Amines, C16-22-alkyl

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-02 to 2011-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C16-22-alkyl

EC Number:

268-215-4

EC Name:

Amines, C16-22-alkyl

Cas Number:

68037-92-3

Molecular formula:

CnH2n+3N with n={16; 18; 20; 22}

IUPAC Name:

C16-22-(even numbered)-alkyl-1-amine

Details on test material:

Name: C16-22-(even numbered)alkylamines
CAS No.: 68037-92-3
Chemical Name: C16-22-(even numbered)alkylamines
Batch No.: S001453
Physical State at RT: solid
Colour: white to yellow
pH: 11.5 at 5% in ethanol:water (1:1) DIN EN 1262
Active Components: (for further details see CoA dated 12.07.2011, attached in the appendix of the report)
Date of Analysis: 12 July 2011
Storage Conditions: put nitrogen gas into bottle before closing it to prevent test item from oxidizing
Expiry Date: at least to 20180421

Method

Target gene:

The five strains of Salmonella typhimurium (a): TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
• the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of
the bacteria and increases permeability to large molecules that do not penetrate the normal
bacteria cell wall,
• the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which
renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
• the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their
sensitivity of detection to some mutagens,
• in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Determination of dose was determined in pre-experiment of r toxicity.
Experiment I:
3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
Experiment II:
1.58, 5.00, 15.8, 50.0, 158 and 500 µg/plate
(TA 98 without metabolic activation)
1.58, 5.00, 15.8, 50.0, 158, 500 and 1000 µg/plate
(all tester strains except TA 98 without metabolic activation)
The toxicity of the test item was determined with testerstrainsTA98andTA100inapre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background lawn or are duction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control. The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol ApplichemLotNo.1R002540
- Negative controls (A.dest., BSL BIOSERVICE Lot No. 110728, 110927,111010)

Controls

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 110728, 110927, 111010 - All tester strains

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol, Lot No. Applichem Lot No. 1R002540 - All tester strains

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used (in three cases only two plates were evaluated)


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs 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.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
SD of means derived for all test items and controls.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was observed in all tester strains used in experiment I and II. In experiment I precipitation of the test item was found at a concentration of 316 ug/plate and higher (with and without metabolic activation), in experiment II precipitation of the test item was found at a concentration of 500 ug/plate and higher (with and without metabolic activation).
Toxic effects of the test item were noted in all tester strains evaluated in experiment I and ll.
In experiment I toxic effects of the test item were observed in tester strain TA 98 at a concentration of 1000 ug/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were seen at a concentration Of 1000 ug/plate (without metabolic activation). In tester strains TA 1535, TA 1537 and TA 102 toxic effects of the test item were noted at concentrations of 316 ug/plate and higher (without metabolic activation).
In experiment Il toxic effects of the test item were noted in tester strain TA 98 at a concentration Of 500 ug/plate (without metabolic activation) and at a concentration Of 1000 pg/plate (with metabolic activation). In tester strains TA 100 and TA 1535 toxic effects of the test item were noted at concentrations of 158 ug/plate and higher (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 500 ug/plate and higher (without metabolic activation) and at a concentration of 1000 ug/plate (with metabolic activation). In tester strain TA 102 toxic effects Of the test item were observed at a concentrations Of 1000 ug/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment II in tester strain TA 98 at a concentration of 50.0 ug/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship, No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with C16-22-(even numbered)alkylamines at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a ,distinct increase of revertant colonies indicating the validity of the experiments.

Experiment 1:

TA 98 untreated controls, A. dest., showed a mean revertant count per plate of 22 (SD 7.5) and 18 (SD 4) with and without S9 respectively. TA 98 negative controls, EtOH, showed a mean revertant count per plate of 27 (SD 1) and 21 (SD 4) with and without S9 respectively. The highest mutation factor was 1.2 (3.16, 10 31.6 ug) and 1.1 (3.16 ug) with and without S9 respectively. Positive control of 4-NOPD without S9 resulted in a mutation factor of 16.0. Positive control of 2-AA with S9 resulted in a mutation factor of 93.5.

TA 100 untreated controls, A. dest., showed a mean revertant count per plate of 113 (SD 7) and 110 (SD 13.2) with and without S9 respectively. TA 100 negative controls, EtOH, showed a mean revertant count per plate of 102 (SD 12.7) and 97 (SD 21.7) with and without S9 respectively. The highest mutation factor was 1.1 (316 ug) and 1.3 (100 ug) with and without S9 respectively. Positive control of NaN3 without S9 resulted in a mutation factor of 12.6. Positive control of 2-AA with S9 resulted in a mutation factor of 26.5.

