Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

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:
From October 16, 2016 to November 02, 2016
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
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
Specific details on test material used for the study:
Batch no.: 3837
Purity / composition correction factor: correction factor is 3.9 according to active ingredient
Appearance: clear colourless liquid
Target gene:
Histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: see remarks
Remarks:
rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galactose metabolism; chl: mutation in nitrate reductase; bio defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: see remarks
Remarks:
rfa: deep rough (defective lipopolysaccharide cellcoat); gal: mutation in galactose metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced Aroclor 1254
Test concentrations with justification for top dose:
0.55, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate (based on range finding dose study and cytotoxicity).
The highest concentration of the test substance used in the mutation assays was 5000 μg/plate or the level at which the test substance inhibited bacterial growth.
Vehicle / solvent:
Milli-Q water for the test substance
DMSO or saline for positive controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO or saline
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 (without metabolic activation) and 2-aminoanthracene (with metabolic activation)
Details on test system and experimental conditions:
- Following dose range findings studies, the test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix.
- Exposure: 48 ± 4 h (+ a pre-incubation of 30 min if needed)
Rationale for test conditions:
- Based on the most recent OECD and EC guidelines.
- Dose range finding studies
- First mutation experiment
Evaluation criteria:
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test substance was considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control. b) The negative response should be reproducible in at least one follow up experiment.
- A test substance was considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.
- Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence and presence of S9-mix. No toxicity was observed in tester strain WP2uvrA.
- In the first mutation experiment, the test substance was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at the dose level of 5000 μg/plate in tester strain TA1535. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in tester strains TA1535 and TA1537 in the absence and presence of S9-mix. No biologically relevant toxicity was observed in tester strain TA98.
- In the second mutation experiment, the test substance was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test substance precipitated on the plates at the dose level of 5000 μg/plate in tester strains TA1535 and TA1537 (absence and presence of S9-mix), TA98 (absence of S9-mix) and TA100 (presence of S9-mix). Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strains TA98 in the presence of S9-mix, and WP2uvrA in the absence and presence of S9-mix.
- In this study, acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.
- Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Conclusions:
Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 0.55, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate, and also exposed to the negative or positive control substances for 48 ± 4 h (plus a pre-incubation time of 30 min if needed). In the dose range finding study, the test substance was initially tested up to 5000 µg/plate in the tester strains TA100 and WP2uvrA through a direct plate assay. The test substance showed precipitation at the highest dose level. Cytotoxicity was evidenced in TA100 in the absence and presence of S9 -mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. In the first mutation experiment, the test substance was tested up to concentrations of 1600 and 5000 µg/plate in the absence and presence of S9-mix, respectively in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Since the test substance was severely cytotoxic in the first mutation experiment, an additional dose range finding test was performed with strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test substance was initially tested up to concentrations of 512 and 5000 µg/plate in the tester strains TA100 and WP2uvrA, respectively. The test substance precipitated at dose levels of 1600 and 5000 μg/plate and therefore the number of revertants of this dose level could not be determined. Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed up to the dose level of 1600 μg/plate. In the second mutation experiment, the test substance was tested up to concentrations of 164 and 512 µg/plate (in absence and presence of S9-mix, respectively) in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verspeek-Rip, 2006).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
From March 23, 1994 to May 31, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
RA study
Justification for type of information:
The in vitro genetic toxicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four bacterial strains were tested. But these were two strains each with base pair substitution and frameshift mutation and thus did not influence the quality and validity of the study
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix induced Aroclor 1254
Test concentrations with justification for top dose:
0, 4, 20, 100, 500, 2500 and 5000 µg/plate (all doses were tested in triplicates)
The test substance proved to be toxic for the bacterial strain TA 100 in the absence of a metabolizing system at a dose of 5000µg/plate only in the cytotoxic experiment. Therefore 5000 µg/plate was chosen as the highest dose in the experiment.
Vehicle / solvent:
Bi-distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2- aminoanthracene
Details on test system and experimental conditions:
Test group:
Top agar was prepared for the Salmonella strains by mixing 100 mL agar (0.6 % agar, 0.5 % NaCI) with 10 mL of a 0.5 mM histidine-biotin solution.The following ingredients were added (in order) to 2 mL of molten top agar at approx. 45 °C:
- 0.1mL of an overnight nutrient broth culture of the bacterial tester strain
- 0.1 mL test compound solution
- 0.5 mL S-9 Mix (if required) or buffer
After mixing, the liquid was poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for approximatly 48 hours at approx. 37 °C in the dark, colonies (his* revertants) were counted.
Two independent experiments were performed.
Evaluation criteria:
A test substance is classified mutagenic if either of the following conditions under a) and b) is achieved:
a) a test substance produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test substance induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test article at complete bacterial background lawn.The test results must be reproducible.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Water solubility: the test substance did not precipitate on the plates up to the highest investigated doses
- Precipitation: no
Conclusions:
Based on the results of the read across study, the test substance was not mutagenic in S. typhimurium with and without metabolic activation.
Executive summary:

