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Genetic toxicity: in vitro

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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:
from 25 February 2011 to 05 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): PEX-2 (previous public name of the registered substance)
- Storage condition of test material: at room temperature

Method

Target gene:
Histidine gene
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 from the liver of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
The dose-levels selected for the preliminary (cytotoxicity) test were 50, 150, 500, 1500 and 5000 µg/plate. The preliminary test was conducted on S. typhimurium TA100 in the absence of metabolic activation system.
Based on the results of the cytotoxicity test, the dose selected for the main test were 50, 150, 500, 1500 and 5000 µg/plate) for the five strains in the presence (S9 mix) and in the absence of metabolic activation.
Vehicle:
Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen as the solvent vehicle due to its known lack of toxicity to the bacteria.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
mitomycin C
other: ICR 191-Acridine
Remarks:
without S9 mix
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation): For the direct plate incorporation test without metabolic activation, 0.1 mL of test substance and 0.1 mL of a fresh bacterial culture grown overnight were added to 2.0 mL of top agar. For tests with metabolic activation, 0.5 mL of S9 mix was added to the top agar after the addition of test substance and bacteria.

DURATION
- Incubation period: 72 hours at 37°C after solidification of the plates

NUMBER OF REPLICATES: triplicate for the solutions of test substance and the negative control and duplicate for the positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the cytotoxicity was performed on the basis of the observation of the inhibition of the background bacterial lawn (transparent instead of opaque and rough) and inhibition or reduction in the number of colonies.

OTHER:
Positive controls:
Without S9 mix:
- sodium azide (SA) for strains TA 1535 and TA 100 (5 µg/plate),
- ICR 191-Acridine (ICR) for strain TA 1537 (1 µg/plate),
- 2-Nitrofluorene (2-NF) for strain TA 98 (2 µg/plate),
- Mitomycin C (MMC) for strain TA 102 (0.5 µg/plate).

With S9 mix:
- 2-Aminoanthracene (2-AA) for strains TA98, TA100, TA102, TA 1535 and TA 1537 (2.5 µg/plate).



Evaluation criteria:
A 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls and a clear dose-relationship are considered as a positive result.
Statistics:
To confirm the positive result the analysis of variance of the data set should indicate significant results (pANOVA <0.05) and a clear dose-related increase in the number of revertants should be observed. The analysis of variance indicates statistically significant differences among the average number of revertants in different concentrations. The dose-response effect is evaluated by means of simple linear regression, where the model Revertants (dose) = intercept +slope.dose is adjusted by least-squares method. The regression indicates the probability of the number of revertants observed in the different concentrations by increased (mutagenicity) or decreased (toxicity).

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/1
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
To assess the toxicity of the test item to the bacteria, five dose-levels were tested in the TA 100 strain, without S9 mix. Cytotoxicity is characterised by inhibition of the background bacterial lawn (transparent instead of opaque and rough) and inhibition or reduction in the number of colonies.
No background bacterial lawn inhibition was observed at the tested concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA:
The counts recorded on negative vehicle control plates showed appropriate results with all tester strains in the range finding and definitive tests. Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains, confirming the sensitivity of cultures and activity of S9 mix. The assays were therefore considered valid.

Any other information on results incl. tables

Table 7.6.1/1:Mean revertant colony counts in the definitive study

 

TA 98

TA 100

TA 102

TA 1535

TA 1537

Conc.
(µg/plate)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

26

33

no

183

127

no

308

307

no

22

28

no

10

19

no

50

27

37

no

193

131

no

311

311

no

25

25

no

12

16

no

150

33

31

no

185

145

no

315

308

no

27

22

no

8

23

no

500

24

31

no

169

135

no

293

290

no

31

23

no

10

13

no

1500

21

33

no

176

131

no

312

240

no

29

23

no

9

14

no

5000

28

31

no

185

138

no

303

223

yes#

27

23

no

9

14

no

2-NF

453

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2-AA

 

1792

 

 

1610

 

 

1222

 

 

1771

 

 

190

 

SA

 

 

 

2170

 

 

 

 

 

331

 

 

 

 

 

MMC

 

 

 

 

 

 

2449

 

 

 

 

 

 

 

 

ICR

 

 

 

 

 

 

 

 

 

 

 

 

1267

 

 

 

 

*negative control: DMSO (100 µL/plate)

MA: metabolic activation

2-NF: 2-Nitrofluorene

2-AA: 2-Aminoanthracene

SA: Sodium Azide

MMC: Mitomycin C

ICR: ICR 191-Acridine

#: with metabolic activation only

Applicant's summary and conclusion

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

Under the condition of this study, the test item 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol did not induce gene mutations by base pair changes or frameshifts in the genome of Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 at the employed range of concentrations both with and without metabolic activation.
Executive summary:

This study was performed to investigate the potential of the test item, 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol, to induce reverse mutation inSalmonella typhimurium. The study was performed according to OECD guideline no. 471 and in compliance with the Principles of Good Laboratory Practice. 

 A preliminary toxicity test was performed to define the dose-levels of 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol to be used for the mutagenicity study. The test item was then tested according to the direct plate incorporation, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Five strains of bacteria Salmonella typhimuriumTA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose‑level). After 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in DMSO.

 

Since the test item was non-cytotoxic to strain TA100 in the absence of metabolic activation in the preliminary test, the highest dose-level selected for the main experiment was 5000 µg/plate, according to the criteria specified in the international guidelines. The selected treatment-levels were 50, 150, 500, 1500 and 5000 µg/plate for the five strains, in the presence and in the absence of metabolic activation. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid. 

No noteworthy toxicity was noted at any dose-levels in the absence or in the presence of metabolic activation except with strain TA102 with S9 mix were toxicity was noted at the dose-level of 5000 µg/plate. The test item did not induce any substantial increase in the number of revertants, either with or without S9 mix, in any of the five strains.

 

In conclusion, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the absence or in the presence of a rat metabolising system therefore 2-isobutyl-2-methyl-1,3-dioxolane-4-methanol is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).