Amines, N-(3-aminopropyl)-N'-C12-18-alkyltrimethylenedi-, reaction products with N-C12-18-alkyltrimethylenediamines and formic acid

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:

2002-10-01 to 2002-11-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amines, tallow alkyl[3-(tetrahydropyrimidinyl)propyl]

EC Number:

296-557-4

EC Name:

Amines, tallow alkyl[3-(tetrahydropyrimidinyl)propyl]

Cas Number:

92797-22-3

IUPAC Name:

92797-22-3

Constituent 2

Reference substance name:

Amines, suifalkyl [(tetrahydropyrimidinyl)3-propyl]

IUPAC Name:

Amines, suifalkyl [(tetrahydropyrimidinyl)3-propyl]

Details on test material:

- Name of test material (as cited in study report): POLYRAM L200
- Chemical name : Amines, suifalkyl [(tetrahydropyrimidinyl)3-propyl]
- CAS number : 92797-22-3
- Physical state: clear brown liquid
- Analytical purity: > 95% expressed in amines content
- Purity test date: 2002-06-20
- Lot/batch No.: 94052206
- Expiration date of the lot/batch: July 2003.
- Storage condition of test material: in dark and at room temperature

Method

Target gene:

TA1535 and TA100: his G 46
TA1537: his C 3076
TA98: his D 3052
TA102: his G 428

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver post mitochondrial fraction of rats induced with Aroclor 1254)

Test concentrations with justification for top dose:

first assay without activation: 0, 0.5, 1.5, 5, 15, 50, 100 μg/plate
first assay with activation: 0, 1.5, 5, 15, 50, 150, 300 μg/plate
second assay with and without activation 0, 1.5, 5, 15, 50, 100 μg/plate, for all strains except for TA102 without activation: 0, 0.5, 1.5, 5, 15, 50 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the test system
- Lot: DMSO for chromatography MERCK, batch # k30049278211

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: different for the respective strains, see below under "Positive test substances"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation): first mutagenicity test and second mutagenicity test without S9 mix
- Preincubation (60 minutes at 37°C ) only in second mutagenicity test with S9 mix
- Exposure duration: 48h

SELECTION AGENT (mutation assays): agar containing traces of histidine and biotin, maintained at 45°C

NUMBER OF REPLICATIONS: two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: the evaluation of the toxicity was based on the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
Reference to historical data or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

In parallel to criteria based on biological significance, data were analysed by means of Dunnett's method (Mahon G.A.T. et al, 1989) allowing the comparison of the mean value for each dose with the mean value for the corresponding solvent control.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 50 - 300 µg/plate, with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50 - 300 µg/plate, with and without activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

other: yes, even though in assay 1 the positive reference compound induced a statistically and biologically significant increase in the number of revertants compared to the control with a ratio of only 2.8.

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: occurred at 150 µg/plate and higer concentrations

RANGE-FINDING/SCREENING STUDIES:
doses of 0, 50, 150, 500, 1500, 5000 µg/plate were tested, bacterial growth was completely inhibited at 150 - 500 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
yes, historical data available in the study report

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Toxicity assay

Strain	Dose µg/plate	without S9-mix			with S9-mix
		T	P	Revertants/ plate	T	P	Revertants/ plate
TA 1535	0	-	-	10	-	-	12
	50	N	-	0	-	-	6
	150	N	+	0	+	+	8
	500	N	++	0	N	++	0
	1500	N	++	0	N	++	0
	5000	N	++	0	N	++	0
TA 1537	0	-	-	3	-	-	6
	50	N	-	0	-	-	7
	150	N	+	0	+	+	3
	500	N	++	0	N	++	0
	1500	N	++	0	N	++	0
	5000	N	++	0	N	++	0
TA 98	0	-	-	11	-	-	9
	50	N	-	0	-	-	13
	150	N	+	0	+	+	13
	500	N	++	0	N	++	0
	1500	N	++	0	N	++	0
	5000	N	++	0	N	++	0
TA 100	0	-	-	106	-	-	111
	50	N	-	0	-	-	99
	150	N	+	0	+	+	53
	500	N	++	0	N	++	0
	1500	N	++	0	N	++	0
	5000	N	++	0	N	++	0
TA 102	0	-	-	184	-	-	243
	50	N	-	0	-	-	277
	150	N	+	0	+	+	270
	500	N	++	0	N	++	0
	1500	N	++	0	N	++	0
	5000	N	++	0	N	++	0

T=Toxicity

(- non toxic; + slightly toxic; ++ moderately toxic; +++ strongly toxic; N no bacterial growth)

P = Precipitate

(- absence; + slight precipitate; ++ moderate precipitate; +++ important precipitate hindering scoring)