A supporting study was conducted to determine the in vitro genetic toxicity of the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were exposed to the test substance at concentration levels of 0, 4, 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation (S9-mix induced Aroclor 1254), and to negative or positive control substances for 48 h. In the dose range finding study, the test substance proved to be toxic for the bacterial strain TA 100 in the absence of a metabolizing system at a dose of 5000 µg/plate. Therefore 5000 µg/plate was chosen as the highest dose in the experiment. The test substance did not precipitate on the plates up to the highest investigated doses. The test substance did not induce toxicity or an increase in number of revertants at any concentration level. Under the study conditions, the substance was not mutagenic in S. typhimurium with and without metabolic activation (Hoechst, 1994).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
RA study
Justification for type of information:
Refer to the section 13 of the IUCLID dataset for details on the read across justification. The in vitro genetic toxicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
From 0 to 1600 µg/mL
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
- Exposure of 4 or 24 hours
- Concentrations from 0 to 1600 µg/mL
- With or without metabolic activation (S9 mix)
- Negative (vehicle) and positive controls included
Rationale for test conditions:
- Guideline
- Cytotoxicity
Evaluation criteria:
- Chromosome damages
- Number of chromosomal aberrations
Statistics:
According to guideline
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1. Summary of results – experimental parts without S9 mix

Exp.

Exposure

Test groups

S9

Prec.*

Genotoxicity

Cytotoxicity

 

period

 

mix

 

Micronucleated
cells**

Proliferation index (PI)

RICC***

 

 

 

 

 

[%]

absolute

[%]

1

4 hrs

Negative control

-

n.d.

0.7

2.77

100.0

 

 

10.94 µg/mL

-

-

n.d.

n.d.

941.5

 

 

21.88 µg/mL

-

-

n.d.

n.d.

91.3

 

 

43.75 µg/mL

-

-

n.d.

n.d.

91.1

 

 

87.50 µg/mL

-

-

n.d.

n.d.

89.1

 

 

175.00 µg/mL

-

-

0.6

2.73

88.9

 

 

350.00 µg/mL

-

-

0.8

2.58

86.1

 

 

700.00 µg/mL+

-

-

4.3S

2.28

41.4

 

 

Positive control2

-

n.d.

2.7S

2.52

n.t.

3

4 hrs

Negative control

-

n.d.

0.8

2.32

100.0

 

 

200.00 µg/mL

-

-

n.d.

n.d.

107.0

 

 

400.00 µg/mL

-

-

0.5

2.16

94.5

 

 

600.00 µg/mL

-

-

0.7

2.38

85.4

 

 

700.00 µg/mL+

-

-

1.0

2.23

78.1

 

 

800.00 µg/mL

-

-

n.s.

n.s.

-0.5

 

 

Positive control1

-

n.d.

2.4S

2.30

n.t.

2

24 hrs

Negative control

-

n.d.

1.2

2.35

100.0

 

 

200.00 µg/mL

-

-

n.d.

n.d.

93.3

 

 

400.00 µg/mL

-

-

n.d.

n.d.

