic toxicity in vitro of the structurally related substance (Z)-Docos-13-enamide (CAS 112-84-5), which is used for read across based on the analogue approach.

According to Regulation (EC) No 1907/2006, Annex IX, column 2, testing for genetic toxicity in vivo is not indicated if no genotoxic activity is demonstrated in bacteria or mammalian cells in vitro. Due to the availability of reliable negative in vitro studies for the structurally related substance (Z)-Docos-13-enamide (CAS 112-84-5), in vivo testing is not indicated for docosanamide (CAS 3061-75-4), either.

 

Based on the negative results of the available in vitro studies on the structural analogue (Z)-Docos-13-enamide (CAS 112-84-5), it may be concluded that the substance docosanamide (CAS 3061-75-4) does not induce genetic toxicity in vitro and in vivo, either.

Justification for classification or non-classification

The available data on genetic toxicity of erucamide (CAS 112-84-5), which is structurally very closely related to docosanamide (CAS 3061-75-4) according to Regulation (EC) No 1907/2006, Annex XI, 1.5, do not meet the criteria for classification according to Regulation (EC) No 1272/2008; therefore, docosanamide (CAS 3061-75-4) is not expected to exert a genotoxic potential, either, and the data are thus conclusive but not sufficient for classification.