Table 2: MUTAGENICITY ACTIVITY ASSAY, RECAPITULATION : ASSAY 1

	TA 1535		TA 1537		TA 98		TA 100		TA102
	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate
Postive control	(a)	524.7	(a)	349.0	(a)	707.0	(a)	1411.7	(a)	1256.0
TEST COMPOUND without S9-mix	0	11.8	0	3.2	0	17.7	0	68.0	0	208.3
	0.5	14.0	0.5	4.0	0.5	19.0	0.5	83.7	0.5	194.7
	1.5	13.7	1.5	6.0	1.5	19.7	1.5	98.3	1.5	196.3
	5	18.0	5	6.0	5	15.7	5	79.7	5	192.7
	15	13.0	15	7.3	15	18.3	15	76.3	15	191.3
	50	11.7	50	4.3	50	12.0	50	55.7	50	67.3
	100	0.0	100	0.0	100	0.0	100	0.0	100	0.0
Postive control	(b)	474.0	(b)	216.7	(b)	1932.0	(b)	2232.7	(b)	677.3
TEST COMPOUND with S9-mix without pre-incubation	0	6.2	0	4.0	0	11.7	0	81.2	0	252.5
	1.5	7.7	1.5	4.0	1.5	9.7	1.5	105.7	1.5	252.3
	5	3.7	5	5.7	5	19.0	5	100.3	5	282.3
	15	6.3	15	3.7	15	19.0	15	100.0	15	270.7
	50	8.7	50	3.0	50	12.3	50	112.3	50	269.0
	150	5.0	150	4.7	150	8.0	150	110.0	150	196.0
	300	0.0	300	0.0	300	0.0	300	0.0	300	0.0

Reference positive compounds (µg/plate) :

TA1535 and TA100 : Sodium azide 1 ; TA1537 : 9-amino-acridine 50 ; TA98 : 2-nitrofluorene 2 TA102: Mitomycin C 0.125

TA1535, TA1537, TA98, TA100 : 2-anthramine 2 ; TA102: benzo(a)pyrene 2

Table 3: MUTAGENICITY ACTIVITY ASSAY, RECAPITULATION : ASSAY 2

	TA 1535		TA 1537		TA 98		TA 100		TA102
	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate	DOSE µg/plate	Revertants /plate
Positive control	(a)	762.0	(a)	386.3	(a)	408.0	(a)	476.7	(a)	891.3
	0	10.7	0	3.3	0	11.0	0	102.5	0	187.3
TEST COMPOUND	1.5	9.0	1.5	2.3	1.5	13.0	1.5	104.0	0.5	195.7
	5	13.3	5	5.0	5	15.3	5	101.7	1.5	187.0
without	15	8.7	15	5.0	15	8.7	15	104.7	5	165.3
S9-mix	50	7.7	50	3.0	50	4.3	50	58.7	15	183.0
	100	0.0	100	0.0	100	0.0	100	0.0	50	55.3
Positive control	(b)	114.3	(b)	183.0	(b)	1335.0	(b)	1358.0	(b)	716.7
	0	7.7	0	8.8	0	12.5	0	71.5	0	233.8
TEST COMPOUND	1.5	5.0	1.5	5.0	1.5	14.0	1.5	86.3	1.5	173.7
with	5	4.3	5	7.0	5	13.0	5	75.0	5	200.0
S9-mix	15	3.7	15	2.7	15	4.3	15	65.0	15	179.0
with	50	7.0	50	8.7	50	10.7	50	92.0	50	236.0
pre-incubation	150	5.0	150	6.3	150	7.7	150	87.3	150	198.3

Reference positive compounds (µg/plate) :

TA1535 and TA100 : Sodium azide 1 ; TA1537 : 9-amino-acridine 50 ; TA98 : 2-nitrofluorene 2 TA102: Mitomycin C 0.125

TA1535, TA1537, TA98, TA100 : 2-anthramine 1 ; TA102: benzo(a)pyrene 2

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results of this study, the test substance did not show any mutagenic activity in the five Salmonella typhimurium strains tested with and without metabolic activation.

Executive summary:

The potential of the test substance to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium according to the OECD guideline 471.The study was conducted in compliance with the principles of Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post- mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). After 48 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 The preliminary assays without metabolic activation showed that the test substance demonstrated a potent toxicity from 5000 to 50 µg/plate in all strains tested with a total absence of bacterial growth. The immediately lower dose of 15 µg/plate induced only a slight toxicity. Under these conditions, the dose of 100 µg/plate was retained as the maximum dose tested for the first mutagenicity assay in the absence of metabolic activation in all strains TA1535.

In the preliminary assays with metabolic activation, the test substance also demonstrated a potent toxicity from 5000 µg/plate to 500 µg/plate in all strains tested with a total absence of bacterial growth. The immediately lower dose of 150 µg/plate induced only a slight toxicity in all strains tested.

Under these conditions, the dose of 300 µg/plate was retained as the maximum dose tested for the first mutagenicity assay in the presence of metabolic activation in all strains.

In the main experiment, the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. Nevertheless,

in the first assay in the presence of S9, the number of revertants observed for the positive control of strain TA102 was slightly lower than the lowest limit for historical values. However, the positive reference compound induced a statistically and biologically significant increase in the number of revertants compared to the control with a ratio of 2.8. The study was therefore considered valid.

In the two independent assays, no significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains tested in the presence of the test substance neither with nor without metabolic activation.

During the first assay, a statistically significant increase in the number of revertants was found in strain TA100 at the intermediate dose of 1.5 µg/plate without metabolic activation, and at the two highest doses of 50 and 150 µg/plate with metabolic activation. However, the biological threshold (ratio 2) was not reached, as the calculated ratio was 1.4 in all cases. Moreover, the effect observed without metabolic activation was neither dose-related nor reproduced in the second assay. Concerning the effect noted with metabolic activation, an increase was indeed observed in the second assay (performed with the preincubation protocol) but (i) it was not statistically significant and (ii) the ratio was not higher than 1.3. Therefore, this increase could not be attributed to a mutagenic effect

Under these experimental conditions the test substance did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimurium.