Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 17 to March 14, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
no
Remarks:
the available study was originally run not for REACH purposes; however, it is scientifically valid and well documented.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomial fraction.
Preparation of the S9 mix
The S9 mix contains an lyophilized homogenate of liver enzymes reconstituted with sterile water, prepared from adult male Sprague Dawley rats liver induced with Aroclor 1254 (Moltox). The ability of the S9 mix to metabolically activate an indirect mutagen (2-aminoanthracene) was assayed by the Ames test. The S9 mix was prepared immediately before use.
S9 mix composition (50 ml):
- 2 ml rat liver S9 (4 %)
- 1ml MgCl2 0,4M and KCl 1.65 M
- 0,25 ml glucose-6-phosphato 1 M
- 2 ml NADP 0.1M
- 25 ml buffer sodium phosphate 0.2 M pH 7.4
- 19.75 ml sterile deionised water
Test concentrations with justification for top dose:
Test concentrations with and without S9:
5, 1.6, 0.512, 0.164, 0.052 mg/plate

Negative control: 100 µl/plate

Positive controls:
STRAINS POSITIVE CONTROLS DOSAGE LEVELS
without S9
TA 1535 sodium azide (NaN3) in water 1.5 μg/plate
TA 1537 9-aminoacridine (9AA) in water 75 μg/plate
TA 100 sodium azide (NaN3) in water 2.0 μg/plate
TA 98 4-nitroquinoline-N-oxide (4NQO) in DMSO 0.5 μg/plate
TA 102 4-nitroquinoline-N-oxide (4NQO) in DMSO 1.5 μg/plate

with S9
TA 1535 2-aminoanthracene (2AAn) in DMSO 10 μg/plate
TA 1537 2-aminoanthracene (2AAn) in DMSO 3.0 μg/plate
TA 100 2-aminoanthracene (2AAn) in DMSO 3.0 μg/plate
TA 98 2-aminoanthracene (2AAn) in DMSO 3.0 μg/plate
TA 102 2-aminoanthracene (2AAn) in DMSO 10 μg/plate
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
AIM OF THE TEST
Ames test allows to detect the induction of point mutations in nucleotidic bases, such as deletions, insertions, transversions and frameshift errors by using modified Salmonella typhimurium strains. These strains carry a defective gene in the histidine operone making them auxotrophe for this aminoacid (mutants His- which require histidine in the culture medium for growth). The method guiding principle is based on the backmutation: phenomenon by which bacteria exposed to a mutagenic substance may change back and become again prototrophe concerning histidine (His+), so revertant bacteria become histidine-independent.
The bacterial cells, in growth phase, are exposed to different concentrations of the test agent and mutagenic activity is determined by the capacity of the test substance to induce a significant increase in the number of reverted colonies (histidine-indipendent mutant, His+) in comparison to the spontaneous reversions occurring in the control cultures.
Some chemical agents are not directly mutagen but become so following transformation and metabolic activation occurring in the organism by liver enzyme activity. Therefore, rat liver microsomial fraction (S9) has been added to identify indirect mutagen substances.

BACTERIAL STRAIN
Each tester strains contains a different type of mutation in the histidine operon. TA 1535 and TA 100 strains are specific testers for mutagens causing base substitutions. The TA 102 strain is used for detecting mutagens that require an intact excision repair system. The sensitivity of TA 100 and TA 102 is greatly enhanced by the introduction of an R factor, pKM101, which confers ampicillin resistance. Furthermore the TA 102 strain contains the multicopy plasmid, pAQ1, which confers tetracycline resistance. The frameshift tester strains used are TA 1537 and TA 98. TA 98, like TA 100, is ampicillin resistant. All S. typhimurium strains carry, along with the defect in the histidine gene (His-), a deep rough (rfa) character, a mutation that causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increase permeability to large molecules. At the end in these strains, except TA 102 strain, there is a delection of a gene coding for the DNA excision repair system (uvrB-), resulting in greatly increased sensitivity in detecting many mutagens. For technical reason, the delection excising the uvrB gene extends through the bio gene and, as a consequence, these bacteria also require biotin for growth.

TEST DESCRIPTION
In each Ames test assay the following parameters must be considered:
- the negative control (or blank) represented by dishes used to detect so called spontaneous revertant: Salmonellae that spontaneously, without any induction by the sample, revert to a normal condition. These dishes contain mutagens solvents (water or dimethyl sulphoxide) and test agent solvents (in this case water).
- the positive control given by standard mutagen to check strains functionality.
Direct test: Mutagens without S9
S. typhimurium TA 1535 sodium azide (NaN3)
S. typhimurium TA 1537 9-aminoacridine (9AA)
S. typhimurium TA 100 sodium azide (NaN3)
S. typhimurium TA 98 4-nitroquinoline-N-oxide (NQNO)
S. typhimurium TA 102 4-nitroquinoline-N-oxide (NQNO)

Indirect test: Mutagens with S9
All strains 2-aminoanthracene (2AAn)