TA 1535 untreated controls, A. dest., showed a mean revertant count per plate of 8 (SD 1.7) and 11 (SD 1.5) with and without S9 respectively. TA 1535 negative controls, EtOH, showed a mean revertant count per plate of 13 (SD 6.4) and 9 (SD 2) with and without S9 respectively. The highest mutation factor was 1.4 (3.16 ug) and 1.4 (10 ug) with and without S9 respectively. Positive control of NaN3 without S9 resulted in a mutation factor of 111.7. Positive control of 2-AA with S9 resulted in a mutation factor of 15.2.

TA 91537 untreated controls, A. dest., showed a mean revertant count per plate of 7 (SD 2.6) and 5 (SD 1.5) with and without S9 respectively. TA 1537 negative controls, EtOH, showed a mean revertant count per plate of 7 (SD 3.5) and 7 (SD 3.1) with and without S9 respectively. The highest mutation factor was 1.9 (100 ug) and 1.6 (10 ug) with and without S9 respectively. Positive control of 4-NOPD without S9 resulted in a mutation factor of 11.3. Positive control of 2-AA with S9 resulted in a mutation factor of 43.2.

TA 102 untreated controls, A. dest., showed a mean revertant count per plate of 340 (SD 21.1) and 268 (SD 19.0) with and without S9 respectively. TA 102 negative controls, EtOH, showed a mean revertant count per plate of 306 (SD 35.7) and 272 (SD 19.3) with and without S9 respectively. The highest mutation factor was 1.3 (10 and 31.6 ug) and 1.1 (3.16 ug) with and without S9 respectively. Positive control of MMS without S9 resulted in a mutation factor of 5.5. Positive control of 2-AA with S9 resulted in a mutation factor of 2.7.

Experiment 2:

TA 98 untreated controls, A. dest., showed a mean revertant count per plate of 34 (SD 6.4) and 28 (SD 7.5) with and without S9 respectively. TA 98 negative controls, EtOH, showed a mean revertant count per plate of 41 (SD 15.0) and 32 (SD 4.9) with and without S9 respectively. The highest mutation factor was 1.1(15.8 ug) and 1.0 (1.58 and 5 ug) with and without S9 respectively. Positive control of 4-NOPD without S9 resulted in a mutation factor of 12.2. Positive control of 2-AA with S9 resulted in a mutation factor of 63.7.

TA 100 untreated controls, A. dest., showed a mean revertant count per plate of 127 (SD 13.9) and 122 (SD 4.9) with and without S9 respectively. TA 100 negative controls, EtOH, showed a mean revertant count per plate of 131 (SD 10.8) and 105 (SD 4.7) with and without S9 respectively. The highest mutation factor was 1.1 (158 ug) and 1.2 (1.58 ug) with and without S9 respectively. Positive control of NaN3 without S9 resulted in a mutation factor of 7.4. Positive control of 2-AA with S9 resulted in a mutation factor of 11.9.

TA 1535 untreated controls, A. dest., showed a mean revertant count per plate of 10 (SD 4.4) and 10 (SD 2.1) with and without S9 respectively. TA 1535 negative controls, EtOH, showed a mean revertant count per plate of 11 (SD 1.2) and 9 (SD 1.2) with and without S9 respectively. The highest mutation factor was 1.5 (158 ug) and 1.6 (5 ug) with and without S9 respectively. Positive control of NaN3 without S9 resulted in a mutation factor of 117.2. Positive control of 2-AA with S9 resulted in a mutation factor of 13.5.

TA 91537 untreated controls, A. dest., showed a mean revertant count per plate of 10 (SD 2.1) and 11 (SD 4.7) with and without S9 respectively. TA 1537 negative controls, EtOH, showed a mean revertant count per plate of 13 (SD 3.5) and 6 (SD 2.6) with and without S9 respectively. The highest mutation factor was 1.4 (158 ug) and 1.6 (50 ug) with and without S9 respectively. Positive control of 4-NOPD without S9 resulted in a mutation factor of 20.3. Positive control of 2-AA with S9 resulted in a mutation factor of 27.1.

