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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:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, the protocol and the results were described with details. Substance analytical certificate not available
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, the protocol and the results were described with details. Substance analytical certificate not available
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
See tables 7.6.1/3, 7.6.1/4, 7.6.1/5 and 7.6.1/6
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity assay was conducted with strains TA98 and TA100 at concentrations between 1 and 5000 µg/plate. The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA98 and TA100.

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/3: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Standard deviation

Factor +

Negative* control

12

1.2

-

8

0.6

-

16

1.0

-

77

10.0

-

Solvent control**

10

0.6

1.0

7

0.6

1.0

19

5.1

1.0

83

7.4

1.0

10

10

2.6

1.0

8

1.7

1.0

14

6.4

0.7

78

8.5

0.9

100

11

3.8

1.1

8

1.7

1.1

16

2.1

0.8

73

12.1

0.9

333.3

10

4.4

1.0

7

1.5

1.0

19

4.2

1.0

78

12.4

0.9

1000.0

11

3.6

1.1

7

1.0

17

1.2

0.9

79

4.7

0.9

5000.0

13

4.6

1.3

5

0

0.7

22

2.9

1.1

73

7.4

0.9

Positive control***

839

20.2

86.8

228

9.8

31.1

1694

244.4

87.6

1065

48.5

12.8

Negative control*: concurrent untreated control

Solvent control**: ethanol

Positive control***: see table 7.6.1/2

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Negative control*

10

3.1

-

9

1.5

-

32

2.0

-

91

10.4

-

Solvent control**

   11

2.6

1.0

7

3.0

1.0

38

6.1

1.0

85

8.5

1.0

10

12

4.7

1.1

11

4.4

1.6

26

5.3

0.7

77

5.5

0.9

100

12

2.0

1.1

8

3.5

1.2

20

4.0

0.5

72

5.0

0.8

333.3

12

3.8

1.1

8

2.0

1.1

26

0.6

0.7

73

13.6

0.9

1000.0

10

3.0

0.9

12

4.0

1.7

28

5.0

0.7

81

7.6

1.0

5000.0

12

3.2

1.1

11

1.7

1.6

36

6.7

0.9

80

5.5

0.9

Positive control***

284

11.1

25.8

252

28.0

36.0

1916

60.5

50.4

1681

150.2

19.7

Negative control*:concurrent untreated control

Solvent control** ethanol

Positive control***: see Table 7.6.1/2

Table 7.6.1/5: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (second test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Negative control*

6

1.2

-

5

1.0

-

15

0.6

-

80

11.0

-

Solvent control**

    9

2.6

1.0

5

0.6

1.0

16

2.1

1.0

86

9.6

1.0

10

9

2.5

1.1

4

1.5

0.9

11

1.2

0.7

73

8.9

0.8

100

7

2.9

1.1

4

1.2

0.9

12

4.5

0.5

74

4.0

0.9

333.3

8

2.6

1.1

5

2.1

1.0

14

2.6

0.7

74

11.1

0.9

1000.0

7

1.0

0.9

4

0.6

0.9

14

2.6

0.7

82

4.7

1.0

5000.0

7

1.2

1.1

4

1.5

0.8

18

2.6

0.9

72

6.0

0.8

Positive control***

675

33.6

75.0

162

27.2

34.6

1068

370.0

65.4

967

150.2

11.2

Negative control*: concurrent untreated control

Solvent control**: ethanol

Positive control***: see table 7.6.1/2

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (second test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Negative control*

7

3.5

-

5

1.0

-

27

1.2

-

82

9.6

-

Solvent control**

    6

1.5

1.0

10

1.2

1.0

34

6.9

1.0

86

4.5

1.0

10

9

2.6

1.4

7

1.0

0.7

29

1.7

0.9

70

3.2

0.8

100

6

3.1

0.9

6

1.5

0.7

35

3.0

1.0

79

8.5

0.9

333.3

6

1.2

1.0

4

1.2

0.4

32

7.2

0.9

83

10.1

1.0

1000.0

7

3.5

1.2

6

2.1

0.6

31

6.0

0.9

81

9.2

0.9

5000.0

6

1.0

0.9

6

1.5

0.7

31

6.8

0.9

76

6.7

0.9

Positive control***

199

9.8

31.4

211

42.7

21.8

1250

102.1

36.8

1515

116.3

17.7

Negative control*: concurrent untreated control

Solvent control**: ethanol

Positive control***: see table 7.6.1/2

Conclusions:
Interpretation of results:
negative TA1535; TA1537; TA98 and TA 100

Under the conditions of the assay, Isohexadecan did not demonstrate in vitro mutagenic activity in the Salmonella test system with and without S9 mix activation system.
Executive summary:

This data is being read across from the source study that tested Isohexadecane based on analogue read across.

In a reverse gene mutation assay in bacteria (Poth, 1990) and in compliance with Good Laboratory Practice, strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to Isohexadecan at concentrations of 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate in the presence and absence of mammalian metabolic activation. No cytotoxicity was observed with all the dose tested. Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used (+/- S9). The positive controls induced the appropriate responses in the corresponding strains. Under the test conditions, Isohexadecan did not induce in vitro mutagenic activity in the bacterial test system in the presence and the absence of S9 activation system.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 bacterial strains were used instead of 5
Principles of method if other than guideline:
Guideline study
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,4,4,6,8,8-heptamethylnonane
EC Number:
224-506-8
EC Name:
2,2,4,4,6,8,8-heptamethylnonane
Cas Number:
4390-04-9
Molecular formula:
C16H34
IUPAC Name:
2,2,4,4,6,8,8-heptamethylnonane
Details on test material:
- Name of test material (as cited in study report): BP Solvent IH/Isohexadecan
- Substance type: petroleum product, UVCB
- Analytical purity : 100% commercial product

