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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
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 journal

Data source

Reference
Reference Type:
publication
Title:
Salmonella Mutagenicity Tests: V. Results from the Testing of 311 Chemicals
Author:
Errol Zeiger, Beth Anderson, Steve Haworth, Timothy Lawlor, and Kristien Mortelmans
Year:
1992
Bibliographic source:
Environmental and Molecular Mutagenesis Volume 19, Supplement 21:2-141 (1992)

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:
Gene mutation toxicity study was performed to evaluate the mutagenic nature of Citral diethyl acetal
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: 1,1-diethoxy-3,7-dimethylocta-2,6-diene
- Molecular formula: C14H26O2
- Molecular weight: 226.35 g/mol
- Smiles notation: O(C(OCC)C=C(CCC=C(C)C)C)CC
- Substance type: Liquid
- Physical state: Organic
- Impurities (identity and concentrations): No data
Specific details on test material used for the study:
- Name of test material: Citral diethylacetal
- IUPAC name: 1,1-diethoxy-3,7-dimethylocta-2,6-diene
- Molecular formula: C14H26O2
- Molecular weight: 226.357 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: approx. 79%
- Impurities (identity and concentrations): approx. 21 %

Method

Target gene:
Histidine
Species / strain
Species / strain:
other: TA100, TA98, TA97 and TA 1535
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers
Test concentrations with justification for top dose:
0.0, 0.3, 1.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/Plate
Vehicle:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in ethanol
Controls
Negative controls:
not specified
Solvent controls:
yes
Remarks:
Ethanol
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (TA98 and TA1538; -S9) and 2-aminoanthracene (all atrains; +S9)
Details on test system and conditions:
The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA 1537), 4-nitro-o-phenylenediamine (TA98 and TA1538), mitomycin C (TA102), and methyl methanesulfonate (TA 104). The positive control for metabolic activation with all strains was 2-aminoanthracene, and either sterigmatocystin or 2-aminoanthracene was used for TA102.

Details on test system and conditions

METHOD OF APPLICATION: pre incubation
DURATION
- Pre incubation period: 20 minutes
- Exposure duration: No data available
- Expression time (cells in growth medium): two days
- 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: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: No data available
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: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
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.

Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Statistics:
Mean ± SEM

Results and discussion

Test results
Species / strain:
other: TA97, TA98, TA100, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes

Any other information on results incl. tables

Table: Mutagenic responses (mean ± SEM; three plates) of Salmonella tester strains to test chemicals.

Dose

                                          TA100

 NA(-)

10% HLI        (-)

30% HLI

  (-)

10% RLI

   (-)

30 % RLI

   (-)

µg/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.000

83

2.4

99

6.2

154

5.5

99

7.9

147

10.7

0.300

95

3.4

102

9.3

 

 

98

2.8

 

 

1.000

73

13.6

99

18.0

 

 

100

13.3

 

 

3.300

85

5.6

86

4.1

138

8.9

100

4.9

126

1.9

10.000

79

4.6

79

3.5

132

8.7

89

1.7

142

7.0

33.000

72

5.2

44s

8.2

135

6.3

54s

12.4

138

11.7

100.00

 

 

 

 

126

1.5

 

 

125

8.2

333.000

 

 

 

 

84s

5.1

 

 

102s

8.0

POS

424

10.1

214

5.2

588

25.5

972

29.6

1223

8.1

 

Dose

                                          TA1535

 NA(-)

10% HLI        (-)

30% HLI

  (-)

10% RLI

   (-)

30 % RLI

   (-)

µg/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.000

20

3.8

21

1.2

23

4.9

22

0.7

30

0.7

0.300

17

0.7

22

2.3

 

 

28

0.3

 

 

1.000

17

0.9

20

3.2

28

3.9

23

4.6

26

5.9

3.300

17

5.4

18

5.5

23

4.2

26

4.1

27

2.3

10.000

18

0.7

15

2.0

20

2.3

19

1.7

33

3.8

33.000

15

2.5

6s

3.1

23

0.3

16s

2.7

32

2.7

100.00

 

 

 

 

23

4.1

 

 

30

2.6

333.000

 

 

 

 

 

 

 

 

 

 

POS

310

2.3

103

4.1

114

3.5

333

33.3

128

11.9

 

Dose

                                          TA97

 NA(-)

10% HLI        (-)

30% HLI

  (-)

10% RLI

   (-)

30 % RLI

   (-)

µg/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.000

92

10.0

119

11.5

145

7.8

126

9.7

203

10.0

0.300

120

6.4

128

10.7

 

 

152

10.0

 

 

1.000

90

3.8

108

10.4

124

4.4

151

6.2

176

7.3

3.300

101

6.5

113

6.7

158

4.1

145

19.9

156

11.3

10.000

102

4.9

94

5.7

157

8.5

99

13.1

133

5.0

33.000

74

8.2

67s

7.8

141

7.4

60s

12.7

133

5.0

100.00

 

 

 

 

157

4.2

 

 

154

3.8

333.000

 

 

 

 

 

 

 

 

 

 

POS

391

4.1

637

4.4

947

21.8

2067

124.4

1086

26.7

 

 

 

Dose

                                          TA98

 NA(-)

10% HLI        (-)

30% HLI

  (-)

10% RLI

   (-)

30 % RLI

   (-)

µg/plate

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0.000

26

4.2

42

4.4

39

2.7

37

3.5

44

0.6

0.300

22

3.6

28

12.0

 

 

38

5.8

 

 

1.000

28

2.6

39

4.2

 

 

37

3.7

 

 

3.300

23

4.3

41

4.3

41

4.1

35

1.7

39

2.3

10.000

21

2.6

39

4.9

52

4.0

33

5.5

42

3.8

33.000

19

2.9

21s

0.9

46

1.5

16s

1.8

40

3.8

100.00

 

 

 

 

43

2.6

 

 

36

1.2

333.000

 

 

 

 

28s

4.4

 

 

31s

2.9

POS

337

10.1

177

11.9

441

9.8

159

10.2

375

3.1

ET95,95% ethanol (solvent), POS, positive control; NA, not activated;

HLI, Aroclor 1254-induced hamster liver S-9; RLI, Aroclor 1254-induced rat liver S-9; s, slight clearing of background lawn; t, complete clearing of background lawn (colonies not counted); p, precipitate present in plates; x, precipitate present with toxicity; +, mutagenic; +W, weakly mutagenic; ?, questionable response; - , nonmutagenic.

Applicant's summary and conclusion

Conclusions:
Citral diethy acetal did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of citral diethyl acetal. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in ethanol and used at dose levels 0.0, 0.3, 1.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. Citral diethy acetal did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.