pentasodium 3,7,11-tris(carboxylatomethyl)-15-(C12-18)-alkyl-3,7,11,15-tetraazaheptadecane-1,17-dioate

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:

3 - 20 September 1993.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was performed under GLP and according to OECD guideline from 1983. With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Principles of method if other than guideline:

This study was performed according to OECD guideline from 1983. With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-C8-22-alkylpolytrimethylenepoly-, carboxymethyl derivs., sodium salts

EC Number:

307-458-3

EC Name:

Amines, N-C8-22-alkylpolytrimethylenepoly-, carboxymethyl derivs., sodium salts

Cas Number:

97659-53-5

Details on test material:

Identity: Arnpholak 7CX
Chemical name: Amines, N-cocoalkyl-polytrimethylene poly, carboxymethyl-sodium salts
Batch number: FP91327
Purity: Active about 27 %
Sodium glycolate max 3 %
Sodium chloride max 12%
Sodium monochloroacetate max 100 ppm
Water ad 100%
Appearance: clear yellow liquid
Storage conditions: room temperature

Method

Target gene:

mutation in the gene coding for histidine

Species / strain

Species / strain / cell type:

other: see below

Details on mammalian cell type (if applicable):

S. typhimurium TA 1535 hisG46 rfa uvrB
S. typhimurium TA 1537 hisC3076 rfa uvrB
S. typhimurium TA 1538 hisD3052 rfa uvrB
S. typhimurium TA 98 hisD3052 rfa uvrB pKM 10 1
S. typhimun'um TA 100 hisG46 rfa uvrB pKM101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: As the test substance was a mixture of solids dissolved in water the solvent chosen for this assay was
water which was confirmed in a solubility test.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

Positive control substances:
without S9 mix:
Identity: N-Ethyl-N'-nitro-N-nitrosoguanidine
Supplier: Sigma
Batch: 67F-3700
Solvent: DMSO
Concentration: 5 microgram/plate for strain TA 1535
3 microgram/plate for strain TA 100

Identity: 9-Aminoacridine
Supplier: Sigma
Batch: 96F-05641
Solvent: DMSO
Concentration: 80 microgram/plate for strain TA 1537

Identity: 2-Aminoanthracene
Supplier: Aldrich
Batch: 61896
Solvent: DMSO
Concentration: 2 microgram/plate for strain TA 1538
1 microgram/plate for strain TA 98

With S9 mix:
dentity: 2-Aminoanthracene
Supplier: Aldrich
Batch: 0013406
Solvent: DMSO
Concentration: 2 microgram/plate for strain TA 1535 and TA 1537
0.5 microgram/plate for strain TA 1538 and TA 98
1 microgram/plate for strain TA 100

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): histidine defiencient agar

NUMBER OF REPLICATIONS: three petri dishes per dose level and an independent repeat was performed

NUMBER OF CELLS EVALUATED: all colonies were counted

DETERMINATION OF CYTOTOXICITY
- Method: assessment a background lawn

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at my dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this .
test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statisticd procedures used will be those described by Mahon et at. (1989).

Statistics:

Not performed

Results and discussion

Test results

Species / strain:

other: all strains tested

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 examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: sufficient for the test
- Precipitation: not observed
- Other confounding effects: not examined


RANGE-FINDING/SCREENING STUDIES: No cytotoxicty was observed in the preliminary toxicity test up to 5000 microgram/plate. this dose was therefore also selcted as the highest dose in the final study.

COMPARISON WITH HISTORICAL CONTROL DATA: not performed

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with Ampholak 7CX at any dose level, in the presence or absence of S-9 mix, in either mutation test.
The concurrent positive control compounds demonstrated the sensitivity of the assay and the rnehbolising activity of the liver preparations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutation test 1

strain	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts	SD
TA1535	5000	-	16	3.2
	1500	-	10	2.6
	500	-	10	4.2
	150	-	16	2.9
	50	-	17	2.5
	0	-	13	3.2
	solvent	-	18	2.1
	5000	+	18	2.5
	1500	+	15	2.6
	500	+	11	6.1
	150	+	16	5.7
	50	+	13	2.5
	0	+	15	1.5
	solvent	+	13	4.2
TA1537	5000	-	8	3.5
	1500	-	5	2.6
	500	-	8	4.7
	150	-	11	4.0
	50	-	9	2.3
	0	-	10	2.6
	solvent	-	9	2.1
	5000	+	8	1.7
	1500	+	6	3.1
	500	+	7	4.2
	150	+	10	5.9
	50	+	11	1.5
	0	+	14	3.5
	solvent	+	10	6.1
TA1538	5000	-	9	1.5
	1500	-	7	1.2
	500	-	14	2.6
	150	-	12	4.0
	50	-	13	2.1
	0	-	9	6.8
	solvent	-	12	2.3
	5000	+	14	1.2
	1500	+	12	6.7
	500	+	11	1.7
	150	+	14	3.1
	50	+	11	3.1
	0	+	19	3.2
	solvent	+	20	2.1
TA98	5000	-	18	3.6
	1500	-	19	3.2
	500	-	20	1.5
	150	-	23	4.4
	50	-	23	3.6
	0	-	28	2.6
	solvent	-	25	5.5
	5000	+	22	1.5
	1500	+	23	2.0
	500	+	28	3.8
	150	+	27	2.6
	50	+	26	2.0
	0	+	29	2.5
	solvent	+	26	5.6
TA100	5000	-	121	7.4
	1500	-	122	17.3
	500	-	103	9.3
	150	-	119	12.9
	50	-	113	5.3
	0	-	113	7.4
	solvent	-	108	4.6
	5000	+	112	13.6
	1500	+	118	9.8
	500	+	119	17.8
	150	+	106	9.6
	50	+	108	6.1
	0	+	120	4.0
	solvent	+	101	7.6

- Absence

+ Presence

SD Standard deviation

Positive control Mutation test 1:

Strain	Compound	Dose level (µg/plate)	Metabolic activation	Mean revertant colony counts	SD
TA1535	ENNG	5.0	-	223	30.9
TA1537	9 AC	80.0	-	X	-
TA1538	NF	2.0	-	173	28.4
TA98	NF	1.0	-	260	8.9
TA100	ENNG	3.0	-	286	6.0
TA1535	AA	2.0	+	139	20.6
TA1537	AA	2.0	+	77	5.9
TA1538	NF	0.5	+	187	15.6
TA98	AA	0.5	+	221	19.5
TA100	AA	1.0	+	473	72.4

- Absence

+ Presence

X Too many colonies to count accurately

SD Standard deviation

ENNG N-ethyl-N’-nitro-N-nitrosoguanidine

9AC 9-aminoacridine

NF 2-nitrofluorene

AA 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

It is concluded that, when tested in water, the test material (40% active ingredient and 60% water) was not mutagenic in this bacterial system. With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have. Under the given conditions, the active ingredient Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS no 2060541-51-5) is considered to be non mutagenic.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test material, histidine dependent auxotrophic mutanrs of Salmonella typhimunum (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to the test substance, diluted in water which was also used as a negative control. Two independent mutation tests were performed accoriding to OECD guodlien and under GLP, 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, 50 μg/plate. No evidence of mutagenic activity was seen at any dose level of the test material in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in water, the test materal was not mutagenic in this bacterial system. Under the given conditions, the active ingredient Sodium cocoamphopolycarboxyglycinate (Amines, N-(3-aminopropyl)-N’-C12-18-alkyltrimethylenedi-, N-(carboxymethyl)derivs., sodium salts with CAS no 2060541-51-5) is therefore considered to be non mutagenic. With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.