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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21st, 1997
Principles of method if other than guideline:
The evaluation of mutagenicity of the substance was evaluated in the methods found in the following references:
1. Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for Detecting Carcinogens and mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, 347-364.
2. Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcin, C., Nohmi, T., Ohta, T., Vennit, S., Zeiger, E (1994). Raccomendations for the performance of bacterial mutation assays. Mutation Res., 312, 217-233.
3. Maron, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, 173-215.
GLP compliance:
no
Remarks:
not-GLP study but considered as reliable based on the reporting of the study
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
5-methyl-2-(isopropyl)cyclohexyl nicotinate
EC Number:
254-991-1
EC Name:
5-methyl-2-(isopropyl)cyclohexyl nicotinate
Cas Number:
40594-65-8
Molecular formula:
C16H23NO2
IUPAC Name:
5-methyl-2-(isopropyl)cyclohexyl nicotinate

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:
4 % S9 mix
Test concentrations with justification for top dose:
0.06; 0.19; 0.56; 1.67; 5.00 mg/plate
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation.
Details on test system and experimental conditions:
DURATION
- Exposure conditions: 48 hours at 37 °C.

S9 MIX: lyophilized Aroclor 1254 rat liver induced (Moltox) supplemented with different cofactors (glucose-6-phosphate and NADP).

NUMBER OF REPLICATIONS: three.

DETERMINATION OF REVERTANT COLONIES: after incubation

STRAINS
- Cultivation: prepared 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 months. The Master plates medium is particular for each strain: the TA 1535 and TA 1537 strains grow in Minimal medium with histidine (His) and biotine (Bio), 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.
- 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 should be observed on MM + Bio + His plates and it shouldn’t be observed on MM + Bio plates (negative control).
- Check for the rfa mutation (Crystal violet sensitivity): culture (0.1 ml) 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 placed in the centre of agar surface. After 24 h incubation at 37 °C, a neat inhibition zone around the disc should be observed for all strains.
- Check for the uvrB mutation (UV sensitivity): bacterial cultures are streaked on Complete Medium plates. Half plates are exposed to uvrB ray (15W 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 24 h incubation at 37 °C all strains should grow on the non irradiated side of the plate only. The strain TA 102 was used as a control and should grow in all the plate.
- Check for the R factor (ampicillin resistance): bacterial cultures are streaked on Minimal Medium + Bio + His + Amp plates. After 24 h incubation at 37 °C, bacterial growth should be observed for ampicillin resistance strains (TA 100, TA 98 and TA 102) only.
- Check for the pAQ1 plasmid (tetracycline resistance): bacterial cultures were streaked on Minimal Medium + Bio + His + Amp + Tet plates. After 24 h incubation at 37 °C, bacterial growth should be observed for tetracycline resistance strain TA 102 only.

METHOD OF APPLICATION: plate incorporation. An aliquot of medium containing histidine and biotine, the S. typhimurium strain suspension, the sample to be tested at different concentrations and, in the case of metabolic activation, a 4 % S9 mix were added in the Petri dishes containing mimimum medium. 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 stationary growth phase (0.1ml) 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 0.5ml 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 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.

PREPARATION OF MEDIUMS
- Growth medium: this medium was prepared by dissolving 25 g of Nutrient Broth (Oxoid) and 5 g of NaCl in one liter of deionized water and sterilized at 1 atmosphere at 121°C for 15 minutes.
- Complete medium: this medium was prepared by dissolving 25 g di Nutrient Broth (Oxoid), 5 g of NaCl and 15g of agar in one liter of deionized water, and it was sterilized at 1 atmosphere 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 atmosphere at 121 °C for 15 minutes. Thereafter, the temperature of the medium was brought 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: 10 g/l MgSO4·7H2O, 100 g/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 one litre of deionized water and sterilized at 1 atmosphere 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.
- Bacterial culture: bacterial cell suspension were prepared by inoculating one colony 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).
- 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 S9 Mix was prepared immediately before use. S9 Mix composition (50ml): 2 ml Rat liver S9 (4%), 1ml MgCl2 0.4M and KCl 1.65 M, 0.25 ml glucose-6-phosphate 1M, 2 ml NADP 0.1 M, 25 ml buffer sodium phosphate 0.2 M pH 7.4, 19.75 ml sterile deionized water.

VALIDITY CRITERIA
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 the range reported in the literature.
d) The activity of the S9 Mix will be confirmed by its capability to activate the positive control.
Evaluation criteria:
The test substance is considered to have a positive response when the number of reverted colonies increases in comparison 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.
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 results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at TA1537, TA100, TA98 without S9; at TA1537, TA1535 and TA98 with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The mean values of colony forming units (UFC) for revertant as obtained by two replicates for each strain.

Table: UFC without S9.

mg/plate TA 1537 TA1535 TA100 TA98 TA102
Mean SD Mean SD Mean SD Mean SD Mean SD
5.00 5.3* 1.15 38.0 1.00 176.7* 23.86 6.3* 1.53 529.0 30.51
1.67 9.0 1.00 35.7 1.15 208.0* 40.29 7.7* 6.66 551.3 35.73
0.56 8.7 2.08 35.5 2.08 304.0 18.52 12.3 4.93 601.3 36.03
0.19 13.3 2.52 33.3 2.31 318.7 33.31 23.3 7.51 580.7 40.25
0.06 13.7 2.08 34.0 1.73 377.0 16.09 37.0 3.61 622.7 43.66
Positive control 432.3 26.95 579.3 12.66 1392.0 18.68 348.3 19.50 1488.7 59.53
DMSO 14.7 1.53 39.7 1.53 329.3 14.05 43.3 5.51 590.3 87.39

Table: UFC with S9.

mg/plate TA 1537 TA1535 TA100 TA98 TA102
Mean SD Mean SD Mean SD Mean SD Mean SD
5.00 3.7* 1.70 11.0* 7.00 282.3 25.01 15.3* 5.03 329.3 42.50
1.67 7.7 1.25 18.7 2.08 304.3 9.07 29.3 4.16 424.7 54.59
0.56 13.7 2.49 21.0 1.00 282.0 27.07 51.7 2.52 407.7 14.98
0.19 10.7 1.70 22.0 1.73 263.3 40.80 56.0 2.00 493.3 26.16
0.06 14.0 2.94 22.7 2.31 296.7 15.89 60.3 5.13 447.0 81.43
Positive control 251.0 32.57 129.0 2.65 952.0 67.10 225.7 29.48 1624.3 32.50
DMSO 15.7 2.49 28.0 1.73 296.7 5.03 54.7 3.21 516.0 68.56

* cytotoxicity effect

Applicant's summary and conclusion

Conclusions:
Τhe substance did not show any evidence of mutagenicity.
Executive summary:

The mutagenic potential of the substance was evaluated according to the OECD Guideline 471 and according to the method found in Ames, B.N et al. (1975), Gatehouse, D. (1994), Maron, D.M. and Ames, B.N. (1983). The substance was tested in 5 different concentrations ranging from 0.06 to 5 μg/plate in the plate incorporation test with and without metabolic activation (S9 mix). Two plates per dose per strain were incubated for 48 hours at 37 °C, after which the revertant colonies were evaluated for the test plates, negative and positive controls.

Cytotoxicity was observed at the highest concentrations of TA 1537, TA100 and TA98 without metabolic activation and at the highest concentration at TA1537, TA1535 and TA98 with metabolic activation.

The substance did not show any evidence of mutagenicity under the test conditions.

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