TA 102 untreated controls, A. dest., showed a mean revertant count per plate of 345 (SD 7.6) and 271 (SD 30.6) with and without S9 respectively. TA 102 negative controls, EtOH, showed a mean revertant count per plate of 358 (SD 34.3) and 275 (SD 27.3) with and without S9 respectively. The highest mutation factor was 1.1 (1.58 ug) and 1.2 (15.8 ug) with and without S9 respectively. Positive control of MMS without S9 resulted in a mutation factor of 4.3. Positive control of 2-AA with S9 resulted in a mutation factor of 2.1.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For results data see study report. Statistical significance in subsqent tables does not account for reduction in background lawn or observed cytotoxicity.

Table 1.1: Experiment I Mean Revertant Colonies per Plate 

	Treatment	Without S9					With S9
Strain		TA 98	TA 100	TA 1535	TA 1537	TA 102	TA 98	TA 100	TA 1535	TA 1537	TA 102
Experiment I Mean (Standard Deviation)	Aqua destillata   (untreated)	18 (4.0)	110 (13.2)	11 (1.5)	5 (1.5)	268 (19.0)	22 (7.5)	113 (7.0)	8 (1.7)	7 (2.6)	340 (21.1)
	EtOH   (negative control)	21 (4.0)	97 (21.7)	9 (2.0)	7 (3.1)	272 (19.3)	27 (1.0)	102 (12.7)	13 (6.4)	7 (3.5)	306 (35.7)
	Test Item 3.16 µg	24 (8.1)	92 (5.1)	9 (1.5)	5 (4.0)	293 16.2	31 (4.9)	97 (16.5)	18 (6.2)	10 (4.6)	370 39.9
	Test Item 10 µg	18 (1.5)	92 (4.4)	13 (3.0)	11 (4.2)	275 (21.5)	34 (1.5)	106 (11.7)	12 (5.8)	9 (4.6)	406* (20.0)
	Test Item 31.6 µg	22 (1.5)	113 (15.5)	11 (5.0)	7 (2.0)	275 (21.5)	32 (8.2)	100 (23.2)	12 (4.0)	9 (4.6)	404* (12.5)
	Test Item 100 µg	19 (3.1)	128 (8.7)	10 (1.5)	4 (0.6)	266 (34.1)	27 (5.1)	103 (9.0)	15 (5.1)	13 (4.0)	337 (28.0)
	Test Item 316 µg	23 (3.6)	108 (2.9)	4† (3.5)	4 (1.0)	266† (34.1)	31 (3.6)	107 (10.1)	13* (0.0)	9 (2.0)	336 (29.0)
	Test Item 1000 µg	6† (2.1)	80 (15.6)	4† (1.7)	3 (3.5)	218 (27.8)	11 (2.0)	92 (12.3)	10 (2.9)	4 (1.0)	332 (59.5)
	2-AA   (Positive Control)						2525* (251.4) 2.5µg	2696* (188.6) 2.5µg	203* (15.5) 2.5µg	288* (37.7) 2.5µg	839* (159.1) 10µg
	4-NOPD   (Positive Control)	341* (21.0) 10µg			75* (18.5) 40µg
	NaN3   (Positive Control)		1227* (63.1) 10µg	1005* (108.1) 10µg
	MMS   (Positive Control)					1488* (169.7) 1µL

* Statistically significant increase over that of the negative control p≤0.05 

† Statistically significant decrease over that of the negative control p≤0.05 

(#) Standard deviation

 

