Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 October - 20 November 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report) : Nicotinsäurepentylester HF
- Substance type : organic
- Physical state : clear, colourless-yellowish liquid
- Analytical purity : 99,9% (according certificate of analysis 74892, dated July 24th 2003)
- Lot/batch No. : 03030417/005
- Storage condition of test material : The test item was stored in a closed opaque vessel at room temperature and kept away from ignition sources.

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100, TA 102
Details on mammalian cell type (if applicable):
TA97a: hisD6610, TA 98: hisD3052 TA 100 und TA 1535 hisG46, TA102:hisG428, TA97, TA98 and TA100 contain uvrB; TA97, TA98, TA100 and TA102 contain pKM 101 and rfa.
Metabolic activation:
with and without
Metabolic activation system:
S9 ; produced from the livers of male Wistar rats which were treated with 80 mg Phenobarbital/kg body weight intraperitoneally and, on the following day, with 80 mg β-Naphthoflavon/kg body weight orally.
Test concentrations with justification for top dose:
First experiment : 5000, 1500, 500, 150 and 50 µg/plate (Plate incorporation method)
Second experiment : 500, 250, 125, 62.5 and 31.25 µg/plate (pre-incubation method)
Vehicle / solvent:
- Solvent used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent : DMSO was chosen as solvent, because the test item was completely soluble, and this solvent doesn't have any effects on the viability of the bacteria or the number of spontaneous revertants.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine ; 2-Aminoanthracene
Remarks:
See -Any other information on materials and methods incl. tables- for details
Details on test system and experimental conditions:
Bacteria :
Salmonella typhimurium (all strains used) were obtained from Prof. Ames (date of arrival: Feb. 25th 2002 (TA97a, 98, 102), June 23rd 2001 (TA 100) and Jan. 29th, 2001 (TA1535)) and stored as stock cultures at -80°C. On Oct. 13th and on Nov. 17th, one vial per strain to be used was thawed and put into a culture vessel containing approx. 45 ml of nutrient broth. After incubation over night at 37 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Description of the Method
Preparations
In the days before each test, the media and solutions were prepared. Two days before the test, the plates were sterilized and the first batches poured. On the day before the test, the remaining plates were poured.
On the day of the test, the overnight cultures were checked for growth. The incubation chamber was heated to 37 °C. The water bath and the heating block were turned to 45 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Test Data
First Experiment
Date of treatment : Oct. 14th, 2003
Concentrations tested : 5000, 1500, 500, 150, 50 µg/plate
Incubation time : 48 hours
Incubation temperature : 37 °C
Tester strains : TA97a, TA98, TA100, TA102, TA1535
Method : plate incorporation method
References : see below

Second Experiment
Date of treatment : Nov. 18th, 2003
Concentrations tested : 500, 250, 125, 62.5, 31.25 µg/plate
Incubation time : 48 hours
Incubation temperature : 37 °C
Tester strains : TA97a, TA98, TA100, TA102, TA1535
Method : pre-incubation method
References : see below

Description of the Method
Plate incorporation method
Per strain and dose, four plates with and four plates without S9 mix were used. 10 ml of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 ml of histidin-biotin-solution 0,5 mMol per 100 ml basis was added and the bottle was placed in the water bath at 45 °C.
0,1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing with 2 ml Top-Agar, 0,1 ml overnight culture of the respective strain and 0,5 ml phosphate buffer I (only for treatments without S9) or 0,5 ml S9 mix were added. The mixture was gently vortexed, then poured an a minimal glucose plate and evenly distributed, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

Pre-incubation method
Per strain and dose, four plates with and four plates without S9 mix were used. 10 ml of the test solution of the appropriate concentration were membrane filtrated into sterile vessels. Top agar basis was melted in a microwave oven, after melting, 10 ml of histidin-biotin-solution 0,5 mMol per 100 ml basis was added and the bottle was placed in the water bath at 45 °C.
0,1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing with 0,1 ml overnight culture of the respective strain, 0,5 ml phosphate buffer I (only for treatments without S9) or 0,5 ml S9 mix were added. The mixture was incubated in a incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated through careful shaking. Then 2 ml top agar were added. The mixture was vortexed gently, then poured on a minimal glucose plate and evenly distributed, using a Drigalski spatula. The plates were closed, covered with brown paper and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.


