Amines, di-C16-18-alkyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Sep 2021 - 07 Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: All contained the following additional mutations in: rfa: deep rough (defective LPS cellcoat); gal: galactose metabolism; chl: nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair (deletion of UV repair B gene)

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: the strain lacks an excision repair system and is sensitive to agents such as UV

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

- volume of S9 mix and S9 in the final culture medium: 0.5 mL

- quality controls of S9 : Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Test concentrations with justification for top dose:

Dose Range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Six concentrations, 10, 25, 50, 100, 250 and 500 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assays was the level at which the test item exhibited limited solubility.

First Experiment: Direct plate
The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. Based on the results of the dose-range finding test, the following concentrations were selected for the remaining tester strains:
- TA1535, TA1537 and TA98: 10, 25, 50, 100, 250 and 500 μg/plate (with (5% v/v S9 fraction) and without metabolic activation)

Second Experiment: Direct plate
Based on the results of the first mutation assay, the test item was tested up to the dose level of
400 μg/plate in all tester strains:
- TA1535, TA1537, TA98, TA100 and WP2uvrA: 5, 20, 50, 100, 200, 400 μg/plate (with (10% v/v S9 fraction) and without metabolic activation)
The test item was tested beyond a precipitating dose level in all experiments.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colorless solution THF.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: 10^9 cells/mL
- Test substance added in agar (plate incorporation) - Experiment 1and Experiment 2

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 ± 4 h
- Temperature: 37.0 ± 1.0°C (actual range 36.9 – 39.7°C)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The revertant colonies were counted automatically with the colony counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

Rationale for test conditions:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix.
The test item was soluble in the selected vehicle. The number of cultures and the strains selected are in agreement with the OECD TG 471.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:

a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:

a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Exp. 1 + 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested beyond a precipitating dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Exp. 1 + 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested beyond a precipitating dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Exp.1 + 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested beyond a precipitating dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Exp. 1 + 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested beyond a precipitating dose level

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Exp. 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested beyond a precipitating dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
First Experiment (dose-range finding study is reported as part of the first experiment):
In the dose-range finding test, the test item was tested up to concentrations of 500 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 250 μg/plate and upwards.
In the first mutation assay, test item was tested at a concentration range of 10 to 500 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 100 μg/plate and upwards in tester strain TA1535 and at dose levels of 250 μg/plate and upwards in tester strains TA1537 and TA98.
In a follow-up experiment, the test item was tested at a concentration range of 5 to 400 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested beyond a precipitating dose level.

RANGE-FINDING/SCREENING STUDIES:
The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 10, 25, 50, 100, 250 and 500 μg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 10, 25, 50, 100, 250 and 500 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

- Signs of toxicity
First Experiment (dose-range finding study is reported as part of the first experiment):
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Second Experiment:
In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. In strains TA1537 (presence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range were observed at 200 μg/plate. However, since no dose-relationship was observed, this reduction was not considered to be caused by toxicity of the test item. It is more likely that this reduction is caused by an incidental fluctuation in the number of revertant colonies.

- Mean number of revertant colonies per plate:
No increase in the number of revertants was observed upon treatment with the test item under all conditions tested. See Table 2 to 4 in section "Any other information on results incl. tables"


HISTORICAL CONTROL DATA
- Negative vehicle historical control data: Valid; Please see table 5 in "Any other information on results incl. tables" section.
- Positive historical control data: Valid; Please see table 6 in "Any other information on results incl. tables" section.

