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Toxicological information

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 September 04,2009 to October 02,2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

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
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. T408209159
- Manufacturing date: 08 Jun 2009
- Expiration date of the lot/batch: 08 Jun 2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed. Keep in a dry, cool and well ventilated place.
- Storage at testing facility: 5 years

Method

Target gene:
For all Salmonella tester strains: rfa wall (enzyme)
TA1535 and TA100 : DNA base pair
TA1537 and TA98 : DNA frameshift
WP2 uvrA : uvrA DNA repair and uvrB
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary test:
5000 , 1580 , 500 , 158 , 50 µg/plate

Main Assay 1°
Tester strains S9 Dose-levels (ug/plate)
TA1535, TA1537, TA100 - 2500, 1250, 625, 313, 156, 78.1
TA1535, TA100 +
WP2uvrA - 5000, 2500, 1250, 625, 313, 156
TA98 ±
TA1537, WP2uvrA + 5000, 2500, 1250, 625, 313

Main Assay 2°

Tester strains S9 Dose-levels (ug/plate)
WP2uvrA, TA98 + 5000, 2500, 1250, 625, 313
TA1535 + 5000, 2500, 1250, 625, 313, 156
WP2uvrA - 2500, 1250, 625, 313, 156, 78.1
TA1537, TA100 + 1250, 625, 313, 156, 78.1, 39.1
TA98 -
TA1535 - 625, 313, 156, 78.1, 39.1
TA1537, TA100 - 625, 313, 156, 78.1, 39.1, 19.5

Main Assay 3°

Tester strains S9 Dose-levels (ug/plate)
WP2uvrA, TA98 ± 625, 313, 156, 78.1 , 39.1
TA1535 ± 625, 313, 156, 78.1 , 39.1
TA1537, TA100 ± 625, 313, 156, 78.1 , 39.1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, Dimethylsulfoxide (DMSO) (Fluka AG, batch 1364641 54207P08).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: in distilled water.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 Migrated to IUCLID6: in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9 Migrated to IUCLID6: in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene ( in DMSO)
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Type and identity of media:
- The following growth media were used:
- Nutrient Broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use.
This was used for the preparation of liquid cultures of the tester strains.
- Nutrient Agar: Oxoid Nutrient Broth No 2 (25g) and Difco Bacto-agar (15g) were dissolved in distilled water (1 litre) and autoclaved.
The solution was then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
- Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel
Bonner Medium E, with 2% Glucose, and poured into 9 cm plastic Petri dishes.
- Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl
in distilled water. Prior to use 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (Salmonella Typhimurium tester strains) or 0.5 mM tryptophan (Escherichia Coli tester strain) was added to the top agar (100 ml). The trace amount of these aminoacids in the media allows all the pla ted bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed.



DURATION
- Preincubation period: 72 hours at +37°C

NUMBER OF REPLICATIONS:3 replicate for each point

First evaluation:
Toxicity was observed at higher dose levels with all tester strains with the exception of TA1535, WP2uvrA and TA98 tester strains in the presence of S9 metabolic activation.
Therefore a Main Assay III, using the pre-incubation method, was performed with these tester strain/treatment combinations. In order to assay the test item at non cytotoxic dose levels, the following concentrations were employed: 625, 313, 156, 78.1, 39.1 ug/plate.
 
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
E. coli WP2 uvr A
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation :No precipitation of the test item was noted at the end of the incubation period at any concentration tested, in any experiment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that the test item Hexahydro-4 Methylphthalic Anhydride does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

A bacterial reverse mutation assay (Ames test) has been undertaken following OECD/EU test methods. Experiments were performed both in the absence and presence of metabolic activation at concentrations up to 5000 µg/plate. The substance did not induce reverse mutation inSalmonella typhimuriumorEscherichia coli.