90.6

 

 

600.00 µg/mL

-

-

1.4

2.08

84.0

 

 

700.00 µg/mL

-

-

0.3

2.23

88.4

 

 

800.00 µg/mL

-

-

0.6

2.09

48.1

 

 

Positive control1

-

n.d.

9.1S

2.32

n.t.

*      Precipitation in culture medium at the end of exposure period

**     Relative number of micronucleated cells per 1000 cells scored per test group

***   Relative increase in cell count (RICC)

+      To confirm the results an increased sample of 4 000 cells was scorted.

S      Frequency statistically significant higher than corresponding control values

n.d. Not determined     n.t.     Not tested

n.s. Not scorable due to strong cytotoxicity

1      EMS 500 µg/mL            2        EMS 600 µg/mL

Table 2. Summary of results – experimental parts with S9 mix

Exp.

Exposure

Test groups

S9

Prec.*

Genotoxicity

Cytotoxicity

 

period

 

mix

 

Micronucleated
cells**

Proliferation index (PI)

RICC***

 

 

 

 

 

[%]

absolute

[%]

1

4 hrs

Negative control

+

n.d.

1.5

2.70

100.0

 

 

5.47 µg/mL

+

-

n.d.

n.d.

98.3

 

 

10.94 µg/mL

+

-

n.d.

n.d.

96.1

 

 

21.88 µg/mL

+

-

n.d.

n.d.

125.4

 

 

43.75 µg/mL

+

-

n.d.

n.d.

114.8

 

 

87.50 µg/mL

+

-

1.7

2.56

117.6

 

 

175.00 µg/mL

+

-

0.5

2.47

97.2

 

 

350.00 µg/mL

+

-

0.9

2.43

93.6

 

 

Positive control1

+

n.d.

8.5S

2.32

n.t.

2

4 hrs

Negative control

+

n.d.

0.8

2.31

100.0

 

 

100.00 µg/mL

+

-

n.d.

n.d.

81.7

 

 

200.00 µg/mL

+

-

1.0

2.27

70.5

 

 

400.00 µg/mL

+

-

1.1

2.07

54.1

 

 

800.00 µg/mL

+

-

1.3

2.05

39.4

 

 

1 600.00 µg/mL

+

-

n.s.

n.s.

32.7

 

 

Positive control1

+

n.d.

15.2S

2.09

n.t.

3

4 hrs

Negative control

+

n.d.

1.5

2.03

100.0

 

 

75.00 µg/mL

+

-

n.d.

n.d.

107.0

 

 

150.00 µg/mL

+

-

n.d.

n.d.

109.3

 

 

300.00 µg/mL

+

-

n.d.

n.d.

99.5

 

 

600.00 µg/mL

+

-

0.8

1.98

89.2

 

 

1 200.00 µg/mL

+

-

1.3

2.23

91.5

 

 

1 600.00 µg/mL+

+

-

1.6

2.03

76.4

 

 

Positive control1

+

n.d.

12.5S

1.80

n.t.

*      Precipitation in culture medium at the end of exposure period

**     Relative number of micronucleated cells per 1000 cells scored per test group

***   Relative increase in cell count (RICC)

+      To confirm the results an increased sample of 4 000 cells was scorted.

S      Frequency statistically significant higher than corresponding control values