Table 1.2: Experiment II Mean Revertant Colonies per Plate 

	Treatment	Without S9					With S9
Strain		TA 98	TA 100	TA 1535	TA 1537	TA 102	TA 98	TA 100	TA 1535	TA 1537	TA 102
Experiment II Mean (Standard Deviation)	Aqua destillata  (untreated)	28 (7.5)	122 (4.9)	10 (2.1)	11 (4.7)	271 (30.6)	34 (6.4)	127 (13.9)	10 (4.4)	10 (2.1)	345 (7.6)
	EtOH   (negative control)	32 (4.9)	105† (4.7)	9 (1.2)	6 (2.6)	275 (27.3)	41 (15.0)	131 (10.8)	11 (1.2)	13 (3.5)	358 (34.3)
	Test Item 1.58 µg	31 (6.8)	122 (16.0)	10 (1.0)	8 (3.2)	294 (14.3)	30 (4.4)	137 (11.1)	14 (1.2)	12 (0.0)	382* (21.7)
	Test Item 5 µg	32 (6.8)	119 (9.6)	14 (3.8)	14 (3.6)	280 (55.7)	34 (4.2)	130 (8.5)	15 (2.6)	12 (1.0)	343 (4.5)
	Test Item 15.8 µg	27 (4.0)	105† (6.7)	6† (0.0)	7 (2.1)	319 (15.9)	46 (24.9)	136 (27.1)	10 (1.2)	12 (2.5)	319† (9.3)
	Test Item 50 µg	17 (8.3)	102† (7.5)	9 (4.4)	9 (2.5)	270 (35.4)	35 (6.7)	123 (10.1)	15 (3.0)	17* (3.2)	295 (42.0)
	Test Item 158 µg	21 (5.9)	90† (17.5)	3 (3.5)	8 (3.2)	246 (31.0)	32 (6.7)	143 (9.9)	16 (3.5)	18* (4.0)	289† (13.6)
	Test Item 500 µg	17 (2.9)	65† (20.8)	4† (1.5)	5 (1.5)	242 (43.2)	29 (8.1)	130 (17.6)	14 (1.7)	8 (1.0)	316† (10.3)
	Test Item 1000 µg		59† (20.4)	6 (2.8)	6 (3.5)	227 (3.6)	8 (5.0)	125 (14.2)	13 (4.7)	5 (2.0)	256 (37.9)
	2-AA   (Positive Control)						2613* (282.2) 2.5µg	1557* (643.7) 2.5µg	144* (7.9) 2.5µg	361* (7.0) 2.5µg	761* (103.2) 10µg
	4-NOPD   (Positive Control)	385* (72.3) 10µg			122* (4.0) 40µg
	NaN3   (Positive Control)		775* (351.3) 10µg	1015* (27.0) 10µg
	MMS   (Positive Control)					1192* (101.1) 1µL

* Statistically significant increase over that of the negative control p≤0.05 

† Statistically significant decrease over that of the negative control p≤0.05 

(#) Standard deviation

 

Table 3: Mean revertant colonies for negative and untreated controls

	Metabolic activation	Without S9					With S9
	Strain	TA 98	TA 100	TA 1535	TA 1537	TA 102	TA 98	TA 100	TA 1535	TA 1537	TA 102
Experiment I (Mean count)	Aqua destillata (untreated)	18	110	11	5	268	22	113	8	7	340
	EtOH (negative control)	21	97	9	7	272	27	102	13	7	306
Experiment II (Mean count)	Aqua destillata (untreated)	28	122	10	11	271	34	127	10	10	345
	EtOH (negative control)	32	105	9	6	275	41	131	11	13	358

 

Historical control data is as follows:

Table 4: HLC - Negative control without S9

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	23.3	117.9	12.4	9.9	243.3
SD	4.2	31.2	4.8	3.7	51.0
Min	16	77	5	5	164
Max	46	174	29	28	399
RSD %	18.2	26.5	38.4	37.4	21.0
n=	852	871	804	802	531

Table 5: HLC - Positive control without S9

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	500.1	1029.1	1140.8	130.3	1512.9
SD	139.1	260.9	266.9	28.5	336.2
Min	250	316	94	43	391
Max	2613	2307	1850	249	2353
RSD %	27.8	25.4	23.4	21.9	22.2
n=	825	849	777	775	517

Table 6: HLC - Negative control with S9

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	30.8	116.6	9.7	10.4	270.4
SD	5.5	17.4	3.1	3.7	57.9
Min	18	80	5	5	163
Max	56	165	27	34	458
RSD %	17.7	14.9	32.0	36.0	21.4
n=	853	872	804	802	531

Table 7: HLC - Positive control with S9

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	2424.9	1945.7	144.1	252.4	1120.7
SD	589.1	589.0	68.9	86	328.0
Min	423	495	28	58	419
Max	3599	3341	582	474	2422
RSD %	24.3	30.3	47.8	34.1	29.3
n=	826	847	777	775	516

