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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity, in-vitro:

Gene mutation (Bacterial reverse mutation assay/Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA: negative with and without metabolic activation (according to OECD 471)

Chromosome aberration (in-vitro mammalian chromosome aberration test): negative with cultured CHL cells without metabolic activation

Gene mutation (in-vitro mammalian cell gene mutation test): negative with cultured L5178Y mouse lymphoma cells with and without metabolic activation (according to OECD 476).

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
RPMI 1640
Properly maintained:
yes
Periodically checked for Mycoplasma contamination:
yes
Periodically checked for karyotype stability:
no
Periodically "cleansed" against high spontaneous background:
yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose levels used in the Mutagenicity Test was the 10 mM dose limit.
Vehicle and positive controls were used in parallel with the test item. Solvent (R0 media) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch BCBG1395V at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0302605 at 2 µg/mL was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
- Justification for choice of solvent/vehicle:
soluble
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (R0 medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.
The dose range of test item was selected following the results of a preliminary toxicity test and was 23.44 to 750 µg/mL in absence and presence of metabolic activation in Experiment 1. In Experiment 2 the dose range was again 23.44 to 750 µg/mL in the absence and presence of metabolic activation.
The maximum dose levels used in the Mutagenicity Test was the 10 mM dose limit. No precipitate of the test item was observed at any dose level. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
Evaluation criteria:
Please see ''Any other information on materials and methods incl. tables'' section
Statistics:
Please see ''Any other information on materials and methods incl. tables'' section
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
modest toxicity 24 hour only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range of the test item used in the preliminary toxicity test was 2.93 to 750 µg/mL.
In both of the 4 hour (+/- S9) exposure groups there was no evidence of dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure group there was evidence of modest reductions in %RSG when compared to the vehicle control values. No precipitate of the test item was observed at any dose level. The maximum dose level used in the subsequent Mutagenicity Test was the maximum recommended 10 mM dose limit.
Experiment 1
The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
There was no evidence of dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). There was no evidence of a marked reduction in viability (%V), therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).
The test item did not induce and statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in the absence or presence of metabolic activation. No Precipitate of the test item was observed at any dose level.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.
Experiment 2
The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13.
As was seen previously there was no evidence of toxicity following exposure to the test item in the presence of metabolic activation and there was evidence of moderate dose-related toxicity following exposure to the test item in the absence of metabolic activation as indicated by the RTG and %RSG values (Tables 9 and 12). There was no evidence of any reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).
The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation. No precipitate of the test item was observed at any dose level.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Please see Attached ''Tables 1 -13'' Due to the quantity and the size of the tables it was not possible to insert them into this section.

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
T
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedures in accordance with accepted standard methods, sufficiently documented
Qualifier:
no guideline followed
Principles of method if other than guideline:
The chinese hamster fibroblast cells were exposed to test substance at three different concentrations for 24 and 48 hr. In this study, no metabolic activation systems were applied.
GLP compliance:
no
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung fibroblasts (CHL)
Metabolic activation:
without
Test concentrations with justification for top dose:
Maximum dose: 1.0 mg/mL
*No growth inhibition at 10 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
DURATION
-Exposure duration: 24 or 48 hr

NUMBER OF PLATES : no data

NUMBER OF CELLS EVALUATED: 100 per dose
Evaluation criteria:
The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %.
Statistics:
no data
Species / strain:
other: Chinese hamster lung fibroblasts (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed
in-house.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzo(a)pyrene: 5 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-Aminoacridine: 80 µg/plate
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethylN-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Remarks:
without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation), Experiment 2: pre-incubation

DURATION
- Preincubation period for bacterial strains: 20 min at 37°C
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10E09 bacteria per mL.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a
reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be
the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
Dunnett's method of linear regression
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, 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:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item (0.15-5000 µg/plate) was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment
were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

RESULTS

Preliminary Toxicity Test

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without

(-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

88

92

99

88

95

110

115

91

86

79

89

+

TA100

87

74

72

77

81

83

86

74

82

83

82

-

WP2uvrA

42

27

31

44

40

29

38

43

48

43

41

+

WP2uvrA

42

49

42

37

34

43

47

46

40

48

43

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.

A copy of the Certificate of Analysis as provided by the sponsor, is attached as background material.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, were presented in attached background material.