Table 2 - Dose-Range Finding Test: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Dose (µg/plate)		TA100				WP2uvrA
Without S9-mix
Positive control		988	±	43		1191	±	32
Solvent control		111	±	11		21	±	9
10		105	±	10		18	±	5
25		103	±	10		30	±	11
50		96	±	24		18	±	4
100		91	±	12	^NP	20	±	9	^NP
250		80	±	23	^SP	30	±	4	^SP
500		81	±	11	^n MP	22	±	5	^n MP
With S9-mix^1
Positive control		1437	±	192		395	±	28
Solvent control		86	±	25		30	±	8
10		75	±	16		31	±	6
25		78	±	7		29	±	10
50		82	±	9		26	±	7
100		80	±	9	^NP	28	±	11	^NP
250		101	±	14	^SP	29	±	9	^SP
500		75	±	7	^n MP	17	±	4	^n MP
Mean number of revertant colonies (3 replicate plates) ± SD is reported
^1	Plate incorporation assay (5% S9)
MP	Moderate Precipitate
NP	No precipitate
SP	Slight Precipitate
n	Normal bacterial background lawn

 

Table 3 - Experiment 1: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay

Dose (µg/plate)			TA1535				TA1537				TA98
Without S9-mix
Positive control		861		±	17		694	±	95		1053	±	78
Solvent control		16		±	6		5	±	2		15	±	8
10		10		±	5		2	±	2		9	±	4
25		7		±	4		2	±	1		16	±	6
50		14		±	2	^NP	2	±	3		19	±	5
100		13		±	5	^SP	2	±	3	^NP	15	±	3	^NP
250		6		±	2	^MP	8	±	6	^SP	13	±	4	^SP
500		8		±	1	^n MP	4	±	3	^n SP	15	±	5	^n SP
With S9-mix^1
Positive control		286		±	44		236	±	44		578	±	156
Solvent control		19		±	1		4	±	3		12	±	3
10		6		±	3		4	±	3		13	±	6
25		6		±	3		2	±	2		15	±	9
50		9		±	2		2	±	2	^n	22	±	2
100		10		±	7	^SP	5	±	6	^NP	13	±	3	^NP
250		4		±	1	^MP	5	±	4	^SP	11	±	4	^SP
500		5		±	1	^n MP	3	±	2	^n SP	17	±	8	^n SP
Mean number of revertant colonies (3 replicate plates) ± SD is reported
^1	Plate incorporation assay (5% S9)
MP	Moderate Precipitate
NP	No precipitate
SP	Slight Precipitate
n	Normal bacterial background lawn

 

Table 4 - Experiment 2: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Dose		TA1535				TA1537				TA98				TA100				WP2uvrA
(µg/plate)
Without S9-mix
Positive control		851	±	79		1128	±	118		928	±	199		825	±	79		1696	±	52
Solvent control		11	±	6		8	±	4		12	±	2		107	±	11		21	±	8
5		12	±	7		6	±	2		16	±	4		108	±	21		25	±	7
20		12	±	5	^NP	4	±	2	^NP	12	±	4	^NP	118	±	15		21	±	4	^NP
50		10	±	4	^SP	3	±	2	^SP	21	±	5	^SP	111	±	13	^NP	20	±	5	^SP
100		8	±	2	^SP/MP	6	±	4	^SP	14	±	4	^SP/MP	115	±	11	^SP	23	±	5	^SP
200		7	±	4	^MP	3	±	1	^MP	10	±	4	^MP	102	±	16	^SP	22	±	4	^MP
400		8	±	2	^n MP	2	±	2	^n MP	7	±	2	^n MP	111	±	14	^SP	15	±	8	^n MP
With S9-mix^1
Positive control		151	±	14		255	±	40		608	±	66		646	±	159		281	±	40
Solvent control		10	±	3		3	±	2		12	±	3		62	±	11		25	±	6
5		7	±	2		5	±	2		16	±	3		53	±	16		24	±	2
20		9	±	3		6	±	3		23	±	1		60	±	4		20	±	7	^NP
50		10	±	2		4	±	1		19	±	0		64	±	16	^NP	26	±	7
100		7	±	7	^NP	4	±	4	^NP	22	±	6	^NP	66	±	9	^SP	30	±	5	^SP
200		10	±	6	^SP/MP	1	±	2	^n MP	15	±	3	^MP	65	±	6	^SP	32	±	13	^SP
400		6	±	1	^n MP	3	±	2	^n MP	10	±	3	^n MP	75	±	7	^n SP	39	±	8	^n SP
Mean number of revertant colonies (3 replicate plates) ± SD is reported
^1	Plate incorporation assay (10% S9)
MP	Moderate Precipitate
NP	No precipitate
SP	Slight Precipitate
n	Normal bacterial background lawn

