Reaction mass of Amines, coco alkyl and β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and β-Alanine, N-coco alkyl derivs.

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:

2001-07-30 to 2001-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study (OECD 471)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

EC Number:

915-790-0

Molecular formula:

n/a

IUPAC Name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

Test material form:

liquid

Details on test material:

- Purity: 69% (28% isopropanol – 3% water)
- Batch Number: 01652201
- Description: yellow liquid
- Container: one glass flask
- Date of receipt: 28 June 2001
- Storage conditions: at room temperature and protected from light
- Expiry date/stability: June 2002
- Carbon content: 65.8% (weight/weight).

Method

Target gene:

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

In addition, to increase their sensitivity to mutagenic substances, further mutations have been added:
. the rfa mutation causes partialloss 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 (conferring ampicillin resistance) to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to sorne mutagens,
. in addition, the pAQ1 tetracycline resistant plasmidic factor has been added to the TA 102 strain.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route.

Test concentrations with justification for top dose:

Experiments without S9 mix:
3.125, 6.25, 12.5, 25 and 50 µg/plate: for the TA 98, TA 102 strains in the second experiment,
6.25, 12.5, 25, 50 and 100 µg/plate: for all tester strains in the first experiments well as for the TA1537 and TA100 strains in the 2nd experiment,
12.5, 25, 50, 100 and 200 µg/plate: for the TA 1535 strain in the second experiment.

Experiments with S9 mix:
12.5, 25, 50, 100 and 200 µg/plate: for the TA 1537, TA 98 and TA 102 strains in the first experiment,
25, 50, 100, 200 and 400 µg/plate: for the TA 1535 and TA 100 strains in the first experiment as well as for all tester strains in the second experiment.

All the dose-levels were expressed as active substance, taking into account the active material content of 69%.

Vehicle / solvent:

Ethanol
The test substance was dissolved in the vehicle at concentrations of 100 mg/mL for the preliminary toxicity, and 8 mg/mL for the mutagenicity experiments. The preparations were made immediately before use.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method and preincubation method.

DURATION
The experiments were performed according to:
. direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix): test substance solution (0.05 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
. preincubation method (second experiment with S9 mix): test substance solution (0.05 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Artek counter, model 880, OSI, 75015 Paris, France).

ln two independent experiments, five dose-levels of AMPHORAM CP1 (three plates/dose-leve) were tested on each strain, with and without S9 mix.

Treatment of results:
ln each experiment, for each strain and for each experimental point, the number of revertants per plate was scored.
The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Evaluation criteria:

This study is considered valid if the following criteria are fully met:
. the number of revertants in the vehicle controls is consistent with our historical data,
. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

A reproducible two-fold increase 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:

Not reported

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 50 µg/plate (without S9) and at 200 µg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test substance was freely soluble in the vehicle (ethanol) at 100 mg/mL.
Consequently, with a treatment volume of 50 µg/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitae was observed in the Petri plates when scoring the revertants at all the dose-levels tested.
In the three tester strains, a moderate to strong toxicity was induced at dose-levels 100 µg/plate or 500 µg/plate, without and with S9 mix respectively.

ACCEPTANCE CRITERIA :
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

RESULTS OF THE MAIN STUDY :
Experiments without S9 mix:
A slight to strong toxicity was induced in the tester strains at dose-levels 50 µg/plate (TA 98, TA 100 and TA 102 strains), 100 µg/plate (TA 1537 strain) or 200 µg/plate (TA 1535 strain).
The test substance did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five tester strains.

Experiments with S9 mix:
A slight to strong toxicity was induced in the tester strains generally at dose-levels 200 µg/plate.
The test substance did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five tester strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Results table / First experiment without S9

 

strains	Doses* (µg/plate)	T	P	Revertants per plate			mean	SD	ratio
TA 1535	0	0	0	10	8	10	9	1	-
	6.25	0	0	10	14	13	12	2	1.32
	12.5	0	0	11	12	10	11	1	1.18
	25	0	0	12	7	10	10	3	1.04
	50	0	0	15	10	7	11	4	1.14
	100	0	0	11	11	13	12	1	1.25
	NaN3 (1)	-	-	331	315	315	320	9	34.32
TA 1537	0	0	0	10	6	4	7	3	-
	6.25	0	0	2	8	6	5	23	0.80
	12.5	0	0	6	6	8	7	1	1.00
	25	0	0	8	7	8	8	1	1.15
	50	0	0	6	6	4	5	1	0.80
	100	1	0	2	4	5	4	2	0.55
	9AA (50)	-	-	180	192	179	184	7	27.55
TA 98	0	0	0	17	14	15	15	2	-
	6.25	0	0	15	23	20	19	4	1.26
	12.5	0	0	15	25	17	19	5	1.24
	25	0	0	15	9	18	14	5	0.91
	50	1	0	9	9	18	12	5	0.78
	100	2	0	8	7	10	8	2	0.54
	2NF (0.5)	-	-	161	117	174	151	30	9.83
TA 100	0	0	0	64	52	39	52	13	-
	6.25	0	0	82	70	70	74	7	1.43
	12.5	0	0	70	59	49	59	11	1.15
	25	0	0	50	67	69	62	10	1.20
	50	1	0	49	62	66	59	9	1.14
	100	1	0	49	52	58	53	5	1.03
	NaN3 (1)	-	-	366	416	442	408	39	7.90
TA 102	0	0	0	174	164	178	172	7	-
	6.25	0	0	208	148	171	176	30	1.02
	12.5	0	0	191	209	173	191	18	1.11
	25	0	0	136	75	167	126	47	0.73
	50	2	0	129	5	0	45	73	0.26
	100	2	0	67	0	0	22	39	0.13
	MMC (0.5)	-	-	548	483	414	482	67	2.80

