Glycine, N-methyl-, N-coco acyl derivs., sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity (OECD 471), Ames: negative with and without metabolic activation

Mammalian Cytogenicity (WoE, OECD 473), Chromosome Aberration: negative with and without metabolic activation

RA from source substances Sodium N-lauroylsarcosinate (CAS 137-16-6), Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) and N-methyl-N-[C18-(unsaturated)alkanoyl]glycine (EC 701-177-3)

Mammalian Mutagenicity (WoE, OECD 476): negative with and without metabolic activation

RA from source substances Sodium N-lauroylsarcosinate (CAS 137-16-6), Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) and N-methyl-N-[C18-(unsaturated)alkanoyl]glycine (EC 701-177-3)

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:

2006-02-15 to 2006-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umweltschutz und Gewerbeaufsicht

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Additional strain / cell type characteristics:

other: histidin-deficient

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

First experiment:
- 1.5, 5, 15, 50, 149 µg/plate with and without metabolic activation

Second experiment:
- 37.5, 75, 150 µg/plate with and without metabolic activation

Vehicle / solvent:

water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

NPD: 80 μg/plate in water, -S9, TA 97a, TA 98, TA 102; Sodium azide: 6 μg/plate in water, -S9, TA100 and TA1535; 2-AA: 3 μg/plate in DMSO, +S9, TA 98; BaP: 40 μg/plate in DMSO, +S9, TA 97a, TA 100, TA 102, TA 1535

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 12 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:
- dublicates each in two independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test material may be considered positive in this test system if the following
criteria are met:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at one strain with or without S9 mixture or there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels

Statistics:

Mean values and standard deviation were calculated

Key result

Species / strain:

other: S. typhimurium: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 150 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- The highest concentration used in the study was 150 µg/plate, based on the cytotoxicity at higher concentrations.

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA97a
–	0	114 ± 49	9 ± 2.4	9 ± 2.4	12 ± 0.5	130 ± 34.5
–	1.5	71 ± 15	6 ± 4	164 ± 25	8 ± 1	124 ± 36
–	5	97 ± 22	7 ± 2	125 ± 9	8 ± 1	97 ± 38
–	15	86 ± 12	6 ± 2	106 ± 19	6 ± 3	80 ± 7
–	50	90 ± 16	8 ± 1	101 ± 10	4 ± 2	83 ± 31
–	150*	113 ± 41	8 ± 3	139 ± 7	14 ± 6	120 ± 55
Positive controls, –S9	Name	NaN3	NaN3	NPD	NPD	NPD
	Concentrations (μg/plate)	6	6	80	80	80
	Mean No. of colonies/plate (average of 2 ± SD)	366 ± 101	1001 ± 0	657 ± 122	205 ± 98	1001 ± 0
+	0	138 ± 38	6 ± 2.6	158 ± 27.9	15 ± 2.4	101 ± 26.9
+	1.5	74 ± 13	5 ± 2	154 ± 44	12 ± 7	93 ± 42
+	5	127 ± 22	7 ± 1	156 ± 26	9 ± 6	115 ± 38
+	15	104 ± 14	8 ± 3	146 ± 9	8 ± 5	86 ± 31
+	50	103 ± 17	11 ± 3	159 ± 23	12 ± 7	101 ± 21
+	150*	78 ± 9	9 ± 1	115 ± 32	8 ± 5	102 ± 31
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	3	3	3	40	3
	Mean No. of colonies/plate (average of 2 ± SD)	1218 ± 251	130 ± 12	382 ± 42	200 ± 48	718 ± 231

Plate incorporation

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA97a
–	0	145 ± 17	12 ± 3.4	186 ± 3.6	8 ± 2.2	153 ± 27.3
–	37.5	158 ± 45	7 ± 3	136 ± 47	7 ± 3	122 ± 54
–	75	131 ± 19	8 ± 4	128 ± 42	13 ± 5	125 ± 20
–	150*	146 ± 7	6 ± 4	129 ± 28	11 ± 5	115 ± 37
Positive controls, –S9	Name	NaN3	NaN3	NPD	NPD	NPD
	Concentrations (μg/plate)	6	6	80	80	80
	Mean No. of colonies/plate (average of 2 ± SD)	1001 ± 0	1001 ± 0	1001 ± 0	1001 ± 0	1001 ± 0
+	0	153 ± 7	13 ± 1.7	137 ± 43.2	10 ± 3.4	141 ± 23.6
+	37.5	95 ± 16	8 ± 3	86 ± 25	10 ± 4	104 ± 31
+	75	104 ± 21	11 ± 3	117 ± 15	10 ± 4	156 ± 29
+	150*	120 ± 10	5 ± 2	134 ± 44	6 ± 2	89 ± 28
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	3	3	3	40	3
	Mean No. of colonies/plate (average of 2 ± SD)	1001 ± 0	1001 ± 0	1001 ± 0	105 ± 6	1001 ± 0