Table 5 - Historical Control Data of the Solvent Control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 26	3 – 23	2 – 24	2 – 20	3 – 61	5 – 60	58 – 188	46 – 176	9 – 61	9 – 68
Mean	9	10	5	5	13	18	106	98	23	26
SD	3	3	2	2	5	6	20	23	9	10
Total number of plates	2215	2193	2219	2220	2301	2337	2365	2293	2155	2146

SD = Standard deviation

Historical control data from experiments performed between May 2018 and May 2021.

 

Table 6 – Historical Control Data of the Positive Control Items

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	107 – 1425	78 – 1481	64 – 1475	48 – 1843	379 – 2118	272 – 3369	173 – 1852	371 – 2666	93 – 2027	109 – 1968
Mean	924	285	829	282	1310	933	832	1403	1253	415
SD	167	119	367	156	299	396	185	409	472	203
Total number of plates	2073	2072	1710	2094	2209	2155	2178	2156	2062	2032

SD = Standard deviation

Historical control data from experiments performed between May 2018 and May 2021.

 

Conclusions:

The results of an AMES test, performed according to OECD TG 471 and in accordance with GLP principles, showed that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In an Ames test, performed according to OECD TG 471 and in accordance with GLP principles, the test item was assessed for its potential to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and at the tryptophan locus of one Escherichia coli strain (WP2uvrA).

The test was performed in two independent direct plate experiments. The vehicle of the test item was tertrahydrofuran.

In the dose-range finding test, the test item was tested up to concentrations of 500 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 250 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.
Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 10 to 500 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 100 μg/plate and upwards in tester strain TA1535 and at dose levels of 250 μg/plate and upwards in tester strains TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 5 to 400 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
The test item did not induce a dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
In conclusion, based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-10 to 2009-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 1.5, 2.6, 4.6, 8.0, 14.0, 24.5, 42.9, 75.1, 131.4, 230.0 µg/mL
Experiment II: 8.0, 14.0, 24.5, 42.9, 75.1, 131.4, 230.0 µg/mL

Without metabolic activation:
Experiment I: 1.5, 2.6, 4.6, 8.0, 14.0, 24.5, 42.9, 75.1, 131.4, 230.0 µg/mL
Experiment II: 4.6, 8.0, 14.0, 24.5, 42.9, 75.1, 131.4, 230.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion 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 scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item Amines, bis(hydrogenated tallow alkyl), dissolved in THF, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphase plates per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 230 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the solubility properties of the test item in an appropriate solvent (THF).
In Experiment I, visible precipitation of the test item in the culture medium was observed at 42.9 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence and presence of S9 mix, at 24.5 µg/mL and above. No relevant increase in the osmolarity or pH value was observed (Exp. I: solvent control: 382 mOsm, pH 7.4 versus 371 mOsm and pH 7.3 at 230 µg/mL; Exp. II: solvent control: 360 mOsm, pH 7.5 versus 370 mOsm and pH 7.4 at 230 µg/mL).
No relevant cytotoxicity, indicated by reduced mitotic indices could be observed in Experiment I and II, up to the highest applicable concentration.
In both experiments, in the absence and presence of S9 mix, no 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 - 2.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data. A single significant increase was observed in Experiment II, in the presence of S9 mix after treatment with 24.5 µg/mL (2.0 % aberrant cells, excluding gaps). The value is in the range of the laboratory´s historical solvent control data (0.0 – 2.5 % aberrant cells, excluding gaps) and has therefore to be regarded as being biologically irrelevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (660.0 or 825.0 µg/mL) or CPA (7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosomal aberration study with
Amines, bis(hydrogenated tallow alkyl)