A history profile of vehicle/untreated and positive control values (reference items) for 2010 and 2011 were presented in attached background material.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

65

 

13

 

19

 

19

 

7

 

72

(70)

13

(12)

30

(26)

19

(16)

4

(6)

74

 

11

 

28

 

11

 

7

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

81

 

18

 

31

 

18

 

8

 

75

(86)

9

(14)

34

(34)

16

(20)

3

(8)

101

 

14

 

36

 

25

 

12

 


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

Gene mutation in bacteria

Mutagenicity in bacteria was assessed in a GLP-study performed according to OECD 471 (Thompson, 2012). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2uvrA were treated with the test item using both, the plate incorporation and pre-incubation methods at dose levels of 50, 150, 500, 1500, and 5000 µg/plate, in triplicate, both with and without metabolic activation. The dose range was determined in a preliminary toxicity assay. Vehicle and appropriate controls were included into the study design. The vehicle control plates gave counts of revertant colonies generally within the normal range. All of the positive controls induces marked increases in the frequency of revertant colonies, both with and without metabolic acitation, thereby confirming sensitivity of the study and efficacy of the metabolising system. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. No precipitation was observed on the plates at any of the doses tested. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Furthermore, there are three tests available, investigating gene mutation in Salmonella thyphimurium strains TA92, TA 94, TA 98, TA 100, TA 1535, and TA 1537 at dose concentration of 10 up to 35000 µg/plate in pre-incubation assays with and without metabolic acitvation (NTP, 1974a/b, Ishidate et al., 1984). All tests revealed no increases in the frequency of revertant colonies at any dose level and independent of the use of a metbolising system.

Taken all these data into consideration, the test item is considered to be non-mutagenic in bacteria.

Cytogenicity in mammalian cells

The clastogenic potential of the test substance in vitro was assessed in a chromosomal aberration test in chinese hamster fibroblast (CHL) cells (Ishidate et al. 1982). CHL cells were exposed to glycine dissolved in saline at three different doses levels, being 1 mg/mL the maximum dose level, for 24 and 48 hours without metbolic activation. 100 cells per dose were evaluated for chromosomal aberration. Untreated cells served as negative controls. No information on positive controls were given in the study report. No increase in the frequency of chromosome aberration was observed at any dose level when compared to negative controls.

80% of all soluble proteins are N-acetylated at the amino terminus, glycine being the most frequent (Harper et al. 2010). N-acetylpeptide hydrolase releases acetylated amino acids which are then deacetylated by aminoacylases into constituent amino acids. Therefore, and due to the lack of a metabolic activation system in the cytogenicity study performed by Ishidate et al. (1982), an in vivo bone marrow micronucleus assay (BMM) conducted with N-acetylglycine was in addition used for cytogenictiy assessment of glycine (Harper et al. 2010). The assay was conducted in accordance with OECD 474. In the BMM study, groups each comprising five male and female ICR (Crl:CD1) mice, which received a single oral dose of 500, 1000, or 2000 mg N-acetylglycine /kg bw or a negative (0.5% methylcellulose in purified deionized water) or positive (cyclophosphamide monohydrate (50 mg/kg bw)) control substance. Animals were sacrificed 24 h after treatment. Mice in remaining groups, receiving either water or N-acetylglycine at 2000 mg/kg bw, were sacrificed 48 h after treatment. No mortality and adverse clinical signs were observed during the study. Regarding cytogenicity, no increase in the incidence of micronucleated polychromatic erythrocytes (MPCEs) in the bone marrow of mice were observed at 24 or 48 h after treatment with N-acetylglycine. At 24 h following treatment with cyclophosphamide, an incrase in the incidence of MPCEs in both male and female mice was observed, thereby confirming validity of the study.

Taken all these data into consideration, the test item is considered to be non-clastogenic.

Gene mutation in mammalian cells

The mutagenic potential of the test substance in mammalian cells in vitro was assessed in a L5178Y mouse lymphoma assay performed according to OECD 476 under GLP conditions (Brown, 2013). The dose range tested was selected following the results of a preliminary toxicity test. Two independent main tests were performed. In experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells were treated with the test item at six dose levels (23.44 - 750 µg/mL) in duplicate, together with the solvent vehicle and appropriate positive control substances using a 4-hour treatment time both in the abscence and presence of metabolic activation. In experiment 2, the cells were treated with the test item at the same six dose levels using a 4-hour treatment time in the presence of metabolic activation and a 24-hour exposure time in the abscence of metabolic activation. No precipitation of the test substance was observed at any dose level. The solvent controls revealed acceptable mutant frequency values that were within the normal range for this cell line. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. Glycine did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. Therefore, the test substance was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference

Harper M.S., Shen Z.A., Barnett J.F.jr, Krsmanovic L., Dakoulas E.W., Delaney B. (2010) Toxicology studies with N-acetylglycine. Food and Chemical Toxicology 48, 1321 -1327

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.