Glycine hydrochloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2017 - 03 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Glycine hydrochloride

EC Number:

227-841-8

EC Name:

Glycine hydrochloride

Cas Number:

6000-43-7

Molecular formula:

C2H5NO2.ClH

IUPAC Name:

2-aminoacetic acid

Test material form:

solid

Method

Target gene:

his/trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

beta-Naphthoflavone/Phenobarbital induced liver homogenate (S9 mix) from young male Wistar rats, Crl:Wl (HAN)

Test concentrations with justification for top dose:

In accordance with the recommendations guideline OECD TG471 for the selection of the concentrations to be tested, 5000 ug/plate was selected as the maximum concentration (OECD TG 471, 1997)
1st series: 5.0 15.8, 50.0 158.0, 500.0, 1580.0, 5000.0 µg/plate with and without 10% S9 mix
2nd series: 50.0, 158.0, 500.0, 1580.0, 5000.0 µg/plate with and without 20% S9 mix

Vehicle / solvent:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: Solubility properties of the test item, non-toxicity to bacteria.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: Daunomycin DAUN, 1.0 µg/plate; 2-Aminoanthracene 2-AA, 2.0, 5.0, 10.0 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 parallel plates were used for each concentration step of the lest material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.

DETERMINATION OF CYTOTOXICITY
- Method: The presence of a background lawn of non-reverlant cells was checked for each plate.

Evaluation criteria:

Evaluation Criteria:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established (see Table 1).
Interpretations:
A test material was to be defined as negative or non-mutagenic in this assay if
• the assay was to be considered valid, and
• "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)
For valid data, the test material was considered to be positive or mutagenic if:
• a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
• "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.
In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response (OECD TG 471, 1997).
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 2: Summary 1st series

Metabolic Activation Test Material Concentr. [µg/plate] Revertants per plate (Mean ± SD)   TA 98 TA 100 TA 1535 TA 1537 WP2 uvrA   Without Activation H2O   37 ± 8 111 ± 20 21 ± 4 6 ± 3 31 ± 8   Test Item 5.00 39 ± 4 109 ± 13 19 ± 2 9 ± 3 27 ± 9   15.8 43 ± 19 122 ± 12 19 ± 5 8 ± 1 30 ± 8   50.0 34 ± 3 109 ± 12 20 ± 5 6 ± 1 40 ± 4   158 25 ± 4 104 ± 9 18 ± 3 6 ± 3 29 ± 3   500 32 ± 9 115 ± 12 15 ± 3 6 ± 2 32 ± 9   1580 29 ± 5 114 ± 12 19 ± 4 9 ± 6 34 ± 10   5000 32 ± 10 116 ± 12 16 ± 5 5 ± 4 37 ± 12   DAUN 1.00 347 ± 35           NaN3 2.00   1407 ± 124 808 ± 25       9-AA 50.0       777 ± 226     NQO 2.00         1831 ± 77   With Activation H2O   41 ± 7 125 ± 22 18 ± 3 8 ± 3 34 ± 10   Test Item 5.00 45 ± 2 116 ± 19 18 ± 4 5 ± 2 39 ± 7   15.8 36 ± 15 128 ± 11 22 ± 2 9 ± 2 36 ± 7   50.0 35 ± 1 115 ± 8 14 ± 7 10 ± 5 38 ± 14   158 47 ± 5 120 ± 8 17 ± 4 10 ± 3 36 ± 9   500 33 ± 7 120 ± 14 14 ± 1 11 ± 3 42 ± 6   1580 37 ± 7 108 ± 14 21 ± 3 8 ± 0 34 ± 5   5000 30 ± 6 146 ± 8 24 ± 10 5 ± 3 44 ± 7   2-AA 2.00 1166 ± 163 1726 ± 226         2-AA 5.00     157 ± 16 395 ± 23     2-AA 10.0         187 ± 171

Table 3: Summary 2nd series

Metabolic Activation Test Material Concentr. [µg/plate] Revertants per plate (Mean ± SD)   TA 98 TA 100 TA 1535 TA 1537 WP2 uvrA   Without Activation H2O   35 ± 6 120 ± 15 18 ± 4 8 ± 3 37 ± 8   Art. G2879 50.0 32±6 124±2 19±5 9±3 34±8   158 29±7 120±8 18±3 8±2 27±9   500 35±10 126±11 18±5 8±4 27±8   1580 33±3 123±8 23±6 7±1 35±8   5000 33±8 122±8 22±3 12±4 40±6   DAUN 1.00 314±46           NaN3 2.00   1601±120 860±35       9-AA 50.0       709±137     NQO 2.00         2008±95   With Activation H2O   38 ± 8 136 ± 17 22 ± 4 13 ± 3 39 ± 6   Art. G2879 50.0 33 ± 8 117 ± 11 16 ± 6 15 ± 1 41 ± 15   158 37 ± 7 136 ± 2 20 ± 6 14 ± 3 39 ± 10   500 35 ± 5 128 ± 1 19 ± 8 11 ± 2 38 ± 3   1580 36 ± 6 129 ± 18 20 ± 3 13 ± 1 39 ± 5   5000 34 ± 2 130 ± 4 18 ± 7 14 ± 2 33 ± 9   2-AA 2.00 349 ± 9 847 ± 53         2-AA 5.00     138 ± 13 148 ± 13     2-AA 10.0         250 ± 14

Key to Positive Controls NaN3             Sodium azide 2-AA             2-Aminoanthracene 9-AA             9-Aminoacridine DAUN             Daunomycin NQO             4-Nitroquinoline-N-oxide

Applicant's summary and conclusion

Conclusions:

In an in vitro bacterial reverse mutation assay (Ames test) according to OECD Guideline 471, the test item did not show a mutagenic potential.

Executive summary:

In an in vitro bacterial reverse mutation assay (Ames test) according to OECD Guideline 471, the mutagenic potential of the test item was determined. Two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms were performed.

The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/beta-Naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used. Treatments of all tested strains were performed using the test item formulations prepared in ultra-pure water in the absence and in the presence of S9 mix, using final concentrations between 5 and 5000 µg/plate, plus vehicle and positive controls. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, and 9-aminoacridine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation, was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning. Thus, the study is considered valid. Following treatment with the test item, no precipitation of the test material on the agar plates occurred. No toxicity to the bacteria was observed.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix.