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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
Zeiger et al
Year:
1987
Bibliographic source:
Environmental Mutagenesis

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Bacterial reverse mutation assay was performed to evaluate the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: N,N'-Bis-(1,4 dimethylpentyl)-p-phenylenediamine
- IUPAC name: 1-N,4-N-bis(5-methylhexan-2-yl)benzene-1,4-diamine
- Molecular formula: C20H36N2
- Molecular weight: 304.518 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, and TA97
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared
Test concentrations with justification for top dose:
0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble and stable in DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-Aminoanthracene (2-AA) (All strains; +S9), 4-nitro-o-phenylenediamine (TA98; -S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose-related increase was judged insufficiently high to justify a call of "+W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen.

The chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials.

It chemical was considered to be questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA98, TA1535, TA100, TA97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA100 or the system developed by Waleh et al. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

 Table: Mutagenicity information

Dose (µg/plate)

TA100

-S9

10% HLI

30% HLI

Mean

SEM

Mean

SEM

Mean

SEM

0

105

4.7

104

2.9

112

2.0

0.03

107

5.3

 

 

 

 

0.1

104

12.8

 

 

 

 

0.3

109

4.2

 

 

 

 

1.0

100

0.6

 

 

 

 

3.0

92

8.1

130

2.8

122

5.3

10

 

 

105

4.0

117

5.3

33

 

 

117

5.2

139

1.9

100

 

 

105

6.2

132

1.9

333

 

 

132

9.0

130

7.8

Positive control

372

9.7

1521

82.5

645

9.0

 

Dose (µg/plate)

TA1535

-S9

10% HLI

30% HLI

Mean

SEM

Mean

SEM

Mean

SEM

0

27

3.2

10

1.7

10

1.2

0.03

 

 

 

 

 

 

0.1

 

 

 

 

 

 

0.3

20

3.2

 

 

 

 

1.0

23

4.2

 

 

 

 

3.0

19

5.3

10

1.0

6

0.7

10

20

2.9

7

0.3

7

1.2

33

17

3.0

7

0.9

8

0.9

100

 

 

7

0.9

6

0.3

333

 

 

6

1.5

5

1.0

Positive control

372

9.7

1521

82.5

645

9.0

 

Dose (µg/plate)

TA1537

-S9

10% HLI

30% HLI

Mean

SEM

Mean

SEM

Mean

SEM

0

4

1.2

5

0.3

6

0.6

0.03

4

0.6

 

 

 

 

0.1

5

1.8

 

 

 

 

0.3

5

1.8

 

 

 

 

1.0

3

1.3

 

 

 

 

3.0

7

0.6

8

0.3

5

0.7

10

 

 

6

1.7

6

0.7

33

 

 

6

1.2

5

1.5

100

 

 

5

0.9

8

0.3

333

 

 

7

1.5

4

1.0

Positive control

192

6.0

342

12.0

108

3.5

 

Dose (µg/plate)

TA98

-S9

10% HLI

30% HLI

Mean

SEM

Mean

SEM

Mean

SEM

0

19

1.0

17

0.9

25

2.3

0.03

 

 

 

 

 

 

0.1

 

 

 

 

 

 

0.3

20

2.3

 

 

 

 

1.0

14

1.2

 

 

 

 

3.0

15

1.8

23

4.4

30

3.2

10

19

3.7

25

3.3

28

5.2

33

16

0.7

24

0.3

30

0.3

100

 

 

23

3.3

33

3.2

333

 

 

21

3.2

27

0.9

Positive control

844

36.7

1655

37.1

541

10.7

 

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs.

 

The test compound was dissolved in DMSO and was used at a dosage level of 0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, 33, 100 or 333 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study.

 

The test chemical did not induce a reproducible, dose-related increase in his+revertants over the corresponding solventin the S. typhimurium tester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.