n.d. Not determined              n.t.     Not tested

n.s. Not scorable due to strong cytotoxicity

1      CPP 2.5 µg/mL

Conclusions:
Under the study conditions, the substance was not clastogenic in Chinese hamster lung fibroblast V79 cells.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read-across substance L-glutamic acid, N-coco acyl derivs., disodium salts according to OECD Guideline 487, in compliance with GLP. Two independent experiments were performed in Chinese hamster lung fibroblasts (V79 cells). Cells were exposed to the test substance (diluted in water) at concentrations from 0 to 1600 µg/mL for either 4 or 24 h with and without metabolic activation (S9 mix).Cytotoxicity, indicated by a clearly reduced proliferation index, cell numbers or a low quality of the slides was observed in the absence of S9 mix at 700 µg/mL (1st experiment) and at 800 µg/mL (2nd and 3rd experiment). In the presence of S9 mix, cytotoxic effects were obtained 800 µg/mL onwards but only in the 2nd experiment. In the 1st experiment a single statistically significant increase in the number of micronucleated cells was observed at 700 µg/mL after 4 h exposure in the absence of metabolic activation. However, this finding was neither confirmed in the repeat experiment under similar conditions (3rd experiment) nor in any additional experiment under modified test conditions (24 h exposure in the absence of metabolic activation or 4 h exposure in the presence of metabolic activation). The finding was considered artifactual due to the strong cytotoxicity in this test group (RICC 41.4%). Under the study conditions, the substance was not clastogenic in Chinese hamster lung fibroblast V79 cells (BASF SE, 2013).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
RA study
Justification for type of information:
Refer to the section 13 of the IUCLID dataset for details on the read across justification. The in vitro genetic toxicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital- and β- naphthoflavone induced rats (S9 mix)
Test concentrations with justification for top dose:
- 1st Experiment
without S9 mix (4-hour exposure period)
0, 25.0, 50.0, 100.0, 200.0, 400.0 and 800.0 μg/mL
with S9 mix (4-hour exposure period)
0, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 μg/mL
- 2nd Experiment
without S9 mix (24-hour exposure period) (discontinued due to lacking cytotoxicity)
0, 10.9, 21.9, 43.8, 87.5, 175.0 and 350.0 μg/mL
with S9 mix (4-hour exposure period)
0, 15.6, 31.3, 62.5, 125.0, 250.0 and 400.0 μg/mL
- 3rd Experiment
without S9 mix (24-hour exposure period)
0, 46.9, 93.8, 187.5, 375.0, 750.0 and 1 500.0 μg/mL
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
- Exposure of 4 or 24 hours
- Concentrations from 0 to 1500 µg/mL
- With and withour metabolic activation (S9 mix)
Rationale for test conditions:
- Guideline
- Cytotoxicity
Evaluation criteria:
Mutant frequencies at the HPRT locus
Statistics:
According to guideline
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Under the study conditions, the substance was not mutagenic at the HPRT locus of Chinese hamster ovary cells.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read-across substance L-glutamic acid, N-coco acyl derivs., disodium salts according to OECD Guideline 476, in compliance with GLP. The potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was evaluated in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β- naphthoflavone induced rats (exogenous metabolic activation). In initial range-finding cytotoxicity test(s), the experimental doses of the main experiments were determined. Cells were exposed for 4 or 24 h to the test substance at concentrations from 0 to 1500 µg/mL. Vehicle and positive controls (ethyl methanesulphonate and 7,12 -dimethylbenzanthracene) were included as well in the experiments. The vehicle control showed mutation frequencies within the range expected for the CHO cell line. Both positive control substances led to the expected increase in the frequencies of forward mutations. In the 1st experiment in the absence and presence of metabolic activation and in the 2nd experiment in the presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd experiment in the absence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd experiment was performed which clearly showed reduced colony numbers at least at the highest concentrations. The test substance did not cause any relevant increase in the mutation frequency either without S9 mix or after the addition of a metabolizing system in the experiments. Under the study conditions, the substance was not mutagenic at the HPRT locus of Chinese hamster ovary cells (BASF SE, 2012).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro:

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 0.55, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate, and also exposed to the negative or positive control substances for 48 ± 4 h (plus a pre-incubation time of 30 min if needed). In the dose range finding study, the test substance was initially tested up to 5000 µg/plate in the tester strains TA100 and WP2uvrA through a direct plate assay. The test substance showed precipitation at the highest dose level. Cytotoxicity was evidenced in TA100 in the absence and presence of S9 -mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. In the first mutation experiment, the test substance was tested up to concentrations of 1600 and 5000 µg/plate in the absence and presence of S9-mix, respectively in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix. Since the test substance was severely cytotoxic in the first mutation experiment, an additional dose range finding test was performed with strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test substance was initially tested up to concentrations of 512 and 5000 µg/plate in the tester strains TA100 and WP2uvrA, respectively. The test substance precipitated at dose levels of 1600 and 5000 μg/plate and therefore the number of revertants of this dose level could not be determined. Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed up to the dose level of 1600 μg/plate. In the second mutation experiment, the test substance was tested up to concentrations of 164 and 512 µg/plate (in absence and presence of S9-mix, respectively) in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verspeek-Rip, 2006).