References
Genotype Confirmation
-Histidine requirement : Each strain was streaked on a biotin and a histidin-biotin-plate, using a sterilized wire loop.
-Ampicilline-Resistance (pKM 101) resp. ampicilline-tetracycline-resistance (pAQ1) The strains were streaked on ampicilline agar, TA102 on ampicilline-tetracycline agar. TA1535 was taking the function of control strain, since it is not ampicilline resistant.
-UV-sensitivity (uvrB) : Two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates were irradiated for 8 seconds with a germicidal lamp (254 nm, 30W), keeping a distance of 33 cm. Incubation over night at 37°C followed.
-Crystal violet sensitivity (deep rough) : For each strain, two plates were used. 0,1 ml of bacteria suspension were mixed with 2 ml Top-Agar and poured on nutrient agar. Sterile paper discs (9 mm diameter), each soaked with 10 μl of crystal violet solution (0,1%) were placed into the middle of each plate, followed by incubation over night.
-Spontaneous revertants : Four replicates, with/without S9, for each solvent which was used in the test.
-Determination of Titre : The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0,1 ml on maximal-soft agar. It should give a density of 10E9 cells/ml .
-Sterility Control : Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar.
-Toxicity Control : Each strain was incubated with the maximum dose of the test item on maximal soft agar.
-Positive Controls : Using diagnostic mutagens, four replicates were prepared. The stock solutions of the substances were diluted to effect an application volume of 0,1 ml/plate.
Evaluation criteria:
The colonies were counted by eye, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
for details see additional information on results
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
for details see additional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
First Experiment
Confirmation of the criteria
The treatments for the confirmation of the genotype and the sterility control didn't show any inconsistencies. The determination of the titre for the strain 98 was below the demanded factor of 100. The determined values for the spontaneous revertants of the negative controls (strain 97a) were lower than the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.
Solubility and Toxicity
The test item was dissolved in DMSO. A stock solution containing 50 g/L was prepared. Signs of toxicity towards the tested strains could be observed in the two highest concentrations. At 5000 µg/plate, no revertants were found. In the concentration 1500 µg/plate, the number of revertants was decreased (strains 97a, 1535) and no background lawn was observed. No growth occurred in the toxicity control.
Mutagenicity
The concentrations 1500 and 150 µg/plate caused an increased number of revertants in the strain 98 with and without metabolic activation. The concentration 150 µg/plate caused an increased number of revertants in the strain 97a with metabolic activation, too. As the determined values for the spontaneous revertants in strain 97a were lower than normal, the increased revertants seem to be false-positive. For strain 98, the increase of revertants is restricted to two concentrations, leaving out the one lying between (500 µg/plate). Additionally, no concentration-related increase over the tested range was found.

Second Experiment
Confirmation of the Criteria
The treatments for the confirmation of the genotype and the sterility control didn't show any inconsistencies. The determination of the titre for the strains 97a and 100 were slightly below the demanded factor of 100. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.
Solubility and Toxicity
The test item was dissolved in DMSO. A stock solution containing 50 g/L was prepared. The toxicity control with 500 µg/plate showed toxicity towards the strains 97a, 98 and 100. In the treatments however, no signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced. Therefore the test item was stated as not toxic in the tested concentrations.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found. Therefore the test item is stated as not mutagenic under the test conditions.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

First experiment : Survey of the findings

strain 97a 98 100 102 1535
induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
water mean 57 72 9 22 106 119 117 117 12 7
sd 75 79,9 3,9 10,8 14 26 13,0 23,5 7,9 2,9
DMSO mean 35 74 12 15 96 97 100 88 8 11
sd 38,1 37,4 1,8 7,9 22,0 16,0 8,7 21,8 4,8 1,3
pos.contr. mean 319 503 814 56 1016 306 716 671 896 282
sd 103 352 264 15 171 82 147 295 221 100
f(I) 9,2 6,8 60,2 3,7 9,6 3,2 7,2 7,7 77,9 26,2
5000 µg/pl. mean 0 0 0 0 0 0 0 0 0 0
sd 0 0 0 0 0 0 0 0 0 0
f(I) 0,00
0
0,00
36
0,00
41
0,00
43
0,00
55
0,00
84
0,00
88
0,00
58
0,00
1
0,00
7
1500 µg/pl. mean
sd 0 25 36 23 14 19 74 29 1 1
f(I) 0,00 0,49 3,42 2,80 0,57 0,87 0,88 0,66 0,16 0,67
500 µg/pl. mean 56 104 9 19 101 106 109 118 7 10
sd 45 20 4 5 9 21 19 15 5 5
f(I) 1,60 1,40 0,75 1,25 1,05 1,08 1,09 1,35 0,88 0,95
150 µg/pl. mean 130 102 71 43 86 105 96 88 5 8
sd 36 38 87 59 30 18 19 16 2 4
f(I) 3,74 1,37 5,94 2,80 0,90 1,08 0,96 1,01 0,66 0,74
50 µg/pl. mean 52 45 9 10 96 79 99 94 6 7
sd 31 27 4 6 14 18 44 9 2 4
f(I) 1,50 0,61 0,73 0,67 1,00 0,81 0,99 1,07 0,72 0,60