Exp.		Preparation			Test item	Mitotic indices	Aberrant cells
		interval			concentration	in %	in %
					in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
	Exposure period 4 hrs without S9 mix
I		22 hrs			Solvent control^1	100.0	1.5	1.5	0.0
					Positive control^2	89.0	11.0	11.0^S	2.5
					14.0	103.5	0.5	0.5	0.0
					24.5	95.3	2.0	1.0	0.0
					42.9^P	97.2	0.0	0.0	0.0
			Exposure period 22 hrs without S9 mix
II		22 hrs			Solvent control^1	100.0	1.5	1.5	0.0
					Positive control^3	22.9	17.5	17.5^S	2.5
					8.0	90.5	1.0	0.5	0.0
					14.0	97.8	1.0	1.0	0.0
					24.5^P	100.4	1.0	1.0	0.0
				Exposure period 4 hrs with S9 mix
I		22 hrs			Solvent control^1	100.0	1.5	1.5	0.0
					Positive control^4	82.1	14.5	14.0^S	3.5
					14.0	100.3	0.5	0.5	0.0
					24.5	91.1	0.0	0.0	0.0
					42.9^P	94.7	2.0	2.0	0.0
II		22 hrs			Solvent control^1	100.0	0.0	0.0	0.0
					Positive control^4	68.2	10.5	10.5^S	0.5
					8.0	99.2	1.0	1.0	0.0
					14.0	94.6	0.0	0.0	0.0
					24.5^P	92.6	2.0	2.0^S	0.5

*  Including cells carrying exchanges

^P  Precipitation occurred

^S  Aberration frequency statistically significant higher than corresponding control values

¹   THF     0.5 % (v/v)

²     EMS 825.0µg/mL
³      660.0µg/mL
⁴   CPA     7.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations.

Executive summary:

The test item Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4), dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations.

The highest applied concentration in this study (230 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The chosen treatment concentrations and the rationale for the dose selection are reported in Table1. The evaluated experimental points and the results are summarised in Table2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applicable concentration being far in the range of test item precipitation.

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment II, in the presence of S9 mix, a single statistically significant increase was observed after treatment with 24.5 µg/mL (2 % aberrant cells, excluding gaps). Since the value is within the laboratory’s historical solvent control data range the finding has to be regarded as being biologically irrelevant.

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix (4 hours treatment): 0.9; 1.8; 3.6; 7.2; 14.4; 28.8 µg/mL
with S9 mix (4 hours treatment): 0.9; 1.8; 3.6; 7.2; 14.4; 28.8 µg/mL
Experiment II:
without S9 mix (24 hours treatment): 0.9; 1.8; 3.6; 7.2; 14.4; 28.8 µg/mL
with S9 mix (4 hours treatment): 1.8; 3.6; 7.2; 14.4; 28.8; 57.6 µg/mL
Following the expression phase of 48 hours the cultures at the lowest concentration of all experimental parts were not continued since a minimum of only four analysable concentrations is required by the guidelines and at least three of the analysed concentrations should be in the soluble range.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF; tetrahydrofurane
- Justification for choice of solvent/vehicle: Solubility properites

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofurane

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofurane

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment I; 24 hours without metaoblic activation and 4 hours with metabolic activation in experiment II
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the corresponding solvent control or negative control, respectively.
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 negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 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:

Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Precipitation:
In the pre-experiment: at 14.4 µg/mL and above with and without metabolic activation at 4 hours and 24 hours treatment
In experiment I without metabolic activation: at 14.4 µg/mL and above
In experiment I with metabolic activation: at 28.8 µg/mL
In experiment II without metabolic activation: at 14.4 µg/mL and above
In experiment II with metabolic activation: at 14.4 µg/mL and above

- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
According to the results of the pre-test at least four adequate concentrations were chosen for the main experiments.
The highest concentration should be 10 mM, but not higher than 5 mg/mL of the pure substance, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20 %.
The highest concentration used in the pre-test was chosen with regard to the solubility of the test item in THF. Test item concentrations between 1.8 and 230 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Relevant cytotoxic effects were solely noted at the two highest concentrations following 24h exposure to the test item without metabolic activation. Heavy precipitation occurred at those test points.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed at 14.4 µg/mL and above in all parts of the pre-experiment.
Therefore, the maximum concentration of experiments I and II was adjusted to the solubility data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.
To overcome problems with possible deviations in toxicity or solubility both main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA:
within the range of the historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

Remarks on result:

other: strain/cell type: in vitro gene mutation assay with L5178Y cells

Remarks:

Migrated from field 'Test system'.