A study was conducted to determine the in vitro genetic toxicity of the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were exposed to the test substance at concentration levels of 0, 4, 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation (S9-mix induced Aroclor 1254), and to negative or positive control substances for 48 h. In the dose range finding study, the test substance proved to be toxic for the bacterial strain TA 100 in the absence of a metabolizing system at a dose of 5000 µg/plate. Therefore 5000 µg/plate was chosen as the highest dose in the experiment. The test substance did not precipitate on the plates up to the highest investigated doses. The test substance did not induce toxicity or an increase in number of revertants at any concentration level. Under the study conditions, the substance was not mutagenic in S. typhimurium with and without metabolic activation (Hoechst, 1994).

A study was conducted to determine the in vitro genetic toxicity of the read-across substance L-glutamic acid, N-coco acyl derivs., disodium salts according to OECD Guideline 487, in compliance with GLP. Two independent experiments were performed in Chinese hamster lung fibroblasts (V79 cells). Cells were exposed to the test substance (diluted in water) at concentrations from 0 to 1600 µg/mL for either 4 or 24 h with and without metabolic activation (S9 mix).Cytotoxicity, indicated by a clearly reduced proliferation index, cell numbers or a low quality of the slides was observed in the absence of S9 mix at 700 µg/mL (1st experiment) and at 800 µg/mL (2nd and 3rd experiment). In the presence of S9 mix, cytotoxic effects were obtained 800 µg/mL onwards but only in the 2nd experiment. In the 1st experiment a single statistically significant increase in the number of micronucleated cells was observed at 700 µg/mL after 4 h exposure in the absence of metabolic activation. However, this finding was neither confirmed in the repeat experiment under similar conditions (3rd experiment) nor in any additional experiment under modified test conditions (24 h exposure in the absence of metabolic activation or 4 h exposure in the presence of metabolic activation). The finding was considered artifactual due to the strong cytotoxicity in this test group (RICC 41.4%). Under the study conditions, the substance was not clastogenic in Chinese hamster lung fibroblast V79 cells (BASF SE, 2013).

A study was conducted to determine the in vitro genetic toxicity of the read-across substance L-glutamic acid, N-coco acyl derivs., disodium salts according to OECD Guideline 476, in compliance with GLP. The potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was evaluated in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β- naphthoflavone induced rats (exogenous metabolic activation). In initial range-finding cytotoxicity test(s), the experimental doses of the main experiments were determined. Cells were exposed for 4 or 24 h to the test substance at concentrations from 0 to 1500 µg/mL. Vehicle and positive controls (ethyl methanesulphonate and 7,12 -dimethylbenzanthracene) were included as well in the experiments. The vehicle control showed mutation frequencies within the range expected for the CHO cell line. Both positive control substances led to the expected increase in the frequencies of forward mutations. In the 1st experiment in the absence and presence of metabolic activation and in the 2nd experiment in the presence of metabolic activation, the highest concentrations tested for gene mutations were clearly cytotoxic. However, in the 2nd experiment in the absence of metabolic activation no cytotoxicity was observed up to the highest applied concentration. Therefore, a 3rd experiment was performed which clearly showed reduced colony numbers at least at the highest concentrations. The test substance did not cause any relevant increase in the mutation frequency either without S9 mix or after the addition of a metabolizing system in the experiments. Under the study conditions, the substance was not mutagenic at the HPRT locus of Chinese hamster ovary cells (BASF SE, 2012).

Justification for classification or non-classification

Based on in vitro genetic toxicity studies with the substance itself and with the read-across substances L-glutamic acid, N-coco acyl derivs., monosodium salts and L-glutamic acid, N-coco acyl derivs., disodium salts

, no classification for genetic toxicity is warranted according to EU CLP (1272/2008) criteria.