Disodium Capryloyl Glutamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Different tests were done in order to assess genetic toxicity.

All were negative.

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:

From 06 March 2019 to 06 May 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

Also Commission Regulation (EC) n° 440/2008 B.13/14 and EPA Health Effects Test Guidelines, OPPTS 870.5100 were kept in consideration.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch 3747 supplied by sponsor
clear liquid
pH as it is 10 (20 °C)
Dry matter 38.5% (in water) as active ingredient, purity 100%
Soluble in water
NaCl 5.1%
Constituents are in the same range used for registration
Storage condition of test material: room temperature (15 °C - 25 °C)
Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable for 12 months minimum
Solubility: soluble and stable in water
Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive
More information can be found on the attached report

Target gene:

Histidine

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa-, uvrB-, R-factor

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomial fraction is obtained from male Wistar rats induced with phenobarbital (80 mg/kg bw) and Beta-naphthoflavone (100 mg/kg bw) for 3 consecutive days by oral route.

Test concentrations with justification for top dose:

The toxicity of the test item was determined with tester strainsTA98 and TA100 in a pre-experiment. The following concentrations were tested:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 microl/plate.
According to the obtained results the below concentrations were chosed for the test.
5.0 microl/plate was selected as the maximum concentration and the concentration range covered two logarithmic decades.
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 microl/plate.

Vehicle / solvent:

Purified water for the substance,
purified water or DMSO for positive controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or purified water

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene

Details on test system and experimental conditions:

Following dose range finding studies, the test was done as descibed below. For the plate incorporation method the following materials are mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL overlay agar.
For the pre-incubation method 100 µL of the test item-preparation is pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, a minimum of three plates is used.
After solidification the plates are inverted and incubated at 37 °C for at least 48 h in the dark.
Two different experiments were performed, plate incorporation test (experiment I) and pre-incubation test (experiment 2). The colonies are counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precludes automatic counting the revertant colonies are counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 are counted manually.

Rationale for test conditions:

Based on OECD 471 (1997) and also on Commission Regulation (EC) n° 440/2008 B.13/14 and EPA Health Effects Test Guidelines, OPPTS 870.5100.
A dose range finding study was performed.

Evaluation criteria:

Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as N or B respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

For soluble, non toxic test compounds the recommended maximum test concentration is 5 microl/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met.

All tables are 

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the substamce is considered to be non-mutagenic in this bacterial reverse mutation assay

Executive summary:

Bacterial reverse mutation assays use amino-acid requiring strains of Salmonella typhimurium
(S. typhimurium) to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of these bacterial reversion assays is that they detect mutations which functionally reverse mutations present in the tester strains and restore the capability to synthesize an essential amino acid.
The purpose of this study is to establish the potential of the test item to induce gene mutations in bacteria by means of a S. typhimurium reverse mutation assay. There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentrations spacing and / or the method of treatment (pre-incubation method). In case of severe
toxicity of the test item or the use of e.g. ethanol, acetone or tetrahydrofuran as the most appropriate solvent, the confirmatory experiment is carried out according to the plate incorporation method with a different spacing between dose levels.
The S. typhimurium histidine (his) reversion system measures his- → his+ reversions. The
S. typhimurium strains are constructed to differentiate between base pair (TA100, TA1535, TA102) and frameshift (TA98, TA1537) mutations.
These assays directly measure heritable DNA mutations of a type which is associated with adverse effects. Point mutations are the cause of many human genetic diseases and
there is substantial evidence that somatic cell point mutations in oncogenes and tumour suppressor genes are involved in cancer in humans and experimental systems.
The tester strains have several features that make them more sensitive for the detection of
mutations. The specificity of the strains can provide useful information on the types of mutations that are induced by mutagenic agents.
According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.
At least five different concentrations of the test item are tested with approximately half log (i.e. √10)
intervals between test points for an initial test. Narrower spacing between dose levels may be
appropriate when a dose response is investigated. For soluble, non-toxic test compounds the
recommended maximum test concentration is 5 mg/plate or 5 µL/plate.
To validate the test, reference mutagens are tested in parallel to the test item. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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^+	-	-	4.3^S	2.28	41.4
		Positive control^2	-	n.d.	2.7^S	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 control^1	-	n.d.	2.4^S	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 control^1	-	n.d.	9.1^S	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 control^1	+	n.d.	8.5^S	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 control^1	+	n.d.	15.2^S	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 control^1	+	n.d.	12.5^S	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 substance 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).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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 substance 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).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From March 23, 1994 to May 31, 1994

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:

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 stdy

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

True negative controls:

no

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:
- 0.1 mL 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 h 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: TA1535, TA2537, 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:

Under the study conditions, the substance was not mutagenic in S. typhimurium strains with and without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the substance 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 strains with and without metabolic activation (Hoechst, 1994).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met.

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 the in vitro genetic toxicity study with the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts, no conclusion on classification for genetic toxicity could be derived according to EU CLP (1272/2008) criteria.