Preincubation

*Remark: 150 µg/plate was the highest concentration of test substance due to toxicity testing

NPD: 4-Nitro-1,2-phenylene diamine

NaN3: sodium azide

2AA: 2-Amino-anthracene

BaP: Benzo-a-pyrene

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to analogue justification report provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: in whole blood treated with heparin

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 30 µg/mL without and from 40 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Source: EC 701-177-3, LAUS, 2010

Additional studies taken into account in the Weight-of-Evidence approach:

CAS 137-16-6, Harlan, 2010: not clastogenic (lymphocytes)

CAS 30364-51-3, Harlan, 2013b: not clastogenic (lymphocytes)

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to analogue justification report provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥4 µg/mL (experiment I, -S9), ≥ 48 µg/mL (experiment I,+ S9); 96 µg/mL (experiment II, -S9, 24 h), 56 µg/mL (experiment II, +S9, 4 h)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Source: EC 701-177-3, Harlan, 2010b

Additional studies taken into account in the Weight-of-Evidence approach:

CAS 137-16-6, Harlan, 2010c: negative (mouse lymphoma L5178Y)

CAS 30364-51-3, Harlan, 2013c: negative (mouse lymphoma L5178Y TK +/- cells)

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Only an in vitro bacterial mutagenicity test (Ames) is available for Glycine, N-methyl-, N-coco acyl derivatives, sodium salts (CAS 61791-59-1). Therefore, a weight of evidence approach referring to the analogue substances Sodium N-lauroylsarcosinate (CAS 137-16-6), Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) and N-methyl-N-[C18-(unsaturated)alkanoyl]glycine (EC 701-177-3) was applied.

 

Genetic toxicity (mutagenicity) in bacteria in vitro

A bacterial gene mutation assay (Ames test) was performed with Glycine, N-methyl-, N-coco acyl derivatives, sodium salts (CAS 61791-59-1) according to OECD TG 471 and in compliance with GLP (LAUS, 2006). The strains Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 were tested according to the plate incorporation procedure in the first and pre-incubation procedure in the second experiment in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 2 repetitions at concentrations from 1.5 to 149 µg/plate (vehicle: water) in the first experiment, and at concentrations from 37.5 to 149 µg/plate (vehicle: water) in the second experiment. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Cytotoxicity was observed in a range finding test at concentrations above 150 µg/plate. Thus, 149 µg/plate was the highest concentration used in this test with and without metabolic activation. No cytotoxicity was observed in the main study. The included positive and negative controls showed the expected results. Under the conditions of the study, the test substance did not induce mutations in the bacterial gene mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was conducted with Sodium N-lauroylsarcosinate (CAS 137-16-6) in accordance with OECD TG 473 under GLP conditions (Harlan, 2010a). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of fresh heparinised human whole blood cultures, incubated for 4 and 24 h with and without a metabolic activation system (S9-mix from rats treated with phenobarbitone and beta-naphthoflavone). Concentrations of 22 - 360 µg/mL (4 h incubation) and 22.5 - 270 µg/mL (24 h incubation) of the test substance in Minimal Essential Media (MEM) were applied. The negative as well as the positive controls showed the expected results and were within the historical control data. In the preliminary toxicity test, haemolysis of the cultures was observed at 732.5 µg/mL and above in the 4 and 24 h exposure groups. In the main experiment, haemolysis was seen at the end of exposure at and above 270 µg/mL in the exposure groups. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

An in vitro mammalian chromosome aberration test was conducted with Sodium N-methyl-N-(1-oxotetradecyl) aminoacetate (CAS 30364-51-3) in accordance with OECD TG 473 under GLP conditions (Harlan, 2013b). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of freshly heparinised human whole blood cultures, incubated for 4 and 24 h with and without a metabolic activation system (S9-mix from rats treated with phenobarbitone and beta-naphthoflavone). Concentrations of 12 - 200 µg/mL (4 h and 24 h incubation) of the test substance in Minimal Essential Media (MEM) with and without metabolic activation were applied. The negative as well as the positive controls showed the expected results and were within the historical control data. Haemolysis was also observed at and above 100.4 µg/mL in the two 4(20) h exposure groups and at above 200.9 µg/mL in the 24 h continuous exposure group. In the first experiment, haemolysis was seen at and above 100 µg/mL in the exposure groups in absence of metabolic activation, and at 200 µg/mL in presence of metabolic activation. The mitotic index data show that 19% mitotic inhibition and 34% mitotic inhibition was achieved at 100 µg/mL in the absence and presence of metabolic activation respectively. In the second experiment haemolysis and reduced cell pellets were observed at the end of the exposure period at 200 µg/mL in the absence of metabolic activation, and at 100 µg/mL in the presence of metabolic activation. The mitotic index data show that 63% mitotic inhibition was achieved at 75 µg/mL in the absence of metabolic activation. In the presence of metabolic activation the response was not marked with a modest 16% mitotic inhibition being observed. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

