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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diisobutylphthalate (test substance No. 02/0233-2)
- Physical state: liquid, colorless, clear
- Analytical purity: ≥99,5%
- Lot/batch No.: SCH382 Palatinol IC #26941 Projekt857
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions: the stability of the test substance under storage conditions throughout the study period was guaranteed
- Storage condition of test material: room temperature
- Other: the homogeneity of the test substance was guaranteed by mixing before preparation of the test substance formulations.

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9 mix prepared from 5 male Sprague-Dawley rats (200 - 300 g) treated with a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw; as a 200 mg/ml solution in corn oil) 5 days before sacrifice.
Test concentrations with justification for top dose:
First experiment: 0, 20, 100, 500, 2500 and 5000 µg/plate for TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA (standard plate test with and without S9 mix); 2nd Experiment: 0, 20, 100, 500, 2500 and 5000 µg/plate for TA 1535, TA 100, TA98 and E.coli; 0, 10, 50, 250, 1250 and 2500 µg/plate for TA 1537, without S9 mix; 0, 2, 10, 50, 250 and 500 µg/plate for TA 1537, with S9 mix (preincubation test with and without S9 mix); third experiment: 0, 312.5, 625, 1250, 2500 and 5000 µg/plate for TA 1537 (preincubation test with and without S9 mix); each 3 test plates per dose or per control
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Controls
Untreated negative controls:
yes
Remarks:
sterility control; additional plates are treated with soft agar, S9 mix, buffer, vehicle or the test substance but without the addition of tester strains
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Remarks:
the Ames test is regularly conducted in the testing facility
Positive controls:
yes
Remarks:
see above
Positive control substance:
other: with S9 mix: 2-aminoanthracene (2-AA; 2.5 µg/plate for TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate for E. coli; dissolved in DMSO); Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 5 µg/plate) for TA 1535, TA 100; continue below
Remarks:
positive control substances without S9 mix (continue): 4-nitro-o-phenylendiamine (NOPD; 10 µg/plate) for TA 98; 9-aminoacridine (AAC; 100 µg/plate) for TA 1537; 4-nitroquinoline-N-oxide (4-NQO; 5 µg/plate) for coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation; standard plate test)

DURATION
- Exposure duration: at 37°C for 48 – 72 hours in the dark,

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: at
37°C for the duration of about 20 minutes using a shaker - Exposure duration: at 37°C for 48 - 72 hours in the dark

SELECTION AGENT (mutation assays): tryptophan/histidine deprivation

DETERMINATION OF CYTOTOXICITY
- Method: toxicity detected by a (1) decrease in the number of revertants; (2) clearing or diminution of the background lawn (= reduced his- or trp- background growth); (3) reduction in the titer, is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.

OTHER: The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
Evaluation criteria:
ACCEPTANCE CRITERIA: generally, the experiment is considered valid if the following criteria are met: (1) the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain; (2) the sterility controls revealed no indication of bacterial contamination; (3) the positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above; and (4) the titer of viable bacteria was ≥108/mL. ASSESSMENT CRITERIA: (1) the test chemical is considered positive in this assay if a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system; (2) a test substance is generally considered non-mutagenic in this test if The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in the number of his+ or trp+ revertants was observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed occasionally from about 500 µg/plate onward in the standard plate test and from 2500 µg/plate in the preincubation test
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was found from about 1250 µg/plate onward with and without S9 mix

COMPARISON WITH HISTORICAL CONTROL DATA: was accurate

ADDITIONAL INFORMATION ON CYTOTOXICITY: a weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 500 µg/plate onward.
In the preincubation assay bacteriotoxicity (reduced his- background growth, slight decrease in the number of his+ revertants, reduction in the titer) was occasionally observed depending on the strain and test conditions from about 2500 µg/plate onward.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to an increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay).

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

MEAN NUMBER OF REVERTANS PER PLATE

1) Standard plate test (first test)

Dosing conditions /test substance dose level (µg/plate)

Mean revertants per plate (negative control value ± SD) and factor of induced revertants compared to control value

TA 1535

TA 100

TA 1537

TA 98

E. coli WP2 uvrA

Without S-9

With S-9

Without S-9

With S-9

Without S-9

With S-9

Without S-9

With S-9

Without S-9

With S-9

DMSO

18±1

18±4

101±8

113±13

10±1

11±1

27±5

31±2

38±1

38±5

20

1.0

1.0

1.0

0.9

0.9

0.8

0.9

1.0

0.8

0.9

100

0.9

1.0

1.0

1.0

0.5

0.9

1.0

0.9

0.8

1.1

500

1.1

0.9

1.1

1.0

0.7

0.5

0.8

0.8

0.9

0.9

2500

0.8

0.8

1.0

0.8

0.6

0.5

1.0

0.8

0.9

1.0

5000

0.8

0.7

0.9

0.6

0.5

0.5

0.9

0.8

0.9

0.9

Positive control

58.8

7.5

10.5

8.7

39.7

13.9

18.8

18.9

17.0

6.1

 

2) Preincubation test (second test)

Dosing conditions /test substance dose level (µg/plate)

Mean revertants per plate (negative control value ± SD) and factor of induced revertants compared to control value

TA 1535

TA 100

TA 1537

TA 98

E. coli WP2 uvrA

Without S-9

With S-9

Without S-9

With S-9

Without S-9

With S-9

Without S-9

With S-9

Without S-9

With S-9

DMSO

17±1

14±1

98±2

115±12

10±2

9±4

33±5

35±9

36±5

30±3

2/10/20

0.9

1.1

1.0

0.8

0.9

1.2

0.9

1.1

1.0

1.2

10/50/100

1.0

0.8

1.1

1.0

0.7

1.1

0.9

1.1

1.0

1.2

50/250/500

1.2

0.8

1.1

0.8

1.0

0.7

0.7

1.0

1.0

1.4

250/1250/2500

0.8

1.0

1.0

0.7

0.8

0.9

0.8

0.9

1.0

1.3

500/2500/5000

0.8

1.0

1.0

0.7

0.7

1.0

0.9

0.6

1.0

1.2

Positive control

59.4

10.2

11.1

7.7

45.4

12.8

23.9

19.6

18.4

8.8

 

3) Preincubation test (third test)

Dosing conditions /test substance dose level (µg/plate)

Mean revertants per plate (negative control value ± SD) and factor of induced revertants compared to control value

TA 1537

Without S-9

With S-9

DMSO

8±2

9±1

20

0.7

1.0

100

1.0

0.8

500

1.1

1.1

2500

0.4

0.6

5000

0.2

 

Positive control

43.4

12.1

 

Applicant's summary and conclusion