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Genetic toxicity in vitro

Description of key information

The mutagenic potential of N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine was investigated in a bacterial reverse gene mutation assay conducted according to OECD 471 with and without metabolic activation. There was no increase of mutant colonies compared to the negative control observed in all strains. Consequently, the target substance N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine is considered to be non-mutagenic.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-27 to 2018-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was dissolved in dimethyl sulfoxide (DMSO) and diluted prior to treatment.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
SOURCE OF CELLS
Source: - Tester strains TA98, TA1535 and TA102: MOLTOX, INC., NC 28607, USA.
Tester strains TA100 and TA1537: Xenometrix AG, Switzerland
Cells were stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.

MEDIA USED
- Type and identity of media: Vogel - Bonner Medium E agar plates were used
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver microsomal fraction S9 mix
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see box "Any other information on materials and methods incl. tables"; Results: see box "Any other information on results incl. tables", Table 2). The concentration range covered two logarithmic decades and contained the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicles/solvents used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535 (10 µg/plate), without S9
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
TA98 (10 µg/plate), TA1537 (40 µg/plate), without S9
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA102 (1 µL/plate), without S9
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All Strains (2.5 µg/plate), except TA102 (10 µg/plate), with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment I), pre-incubation (Experiment II)
- Cell density at seeding (if applicable): approx. 10^9 cells/mL

EXPERIMENTAL PERFORMANCE
- Experiment I:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension, 2000 μL Overlay agar.
- Experiment II:
For the pre-incubation method 100 µL of the test solution was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.

DURATION
- Preincubation period (Experiment II): 60 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/concentration level including the controls

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is considered either as a clearing or diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Evaluation criteria:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2014 - 2016) (see box “Any other information on material and methods incl. tables”, Table 1)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results and a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: To determine the range of concentrations (including the highest concentration) to be tested, the toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control. No toxic effects of the test item were noted in TA98 or TA100 at any of the tested concentrations up to 5000 µg/plate and evaluated either with or without metabolic activation in the pre-experiment (see Table 1 in box “Any other information on results incl. tables).

PRECIPITATION: Precipitation of the test item was observed in all tester strains used in both experiments I (and II at the higher concentrations of 2500 and 5000 µg/plate (with and without metabolic activation).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): See Tables 2-5 in box “Any other information on results incl. tables”.
Remarks on result:
other: Experiment I, Experiment I, precipitation was observed in all tester strains at 2500 and 5000 µg/plate

Table 1: Results of the pre-experiment

Substance

Dose

(µg/plate)

TA98

TA100

Mutation Factor

Mutation Factor

without S9

with S9

without S9

with S9

Solvent Control (DMSO)

 

1.0

 

1.0

 

1.0

 

1.0

 

4-NOPD

10.0

10.4

 

-

 

-

 

-

 

NaN3

10.0

-

 

-

 

6.6

 

-

 

2-AA

2.50

-

 

29.5

 

-

 

19.2

 

Test Item

3.16

0.9

 

1.1

 

1.1

 

1.2

 

10.0

0.9

 

1.1

 

1.1

 

1.0

 

31.6

1.2

 

1.0

 

1.0

 

1.1

 

100

1.1

 

0.9

 

1.0

 

1.2

 

316

1.3

 

0.8

 

1.0

 

1.1

 

1000

1.2

 

1.0

 

1.0

 

1.1

 

2500

1.2

 

0.8

 

1.0

 

1.2

 

5000

1.1

 

0.9

 

1.2

 

1.1

 

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).

Table 2:  Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 (-S9)

 

 

TA98

TA100

TA1535

TA1537

TA102

Mean

24.2

90.7

13.8

8.2

270.4

SD

6.7

15.6

6.7

2.9

55.0

Min

11

49

4

3

141

Max

58

155

41

35

472

RSD [%]

27.7

17.2

48.6

35.3

20.3

n

 

972

1191

929

931

682

 

Table 3:  Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) without S9 (-S9)

 

 

TA98

TA100

TA1535

TA1537

TA102

Substance
Conc./plate

 

4-NOPD
10 µg

NaN3
10 µg

NaN3
10 µg

4-NOPD
40 µg

MMS
1 µL

Mean

430.7

612.1

792.0

94.5

1729.2

SD

155.5

220.0

299.5

22.7

518.8

Min

141

132

38

35

272

Max

1830

1423

1854

273

3321

RSD [%]

36.1

35.9

37.8

24.0

30.0

n

 

971

1188

931

929

682

Table 4:  Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 (+S9)

 

 

TA98

TA100

TA1535

TA1537

TA102

Mean

29.0

96.4

10.5

8.3

339.7

SD

6.8

14.1

4.5

3.1

71.3

Min

15

62

3

3

157

Max

59

160

38

36

586

RSD [%]

23.4

14.6

42.7

37.4

21.0

n

 

967

1189

925

926

676

Table 5:  Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) with S9 (+S9)

 

 

TA98

TA100

TA1535

TA1537

TA102

Substance
Conc./plate

 

2-AA
2.5 µg

2-AA
2.5 µg

2-AA
2.5 µg

2-AA
2.5 µg

2-AA
10 µg

Mean

1880.5

1727.7

133.9

234.1

801.2

SD

708.5

522.0

134.9

101.4

223.7

Min

70

169

22

26

137

Max

3606

3132

1954

682

3588

RSD [%]

37.7

30.2

100.8

43.3

27.9

n

 

966

1184

927

925

678

S9: metabolic activation
Conc.:
 concentration
Mean: mean of revertants/plate
Min.:
minimum of revertants/plate
Max.:
maximum of revertants/plate
SD:
Standard Deviation
RSD:
Relative Standard Deviation
n:
Number of control values

Conclusions:
Under the tested experimental conditions mentioned, N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, conducted according to OECD guideline 471, strains TA98, TA100, TA102, TA1535 and TA1537 of S. typhimurium were exposed to N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine (100% purity) in DMSO at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria conducted according to OECD guideline 471, strains TA98, TA100, TA 102, TA1535 and TA1537 of S. typhimurium were exposed to the target substance N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine dissolved in DMSO at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains and both experiments (plate incorporation and pre-incubation). Based on the results the target substance is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

Justification for classification or non-classification

Based on the available study data and according to the criteria of the CLP Regulation (EC) 1272/2008, N2-Isobutyryl-5’-O-(4,4’-Dimethoxytrityl)-2’-deoxyguanosine does not warrant classification for mutagenicity.