Registration Dossier

Administrative data

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

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

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

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Oct - 07 Nov 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted in 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: S. typhimurium TA 97a, TA 98, TA 100, TA 102, and TA 1535
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, +S9, -S9: 53, 157, 508, 1507, and 5005 µg/plate
Second experiment, +S9, -S9: 323, 635, 1300, 2607, and 4792 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine (NPD); 2-Amino-anthracene (2-AA)
Remarks:
NPD; 20 µg/plate in water, -S9, TA98, TA 97a and TA 102; sodium azide (1 µg/plate in water, -S9, TA1535 and TA100); 2-AA; 1 µg/plate in DMSO, +S9, all strains; benzo-a-pyrene (BaP; 20 µg/plate in DMSO, +S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 48 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: four in two different experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: Four plates per strain were incubated with the corresponding dose of the test item on maximal soft agar, to test the toxicity of the following concentration: 5005 µg/plate.
The titre was determined according to the following criterion: the determination of the titre should give a number of at least 10E9 cells/mL, correlating to 100 colonies/plate after dilution.
The test item was considered non-toxic, if the quotient titre/tox is below 2.


Evaluation criteria:
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed.
A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
Mean and standard deviation
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Concentrations of the test item are stated as nominal concentrations, as suspensions had to be tested due to the poor solubility of the test item. As no complete dissolution was possiblem undissolved particles were visible on the plates.

First Experiment

Confirmation of the criteria and validity

The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity

The test item was suspended in deionised water. All concentrations were weight directly. As no complete dissolution was possible, undissolved particles were visible on the plates.

No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore the test item is stated as not mutagenic under the test conditions.

Second Experiment

Confirmation of the criteria and validity

The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.

Solubility and toxicity

The test item was suspended in deionised water. All concentrations were weights directly. As no complete dissolution was possible, undissolved particles were visible on the plates. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the test conditions.

Table 1 First experiment: mean revertants

Strain

TA97a

TA97a

TA 98

TA 98

TA100

TA100

TA102

TA102

TA1535

TA1535

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

111±7.6

120±4.2

18±2.5

15±1.2

103±7.1

98±9.6

134±5.2

143±9.2

19±3.4

20±2.9

DMSO

112±8.2

105±5.2

18±2.2

16±2.2

94±15.6

87±9.5

134±8.3

132±7.4

18±1.9

22±1.3

Positive controls*

611±72

726±29

199±20

203±4

441±43

437±26

509±27

543±22

220±14

232±17

5005µg/plate

107±8

116±2

16±2

20±1

83±2

80±5

142±12

152±3

18±3

19±3

1507µg/plate

144±7

119±9

16±2

17±2

85±2

89±6

137±14

144±9

16±3

17±1

508 µg/plate

106±10

109±6

15±2

16±1

89±3

93±12

143±9

134±6

17±3

17±2

157 µg/plate

112±2

111±8

15±2

18±2

85±5

83±12

152±3

137±14

20±4

17±2

53 µg/plate

109±5

107±6

16±2

18±3

81±3

78±5

152±10

129±8

14±2

16±2

 

 Table 2 Second experiment: mean revertants

Strain

TA97a

TA97a

TA 98

TA 98

TA100

TA100

TA102

TA102

TA1535

TA1535

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

117±5

103±3.3

17±2.6

17±2.6

110±3.8

105±3.6

154±7.1

144±5.8

21±2.2

21±3.4

DMSO

116±4.6

113±4.9

16±0.6

15±1.4

97±8

104±3

156±4.8

147±8.5

16±3.5

23±3.0

Positive controls*

629±38

570±25

206±14

235±16

538±58

550±33

628±51

598±15

513±39

474±11

4792µg/plate

115±5

109±4

16±2

13±2

84±4

85±5

151±6

138±3

19±5

12±1

2607µg/plate

115±4

114±3

18±2

16±2

81±6

90±2

132±13

127±11

16±4

14±2

1300 µg/plate

105±5

104±3

17±3

17±2

92±6

89±3

141±3

146±9

16±4

17±5

635 µg/plate

107±5

112±3

14±3

17±1

81±8

108±4

146±8

145±5

14±3

17±3

323 µg/plate

117±4

118±4

14±3

12±1

75±3

82±6

134±12

143±6

19±2

15±2

Conclusions:
Interpretation of results: negative
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, 2010c: not clastogenic (lymphocytes)

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

Conclusions:
Interpretation of results: negative
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, 2010d: negative (mouse lymphoma L5178Y)

CAS 30364-51-3, Harlan, 2013b: 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 limited data on genetic toxicity is available for Reaction mass of N-(1-oxooctadecyl)sarcosine and N-hexadecanoyl-N-methylglycine (EC 947-850-7). 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 reliable bacterial gene mutation assay (Ames test) was performed with Reaction mass of N-(1-oxooctadecyl)sarcosine and N-hexadecanoyl-N-methylglycine (EC 947-850-7) according to OECD TG 471 and in compliance with GLP (LAUS, 2012). The strains Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535 were tested in two independent experiments according to the plate incorporation and pre-incubation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiments were conducted each in 4 replications at concentrations from 53 to 5005 µg/plate in the first experiment and 323 to 4792 µg/plate in the second experiment (vehicle: water). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. 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 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 the analogue substance 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.

An in vitro mammalian chromosome aberration test was conducted with the analogue substance 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 the analogue substance 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.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

The in vitro mammalian cell gene mutation study testing 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 701-177-3) did not show gene mutation activity in this test performed in V79 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, the test substance 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.

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 to be expected with respect to the registered substance.

Justification for classification or non-classification

The available genotoxicity and mutagenicity data from the registered substance and from analogue substances are conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008 (CLP). Therefore, applying the Weight-of-Evidence approach, the registered substance Reaction mass of N-(1-oxooctadecyl)sarcosine and N-hexadecanoyl-N-methylglycine (EC 947-850-7) is considered not to meet the criteria for classification for genetic toxicity.