Liturature

(1). Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection Proc. Nat\. Acad. Sci. (USA) 70, 2281-2285
(2). Ames, B.N., McCann, J. and Yamasaki, E. (1977) Methods of detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test In: BJ. Kilbey et a\. (Eds.) "Handbook of Mutagenicity Test Procedures" Elsevier, Amsterdam, 1-17
(3). Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E. and Zeiger, E. (1987). Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189,83-91
(4). Gatehouse D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Ohta, T., Venitt, S., Zeiger, E. (1994) Recommendation for the performance of bacterial mutation assays Mutation Res. 312,217-233
(5). Kier, L.E., Brusick, DJ., Auletta, A.E., von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V., McCann, 1., Mortelmans, K., Prival, M., Rao, T.K. and Ray, V. (1986) The Salmonella typhimurium/mammalian microsomal assay. A report ofthe U.S. Environmental Protection Agency Gene-Tox-Program Mutat. Res. 168, 69-240
(6). Maron, D.E. and Ames, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173-215.
(7). McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 Chemicals. Proc. Nat\. Acad. Sci. (USA) 72, 5135-5139
(8). McCann,1. and Ames, B.N. (1976) Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 Chemicals: Discussion.' Proc. Nat\. Acad. Sci. (USA) 73, 950-954
(9). Mortelsmans, K. and Zeiger, E. (2000) The Ames Salmonella/microsome mutagenicity assay. Mutat. Res. 455, 29-60

(10). Zeiger, E., Haseman, J., Shelby, M., Margolin, B. and Tennant, R. (1988) Salmonella mutagenicity tests IV. Results from testing of 300 Chemicals Environ. Mol. Mutagen., 11, 1-158
(11). Zeiger, E., Anderson, B., Haworth, S., Lawlor, T. and Mortelmanns, K. (1992) Salmonella mutagenicity tests V. Results from the testing of 311 Chemicals Environ. Mol. Mutagen. 19 (Suppl. 21),2-141

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, C16-22-(even numbered)alkylamines did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, C16-22-(even numbered)alkylamines is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Introduction

This study followed the procedures indicated by internal BSL BIOSERVICE SOPs and the following internationally accepted guidelines and recommendations:
OECD Guidelines for Testing of Chemicals, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997
Commission Regulation (EC) No. 440/2008 B.13/14:"Mutagenicity - Reverse Mutation Test using Bacteria", dated May 30, 2008
EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Assay" EPA 712-C-98-247, August 1998

 

This study was conducted to comply with OECD Principles of Good Laboratory Practice (as revised in 1997). 

The princilple of this study was to investigate the potential of CI6-22-(even numbered)alkylamines for its ability to induce gene mutations the plate incorporation test (experiment I and II) was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. 

 

Methods

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 of S. typhimurium were exposed to C16-22-(even numbered)alkylamines at concentrations of 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (experiment I), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I and II).

In experiment II the test item was tested a concentrations of 1.58, 5.00. 15.8, 50.0, 158, 500 and 1000 µg/plate in all tester strains used, except for TA 98 (without metabolic activation - highest concentration: 500 µg/plate). Testing up to the limit concentration of 5000 µg/plate was not possible due to strong precipitation which interfered with evaluation. Positive, negative and sovent controls were conducted to ensure validity of experimental conditions for all tester strains.

 

Results & Discussion 

The positive controls induced the appropriate vaild responses in the corresponding strains. The reference mutagens induced a ,distinct increase of revertant colonies indicating the validity of the experiments. Negative and solvent controls showed appropriate and valid response when compared with historical control data. 

Precipitation of the test item was observed in all tester strains used in experiment I and II. In experiment I precipitation of the test item was found at a concentration of 316 ug/plate and higher (with and without metabolic activation), in experiment II precipitation of the test item was found at a  concentration of 500 ug/plate and higher (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.
In experiment I toxic effects of the test item were observed in tester strain TA 98 at a concentration of 1000 ug/plate (with and without metabolic activation). In tester strain TA100 toxic effects of the test item were seen at a concentration of 1000 ug/plate (without metabolic activation). In tester strains TA 1535, TA 1537 and TA 102 toxic effects ofthe test item were noted at concentrations of 316 j..tg/plate and higher (without metabolic activation). In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 500 ug/plate (without metabolic activation) and at a concentration of 1000 ug/plate (with metabolic activation). In tester strains TA 100 and TA 1535 toxic effects ofthe test item were noted at concentrations of 158 ug/plate and higher (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 500 ug/plate and higher (without metabolic activation) and at a concentration of 1000 ug/plate (with metabolic activation). In tester strain TAI02 toxic effects ofthe test item were observed at a concentrations of 1000 ug/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment II in tester strain TA 98 at a concentration of 50.0 ug/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with C16-22-(even numbered)alkylamines at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, C16-22-(even numbered)alkylamines did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. There was no evidence of induced mutant colonies over background in any of the bacterial strains tested with the test material. Therefore, C16-22-(even numbered)alkylamines is considered to be non-mutagenic in this bacterial reverse mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.