 

0: vehicle control (ethanol)

*: doses expressed as active substance

SD: standard deviation

Ratio = number of revertants with the test substance / number of revertants with the vehicle

T= toxicity, P = precipitate

 

 

 

Table 3: Results table / Second experiment without S9

 

strains	Doses* (µg/plate)	T	P	Revertants per plate			mean	SD	ratio
TA 1535	0	0	0	4	10	8	7	3	-
	6.25	0	0	17	12	12	14	3	1.86
	12.5	0	0	12	8	11	10	2	1.41
	25	0	0	11	9	5	8	3	1.14
	50	0	0	9	6	9	8	2	1.09
	100	2	0	2	1	3	2	1	0.27
	NaN3 (1)	-	-	371	697	408	392	19	53.45
TA 1537	0	0	0	6	6	5	6	1	-
	6.25	0	0	4	4	3	4	1	0.65
	12.5	0	0	4	5	6	5	1	0.88
	25	0	0	7	2	5	5	3	0.82
	50	0	0	8	5	6	6	2	1.12
	100	1	0	4	4	5	4	1	0.76
	9AA (50)	-	-	165	158	170	164	6	29.00
TA 98	0	0	0	21	22	22	22	1	-
	6.25	0	0	18	12	14	15	3	0.68
	12.5	0	0	18	21	22	20	2	0.94
	25	0	0	18	18	12	16	3	0.74
	50	0	0	23	27	15	22	6	1.00
	100	0	0	17	20	13	17	4	0.77
	2NF (0.5)	-	-	120	115	104	113	8	5.22
TA 100	0	0	0	59	65	60	61	3	-
	6.25	0	0	67	70	51	63	10	1.02
	12.5	0	0	58	72	58	63	8	1.02
	25	0	0	42	43	56	47	8	0.77
	50	0	0	66	63	70	66	4	1.08
	100	0	0	55	48	35	46	10	0.75
	NaN3 (1)	-	-	355	339	341	345	9	5.63
TA 102	0	0	0	138	173	135	149	21	-
	6.25	0	0	172	95	93	120	45	0.81
	12.5	0	0	130	158	173	154	22	1.03
	25	0	0	143	92	103	113	27	0.76
	50	0	0	130	116	123	123	7	0.83
	100	0	0	103	91	83	92	10	0.62
	MMC (0.5)	-	-	586	520	490	532	49	3.58

 

0: vehicle control (ethanol)

*: doses expressed as active substance

SD: standard deviation

Ratio = number of revertants with the test substance / number of revertants with the vehicle

T= toxicity, P = precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item is not mutagenic in a bacterial mutation assay.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium.

A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal 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).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 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 test substance was dissolved in ethanol.

All the dose-levels were expressed as active substance, taking into account the active material content of 69%.

The dose-levels of the positive controls were as follows:

without S9mix:

1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

with S9mix:

2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:

The selected treatment-levels were as follows:

3.125, 6.25, 12.5, 25 and 50 µg/plate: for the TA 98 and TA 102 strains in the second experiment,

6.25, 12.5, 25, 50 and 100 µg/plate: for all tester strains in the first experiments well as for the TA 1537 and TA 100 strains in the second experiment,

12.5, 25, 50, 100 and 200 µg/plate: for the TA 1535 strain in the second experiment.

A slight to strong toxicity was induced depending on the dose-levels and the tester strains.

The test substance did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five tester strains.

Experiments with S9 mix:

The selected treatment-levels were as follows:

12.5, 25, 50, 100 and 200 µg/plate: for the TA 1537, TA 98 and TA 102 strains in the first experiment,

25, 50, 100, 200 and 400 µg/plate: for the TA 1535 and TA 100 strains in the first experiment as well as for all tester strains in the second experiment.

A slight to strong toxicity was induced in the tester strains, depending on the dose-levels and the experimental conditions.

The test substance did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five tester strains.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Under these experimental conditions, the test substance does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.