An in vitro mammalian chromosome aberration test was conducted with N-methyl-N-[C18-(unsaturated)alkanoyl]glycine (EC 701-177-3) in accordance with OECD TG 473 under GLP conditions (LAUS, 2010). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of fresh heparinised human whole blood cultures, incubated for 4 h with and without and for 22 h without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 10 - 40 µg/mL (4 h incubation) and 0.5 - 50 µg/mL (22 h incubation) of the test substance in the vehicle DMSO were applied. The negative as well as the positive controls showed the expected results. In the pre-experiment cytotoxicity was observed from 55.3 µg/mL with and without metabolic activation. In the main experiments a reduction in the mitotic index was observed from 30 µg/mL without and from 40 µg/mL with metabolic activation. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

The in vitro mammalian cell gene mutation study of Sodium N-lauroylsarcosinate (CAS 137-16-6) was carried out according to OECD TG 476 under GLP conditions (Harlan, 2010c). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphthoflavone-induced rat liver, S9).In the first experiment, cells were exposed for 4 h to test substance at concentrations of 6.25-70 µg/mL and 12.5-100 µg/mL without and with metabolic activation, respectively. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 3.13 to 80 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. In the short- and long-term exposure experiments, cytotoxicity was observed from 60 and 70 µg/mL (4 h) without and with metabolic activation and from 12.5 µg/mL (24 h), respectively. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, Sodium N-lauroylsarcosinate (CAS 137-16-6) did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

The in vitro mammalian cell gene mutation study of Sodium N-methyl-N-(1-oxotetradecyl) aminoacetate (CAS 30364-51-3) was carried out according to OECD TG 476 under GLP conditions (Harlan, 2013c). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphthoflavone-induced rat liver, S9). In the first experiment, cells were exposed for 4 h to test substance at eight concentrations of 1.25 - 50 µg/mL without and with metabolic activation. Nine concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 5 - 60 µg/mL. Nine concentrations of the second experiment for an exposure of 4 h ranged from 10 - 50 µg/mL in presence of metabolic activation. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. In the first and second experiment there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and % RSG values. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, Sodium N-methyl-N-(1-oxotetradecyl) aminoacetate (CAS 30364-51-3) did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

The in vitro mammalian cell gene mutation study of N-methyl-N-[C18-(unsaturated)alkanoyl]glycine (EC 701-177-3) was carried out according to OECD TG 476 under GLP conditions (Harlan, 2010b). Gene mutations in the HPRT locus were investigated in Chinese hamster lung fibroblasts (V79) in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphthoflavone-induced rat liver, S9). V79 cells were incubated with the test material at 0.25, 0.5, 1, 2 and 4 µg/mL for 4 h in the absence and at 4, 8, 16, 32 and 48 µg/mL in the presence of a metabolic activation system. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 4 to 96 µg/mL, and concentrations with metabolic activation for an exposure period of 4 h ranged from 4 to 56 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. Cytotoxicity was apparent at concentrations from 4 µg/mL and 48 µg/mL in experiment I without and with metabolic activation, respectively. In the second experiment, cytotoxicity was observed from 96 µg/mL and 56 µg/mL without and with metabolic activation, respectively. In the second experiment, precipitation was observed from 237.5 µg/mL after 24 h without metabolic activation. There was no significant increase in the number of forward mutations at the HPRT locus of V79 cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, N-methyl-N-[C18-(unsaturated)alkanoyl]glycine (EC No. 701-177-3) did not show gene mutation activity in this test performed in V79 cells in vitro.

 

Conclusion

Taken together, the available in vitro data on genetic toxicity and mutagenicity from the registered substance and several analogue substances do not indicate any mutagenic and clastogenic potential.

Justification for classification or non-classification

The genotoxicity and mutagenicity data as available from the registered and the analogue substances are conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008 (CLP). Therefore, the registered substance Glycine, N-methyl-, N-coco acyl derivatives, sodium salts (CAS 61791-59-1) is considered not to meet the criteria for classification for genetic toxicity.