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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 10, 2014 - May 22, 2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP, so therefore meets the criteria of Klimisch code 1.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Aminoguanidinium hydrogen carbonate
EC Number:
EC Name:
Aminoguanidinium hydrogen carbonate
Cas Number:
Molecular formula:
N-aminoguanidine; carbonic acid
Constituent 2
Reference substance name:
Aminoguanidinium Hydrogen Carbonate
Aminoguanidinium Hydrogen Carbonate
Details on test material:
Name of test item: Aminoguanidinium hydrogen carbonate
CAS No.: 2582-30-1
Purity: 98.6 g / 100 g (based on acidimetric titration)
Storage Conditions: Room temperature


Target gene:
TK +/-
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The mouse lymphoma L5178Y TK+/- cell line (clone 3.7.2c [6]) is derived from a permanent
cell line of 3-methylcholanthrene induced tumors (thymus) in DBA/2 mice and has a
- high proliferation rate (doubling time of about 9 - 10 hours)
- high plating efficiency (about 90%)
- stable karyotype with a near diploid number of 40 ± 1 chromosomes.
Stocks of the mouse lymphoma L5178Y TK+/- cell line (1-mL portions) in culture medium
supplemented with 7% (v/v) DMSO are stored in liquid nitrogen (about -196°C). Each batch
used for mutagenicity testing was checked for mycoplasma contamination previously.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and β-napthoflavone induced rat liver S9 cells
Test concentrations with justification for top dose:
Experiment 1: 43.75, 87.50, 175.00, 350.00, 700.00 and 1400.00 µg/ml with and without S9-mix for 4 hours exposure
Experiment 2: 43.75, 87.50, 175.00, 350.00, 700.00 and 1400.00 µg/ml without S9-mix for 24 hours exposure
65.63, 131.25, 262.50, 525.00, 1050.00 and 1400 µg/ml with S9 mix for 4 hours exposure.
Vehicle / solvent:
RPMI 1640 medium including stable glutamine supplemented with:
1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
1% (v/v) sodium pyruvate (10 mM) (= RPMI-0)
For treatment medium (with S9 mix):
RPMI-0 supplemented with 5% (v/v) fetal calf serum (FCS) (= RPMI-5)
For treatment medium (without S9 mix) and subculturing cells:
RPMI-0 is supplemented with 10% (v/v) fetal calf serum (= RPMI-10)
For cloning efficiency and selection medium:
RPMI-0 supplemented with 20% (v/v) fetal calf serum (= RPMI-20)
Controlsopen allclose all
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
not specified
Positive controls:
Positive control substance:
with activation
Positive controls:
Positive control substance:
without activation
Details on test system and experimental conditions:
- The test material was formulated in culture media prior to serial dilutions being prepared.
- There was no marked change in pH when the test material was dosed into media.

PB/βNF S9 was prepared from the livers of at least 5 male Sprague-Dawley rats weighing approximately 250 g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. The S9 was stored at approximately -70 to -80 °C.
S9-mix was prepared by mixing S9, NADP (4 mM), glucose-6-phosphate (5 mM), KCl (33 mM) and MgCl₂ (8 mM) in phosphate buffer (pH 7.4, 15 mM)

An exponentially growing stock culture of cells was set up. Cells were counted and processed to give 15 x 10⁶ cells/culture for the 4 hour exposure and 10 x 10⁶ cells/culture for the 24 hour exposure in sterile plastic universals. RPMI-5 medium was used for cultures with S9 and RPMI-10 was used for cultures without S9. The treatments were performed in duplicate, both with and without metabolic activation at eight dose levels, vehicle and positive controls. To each universal was added 0.8 mL of S9-mix if required, 2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.
The treatment vessels were incubated at 37 °C for 4 or 24 hours (without S9) or 4 hours (with S9) with continuous shaking using an orbital shaker within an incubated hood.

At the end of the treatment period, for each experiment, the cells were washed twice then resuspended in R20 medium at a cell density of 2 x 10⁵ cells/mL. The cultures were incubated at 37 °C with 5 % CO₂ in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 10⁵ cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 10⁴ cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (% V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (% RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (% V) data a Relative Total Growth (RTG) value.

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5 % CO₂ in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Large colonies are defined as those that cover approximately greater than 1/4 of the diameter of the well.

Evaluation criteria:
The test substance is considered mutagenic if all following criteria are met:
- The mutation frequency exceeds a threshold of 126 mutant colonies per 106 cells (GEF:
Global Evaluation Factor) above the concurrent negative/vehicle control value.
- Evidence of reproducibility of any increase in mutant frequencies, means the mutagenic
response occurs at least in both parallel cultures of one experiment.
- A statistically significant dose-related increase in mutant frequencies using an appropriate
statistical trend test.
The test substance is considered non-mutagenic if at least one of the following criteria is met:
- The mutation frequency is below a threshold of 126 mutant colonies per 106 cells (GEF)
above the concurrent negative/vehicle control value.
- No evidence of reproducibility of an increase in mutant frequencies is obtained.
- No statistically significant dose-related increase
An appropriate statistical trend test (MS EXCEL function RGP; 9) was performed to assess a
possible dose-related increase of mutant frequencies. The number of mutant colonies
obtained for the test substance treated groups was compared with that of the respective
negative/vehicle control groups. A trend was judged as statistically significant whenever the
one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
However, both, biological and statistical significance have been considered together.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
Range finding / screening studies: 1400 µg/ml aminoguanidinium hydrogen carbonate was used as top concentration both with and without S9 mix at 24 hour exposure time. No cytotoxicity indicated by reduced relative suspension growth of 10-20% was observed up to the highest applied concentration after 4 hours and 24 hours hours exposure in the prescence and absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):

The test material was not genotoxic under the conditions of test.
Executive summary:

No cytotoxicity indicated by either reduced relative cloning efficiency 1 (RCE1) or reduced

relative total growth (RTG) of below 20% of control was observed in both experiments.

The test substance was poorly soluble in culture medium. The highest required concentration

tested for gene mutations showed clear test substance precipitates in culture medium.

Based on the results of the present study, the test substance did not cause any biologically

relevant increase in the mutant frequencies either without S9 mix or after adding a

metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions described, Aminoguanidinium hydrogen

carbonate did not induce forward mutations in vitro in the mouse lymphoma assay with

L5178Y TK+/- cells in the absence and the presence of metabolic activation.

The study is classified as reliable without restriction. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.