BACTERIAL STRAINS MAINTENANCE AND CHECK
The S. typhimurium strains were made from fresh bacterial culture derived from permanent cultures stored at -20 °C in 9 % DMSO. Fresh bacterial cultures were grown in Nutrient Broth (Oxoid) with ampicillin or tetracycline (for ampicillin or tetracycline resistent bacteria), were subcultivated on appropriate medium (Master plate) and stored at + 4°C for up to 2 mont hs. The Master plates medium is particular for each strain: the TA 1535 and TA 1537 strains grow in Minimal medium with histidine and biotine, the TA 100 and TA 98 strains grow in Minimal medium with histidine, biotine and ampicillin. The TA 102 strain grows in Minimal medium with histidine, biotine, ampicillin and tetracycline.
The liquid culture, the permanent cultures and Master plates were checked with the following controls to confirm the tester strain genotype:
a) Check for histidine-requirement
Bacterial cultures are streaked on Minimal Medium (MM) + Bio plates and on MM + Bio + His plates. After 48h incubation at 37 °C, bacterial growth shou ld be observed on MM + Bio + His plates and shouldn’t be observed on MM + Bio plates (negative control).
b) Check for the rfa mutation (Crystal violet sensitivity)
Culture (0.1ml) is added to 2 ml of top agar and plated on Complete Medium plates. When the medium is solidified, a disc with 10 μl of crystal violet solution (1 mg/ml) is put in the centre of agar surface. After 24h incubation at 37 °C, a neat inhibition zone around the disc should be observed for all strains.
c) Check for the uvrB mutation (UV sensitivity)
Bacterial cultures are streaked on Complete Medium plates. Half plates are exposed to uvrB ray (15 W germicidal lamp at 33 cm distance) for 6 seconds (TA 1535 and TA 1537) or 8 seconds (TA 100, TA 98 and TA 102). After 24h incubation at 37 °C all strains should grow on the un-irradiated side of the plate only. The strain TA 102 was used as a control and should grow in all the plate.
d) Check for the R factor (ampicillin resistance)
Bacterial cultures are streaked on Minimal Medium + Bio + His + Amp plates. After 24h incubation at 37 °C, bacterial growth should be observed for ampic illin resistance strains (TA 100, TA 98 and TA 102) only.
f) Check for the pAQ1 plasmid (tetracycline resistance)
Bacterial cultures were streaked on Minimal Medium + Bio + His + Amp + Tet plates. After 24h incubation at 37 °C, bacterial growth should be obse rved for tetracycline resistance strain TA 102) only.

Culture media
Liquid growth medium: prepared by dissolving 25 g of Nutrient Broth (Oxoid) and 5 g of NaCl in 1 l of deionized water and sterilised at 1 atm at 121 °C for 15 minutes.
Complete medium: prepared by dissolving 25 g di Nutrient Broth (Oxoid), 5 g di NaCl and 15 g of agar in 1 l of deionized water, then sterilised at 1 atm at 121 °C for 15 minutes.

Minimal medium
The medium consisted of 15 g of agar in 930 ml of deionized water sterilized at 1 atm at 121°C for 15 minutes. Thereafter, the temperature of the medium was brougth to about 60-65 °C and 50 ml glucose 40 % sterile solution and 20 ml of Vogel Bonner sterile solution 50X were added.
About 20 ml of the medium was poured into each of sterile plastic Petri plates (9 cm diameter).
The Vogel Bonner solution 50X was prepared with:
- 10g/l MgSO4 · 7H2O
- 100g/l citric acid · H2O
- 500 g/l K2PO4 anhydrous
- 175 g/l NaNH4HPO4 · 4H2O.

Top agar
This superficial medium was prepared with 6 g of agar and 5 g of NaCl dissolved in 1 l of deionized water and sterilized at 1 atm at 121 °C for 15 minutes.
For each 100 ml of the top agar was added 10 ml of 0.5 mM Histidine/Biotine solution.

Preparation of the bacterial culture
Bacterial cell suspension were prepared by inoculating one colonie of the Master culture in 25 ml liquid growth medium. The liquid culture was developed for about 16 hours a 37 °C in a shaking thermostatic incubator (overnight culture).

PLATE INCORPORATION TEST WITH AND WITHOUT METABOLIC ACTIVATION
The test substance or positive or negative control solutions was put into sterile test tube containing 2 ml of soft top agar kept liquid in a thermostatic bath at 45 °C. A suspension of Salmonella strains in statio nary growth phase (0.1 ml) was rapidly added. For the test with metabolic activation 0.5 ml of S9 mix was also added, instead for the text without S9 mix 0.5 ml of a physiologic solution (PBS) was added. The test tubes were shaken rapidly and the contends poured onto plates containing solid growth minimal medium. The plates were incubated at 37 °C for 48 hours. Two plates per dose per Salmonella strain were prepared both for the test with and without metabolic activation. The revertant colonies per plate were counted as UFC (Colony forming units) after 48 hours incubation.
Evaluation criteria:
For the test to be considered valid, the following criteria must be met:
a) The sterility check must prove negative for bacterial growth.
b) The growth of all the strains must be inhibited by crystal violet; the growth of strains TA 1535 and TA 1537 must be inhibited by ampicillin, while the growth of strains TA 100, TA 98 and TA 102 must not.
The growth of all strains, except TA 102 must not inhibited by tetracycline.
c) The frequency of spontaneous for reversions for each strain must fall within both the range reported in the literature and the historical range for our laboratory.
d) The activity of the S9 Mix will be confirmed by its capability to activate the positive control (which requires a metabolic transformation in order to exert its mutagenic effect).

The test substance is considered to have elicitated a positive response when:
- the number of reverted colonies increases when compared with the number of revertants in the negative controls in a dose-response related way or in a reproducible way at one or more concentrations in at least one strain with or without metabolic activation. Statistical methods may be used to decide the
significance of the increase.
Statistics:
The mean and the standard deviation will be calculated for reversions read in each dosage group and they will be compared with the spontaneous revertants (in the negative controls).

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest tested concentrations, sample caused high bacterial mortality
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic.
Executive summary:

Method

The test was based on OECD 471, using Salmonella typhimurium strains, i.e. TA 1535, TA 1537, TA 98, TA 100 and TA 102. Studies were done in duplicate both with and without metabolic activation by S9 -mix.

Results

The substance resulted to be not mutagenic under test conditions.