Second experiment : Survey of the findings

strain 97a 98 100 102 1535
induction -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
water mean         109       14  
sd         18       2,9  
DMSO mean 95 74 9 16 86 75 94 87 11 12
sd 19,8 47,2 3,6 3,5 9 5 29,3 15,8 4,5 5,4
pos.contr. mean 896 394 1046 40 736 605 886 446 834 148
sd 245 105 213 4 194 193 250 226 387 25
f(I) 9,5 5,3 116 2,5 6,8 8,1 9,5 5,1 61,7 12,6
500 µg/pI. mean 65 80 10 19 85 95 64 83 13 14
sd 26 21 3 4 19 21 20 10 3 2
f(I) 0,68 1,08 1,06 1,17 0,99 1,27 0,68 0,95 1,20 1,17
250 µg/pI. mean 85 86 7 12 87 100 88 99 11 12
sd 37 51 5 3 13 8 10 5 2 4
f(I) 0,90 1,17 0,81 0,75 1,01 1,34 0,94 1,13 1,00 1,00
125 µg/pl. mean 80 118 8 15 95 87 97 100 14 10
sd 9 22 2 4 14 15 6 8 3 4
f(I) 0,85 1,60 0,83 0,94 1,10 1,16 1,03 1,14 1,25 0,87
62,5 µg/pI. mean 99 75 10 13 68 99 63 105 11 7
sd 4 3 2 4 11 16 15 26 5 3
f(I) 1,05 1,02 1,06 0,84 0,78 1,32 0,67 1,21 1,00 0,62
31,25 µg/pI. mean 73 88 11 15 82 87 92 92 13 12
sd 18 42 3 6 8 10 10 18 9 4
f(I) 0,77 1,19 1,17 0,94 0,96 1,16 0,98 1,05 1,18 0,98

For water only the spontaneous revertants which were needed for the assessment of the positive controls were tested.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD TG471 and EU Method B.13/14 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The study was conducted under GLP compliance and a well documented study report is available.
The test item Nicotinsäurepentylester HF can be stated as not mutagenic under the conditions of the test .
Executive summary:

This study was performed in order to evaluate the mutagenic potential of Nicotinsäurepentylester HF according to the OECD Guidelines for the Testing of Chemicals „Bacterial Reverse Mutation Test" Part 471, adopted July 21 St, 1997 and EU-Guideline B.13/14 "Rückmutationstest unter Verwendung von Bakterien", adopted July 31st 2003.

Two valid experiments were performed.

First Experiment:

Five concentrations of the test item, dissolved in DMSO (ranging from 5000 to 50 µg/plate) were tested. Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.

The concentrations 1500 and 150 µg/plate caused an increase of revertants in strains 97a and 98. As the determined values for the spontaneous revertants in strain 97a were lower than normal, the increased revertants seem to be false-positive, taking into account that neither an increase in revertants for the concentration 500 µg/plate nor a dose-effect-relationship could be observed.

Signs of toxicity towards the bacteria could be observed in the two highest concentrations. The treatments for the confirmation of the genotype and the sterility control didn't show any inconsistencies. The determination of the titre for the strain 98 was below the demanded factor of 100. The determined values for the spontaneous revertants of the negative controls were lower than normal. All positive controls showed mutagenic effects with and without metabolic activation.

Second experiment:

To verify the results of the first experiment, a second experiment was performed, using five concentrations of the test item (500 to 31,25 µg/plate) and a modification in study Performance (pre-incubation method).

The test item didn't show mutagenic effects in the second experiment. The toxicity control with 500 µg/plate showed toxicity towards the strains 97a, 98 and 100. In the treatments, no signs of toxicity (missing background Iawn, decreased number of revertants) towards the bacteria could be observed.

The treatments for the confirmation of the genotype and the sterility control didn't show any inconsistencies. The determination of the titre for the strains 97a and 100 were below the demanded factor of 100. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item showed only insignificant mutagenic effects towards Salmonella typhimurium, strains TA 97a and TA 98 in the first experiment. No dose-effect relationship could be determined. In the second experiment, no mutagenicity was detected.

THEREFORE THE TEST ITEM NICOTINSÄUREPENTYLESTER HF CAN BE STATED AS NOT MUTAGENIC UNDER THE CONDITIONS OF THE TEST.