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with THF		-	100.0	113	239	100.0	83	209
Pos. control with MMS	19.5	-	45.1	269	239	59.5	271	209
Test item	0.9	-	culture was not continued^#			culture was not continued^#
Test item	1.8	-	68.8	130	239	107.9	86	209
Test item	3.6	-	76.2	136	239	89.5	82	209
Test item	7.2	-	76.2	103	239	113.2	114	209
Test item	14.4 (p)	-	72.1	131	239	128.4	77	209
Test item	28.8 (p)	-	65.4	143	239	101.8	83	209
Solv. control with THF		+	100.0	177	303	100.0	173	299
Pos. control with CPA	3.0	+	30.0	412	303	54.6	217	299
Pos. control with CPA	4.5	+	5.8	809	303	21.3	314	299
Test item	0.9	+	culture was not continued^#			culture was not continued^#
Test item	1.8	+	46.4	211	303	121.1	135	299
Test item	3.6	+	65.5	153	303	100.8	160	299
Test item	7.2	+	51.8	248	303	83.1	202	299
Test item	14.4	+	47.5	123	303	99.2	150	299
Test item	28.8 (p)	+	30.8	189	303	67.2	196	299
Experiment II / 24 h treatment			culture I			culture II
Solv. control with THF		-	100.0	123	249	100.0	180	306
Pos. control with MMS	13.0	-	36.0	407	249	20.5	700	306
Test item	0.9	-	culture was not continued^#			culture was not continued^#
Test item	1.8	-	95.0	145	249	58.1	271	306
Test item	3.6	-	79.2	157	249	80.9	271	306
Test item	7.2	-	68.0	161	249	62.6	246	306
Test item	14.4 (p)	-	45.0	210	249	63.6	277	306
Test item	28.8 (p)	-	81.8	119	249	62.5	267	306
Experiment II / 4 h treatment			culture I			culture II
Solv. control with THF		+	100.0	169	295	100.0	173	299
Pos. control with CPA	3.0	+	39.8	299	295	48.5	514	299
Pos. control with CPA	4.5	+	21.8	471	295	24.5	666	299
Test item	1.8	+	culture was not continued^#			culture was not continued^#
Test item	3.6	+	87.0	172	295	104.2	156	299
Test item	7.2	+	109.7	127	295	78.5	251	299
Test item	14.4	+	87.4	159	295	95.8	158	299
Test item	28.8 (p)	+	90.9	159	295	71.1	155	299
Test item	57.6 (p)	+	78.3	194	295	87.7	157	299

Threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#    culture was not continued since a minimum of four concentrations is required by the guidelines

(p)  precipitation visible to the unaided eye

 

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:                       1.8; 3.6; 7.2; 14.4; and 28.8 µg/mL
with S9 mix:                            1.8; 3.6; 7.2; 14.4; and 28.8 µg/mL

Experiment II:

without S9 mix:                       1.8; 3.6; 7.2; 14.4; and 28.8 µg/mL
with S9 mix:                          3.6; 7.2; 14.4; 28.8; and 57.6 µg/mL

Precipitation of the test item visible to the naked eye was noted at 14.4 µg/mL and above in the first experiment without metabolic activation and at 28.8 µg/mL in the first experiment with metabolic activation. In the second experiment precipitation as described above occurred at 14.4 µg/mL and above without and at 28.8 µg/mL and above with metabolic activation.

No relevant toxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were 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 any of the experimental parts. The threshold of 126 above the corresponding solvent control was not reached or exceeded.

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

In this study the range of the solvent controls was from 83 up to 180 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 77 up to 277 mutant colonies per 10⁶cells. The highest solvent control values slightly exceeded the recommended 50 – 170 x 10⁶control range as stated under paragraph 8.12, acceptability of the assay of this report. However, the deviation was rather minor with a maximum of just 10 colonies per 10⁶cells. The absolute values of the mutation frequency still remained within the historical control range and below an upper limit of 200 colonies per 10⁶cells originally recommended by the IWGT (11). The cloning efficiency exceeded the upper limit of 120% in the solvent controls of the first experiment without metabolic activation, in the first culture of the second experiment without metabolic activation, and in the second culture of the second experiment with metabolic activation. This deviation was judged as irrelevant since it either was very minor (124 and 127% in the first experiment without metabolic activation) or occurred in only one of both parallel cultures (in culture I of the second experiment without metabolic activation and in culture II of the second experiment with metabolic activation). Cloning efficiency values above 100% occur since the cell suspension does not form an ideal solution. Even though suspension cell cultures do not readily adhere to solid surfaces, the cells tend to form transient aggregates of several cells that are counted as single cells upon cell counting. These aggregates do not affect the validity of the data however, since the absolute values of the cloning efficiency are used in the calculation of the mutation frequency. The total suspension growth of the solvent control of the first culture of the second experiment without metabolic activation fell short of the acceptable range of 32-180 but the total suspension growth of the parallel culture remained within the acceptable range.

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 in experiment I and II) 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.

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Additional information

Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was tested for genetic toxicity in three types of in vitro tests. All the three tests showed no indications on genetic toxicity while assessing for a varity of genotoxic endpoints.

In a key study (CRL, 2022) conducting the bacterial reverse mutation assay (Ames test) showed that the substnace did not cause any substantial increase in the number of revertant colonies of any of the five strains at any dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experimentThe result from the study is considered to be relevant and is used in the classification of the substance.

The supporting study (Jones and Fenner, 1987) bacterial reverse mutation assay (Ames test) showed that the substance did not cause any substantial increase in revertant colony numbers of any of the five tested strains at any dose level, either in the presence or absence of metabolic activation (S-9 mix). No evidence of mutagenic potential was seen in this study.This study is rated as reliability 2 since the substance is poorly described and no specific certificate of analysis or information on batch is included in the report. Despite this, the result from this study is considered to be reliable, since the product tested has not changed significantly in its composition since the testing was carried out. Therefore the results are considered valid for the current manufactured Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4). Another notation on this study is that not all current required strains are included (E.coli WP2 or S. typhimurium TA102) and that 2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. 

 

In the test on chromosomal aberrations in human lymphocytes, reliability rating 1, Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was not clastogenic, both in absence and presence of metabolic activation by S9 mix.

Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) did not either induce mutations in the mouse lymphoma thymidine kinase locus (TK+/-) assay with reliability rating 1. The assay was performed both in absence or presence of metabolic activiation (S-9 mix).

Based on the results from these three studies, it can be concluded that Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) would not be expected to have any genotoxic hazard to human health.

 

Short description of key information:

Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) was tested in three in-vitro genotoxicty studies. All the three tests showed no indications on genetic toxicity.

 

The first tests on Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) is the bacterial reverse mutation assay (Ames test) compliant to OECD Guideline 471. The primary key study is rated Klemish score of 1 as the test sample used is a direct representitive compostion of Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) and adheres to TG 471 without any major deviations. No evidence of mutagenic potential was seen in this study. The supporting study is rated as reliability 2 since the substance is poorly described and no specific certificate of analysis or information on batch is included in the report. Another notation on this study is that not all current required strains are included (E.coli WP2 or S. typhimurium TA102) and 2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. No evidence of mutagenic potential was seen in this study.

 

The second available study on genetic toxicity is chromosome aberration test in human lymphocytes in vitro, (OECD Guideline 473), which is GLP compliant and has reliability rating 1. No evidence of clastogenisity was seen in this study.

 

The third genetic toxicity study on Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4) is mouse lymphoma assay according to OECD Guideline 476. This study is performed according to GLP and has a reliability rating 1 and did not induce mutations in the mouse lymphoma thymidine kinase locus (TK+/-).

 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the three negative in-vitro tests for genotoxicity, Amines, di-C16-18 (even-numbered) alkyl (CAS No. 308062-60-4), does not require classification as a mutagen according to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008.