Method

Target gene:
Reversion Histidine auxotrophy
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Remarks:
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO in liquid nitrogen.
Metabolic activation:
with and without
Metabolic activation system:
The S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male wistar rats; strain WU (Savo-Ivanovas, med. Versuchstier Zuchten GmbH) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously
Test concentrations with justification for top dose:
10.0; 100.0; 333.3; 1000 and 5000 µg/plate (With and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity for the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
concurrent untreated control
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below Table 7.6.1/2
Remarks:
6 plate for negative control (untreated strains), 6 plate for solvent, 3 plates for positive controls.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
After range-finding test, two independent experiments were conducted in the main test by agar plate incorporation with and without S9 mix. The different controls (negative, solvent and positive controls) were tested in the same conditions.

DURATION
- Preincubation period: the bacterial culture was incubated in a shaking water bath for 6 hours at 37°C.
- Exposure duration: 3 days at 37°C in the dark

SELECTION AGENT (mutation assays):histidine

NUMBER OF REPLICATION: three scoring (3 measurements/plate), the mean number and standard deviation of revertants are calculated for all groups. the means for all treatment groups are compared with those obtained for the solvent control groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Other: the colonies were counted using the BIOTRAN III counter. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

Evaluation criteria:
- A positive response was indicated by a reproducible, dose-related increase, whether it be two-fold over background or not.
- Mutation Factors (MF) (induced/spontaneous revertants) were calculated for all strains at the dose level tested.
Statistics:
A compound is deemed to provide evidence of mutagenic potential if:
- a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and
- the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

Results and discussion

Test results
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
See tables 7.6.1/3, 7.6.1/4, 7.6.1/5 and 7.6.1/6
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity assay was conducted with strains TA98 and TA100 at concentrations between 1 and 5000 µg/plate. The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA98 and TA100.

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY: no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/3: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Standard deviation

Factor +

Negative* control

12

1.2

-

8

0.6

-

16

1.0

-

77

10.0

-

Solvent control**

10

0.6

1.0

7

0.6

1.0

19

5.1

1.0

83

7.4

1.0

10

10

2.6

1.0

8

1.7

1.0

14

6.4

0.7

78

8.5

0.9

100

11

3.8

1.1

8

1.7

1.1

16

2.1

0.8

73

12.1

0.9

333.3

10

4.4

1.0

7

1.5

1.0

19

4.2

1.0

78

12.4

0.9

1000.0

11

3.6

1.1

7

1.0

17

1.2

0.9

79

4.7

0.9

5000.0

13

4.6

1.3

5

0

0.7

22

2.9

1.1

73

7.4

0.9

Positive control***

839

20.2

86.8

228

9.8

31.1

1694

244.4

87.6

1065

48.5

12.8

Negative control*: concurrent untreated control

Solvent control**: ethanol

Positive control***: see table 7.6.1/2

Table 7.6.1/4: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Negative control*

10

3.1

-

9

1.5

-

32

2.0

-

91

10.4

-

Solvent control**

   11

2.6

1.0

7

3.0

1.0

38

6.1

1.0

85

8.5

1.0

10

12

4.7

1.1

11

4.4

1.6

26

5.3

0.7

77

5.5

0.9

100

12

2.0

1.1

8

3.5

1.2

20

4.0

0.5

72

5.0

0.8

333.3

12

3.8

1.1

8

2.0

1.1

26

0.6

0.7

73

13.6

0.9

1000.0

10

3.0

0.9

12

4.0

1.7

28

5.0

0.7

81

7.6

1.0

5000.0

12

3.2

1.1

11

1.7

1.6

36

6.7

0.9

80

5.5

0.9

Positive control***

284

11.1

25.8

252

28.0

36.0

1916

60.5

50.4

1681

150.2

19.7

Negative control*:concurrent untreated control

Solvent control** ethanol

Positive control***: see Table 7.6.1/2

Table 7.6.1/5: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (second test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Negative control*

6

1.2

-

5

1.0

-

15

0.6

-

80

11.0

-

Solvent control**

    9

2.6

1.0

5

0.6

1.0

16

2.1

1.0

86

9.6

1.0

10

9

2.5

1.1

4

1.5

0.9

11

1.2

0.7

73

8.9

0.8

100

7

2.9

1.1

4

1.2

0.9

12

4.5

0.5

74

4.0

0.9

333.3

8

2.6

1.1

5

2.1

1.0

14

2.6

0.7

74

11.1

0.9

1000.0

7

1.0

0.9

4

0.6

0.9

14

2.6

0.7

82

4.7

1.0

5000.0

7

1.2

1.1

4

1.5

0.8

18

2.6

0.9

72

6.0

0.8

Positive control***

675

33.6

75.0

162

27.2

34.6

1068

370.0

65.4

967

150.2

11.2

Negative control*: concurrent untreated control

Solvent control**: ethanol

Positive control***: see table 7.6.1/2

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (second test)

Test substance concentration
(µg/plate)

TA 1535

TA 1537

TA 98

TA 100

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Mean

Standard deviation

Factor +

Negative control*

7

3.5

-

5

1.0

-

27

1.2

-

82

9.6

-

Solvent control**

    6

1.5

1.0

10

1.2

1.0

34

6.9

1.0

86

4.5

1.0

10

9

2.6

1.4

7

1.0

0.7

29

1.7

0.9

70

3.2

0.8

100

6

3.1

0.9

6

1.5

0.7

35

3.0

1.0

79

8.5

0.9

333.3

6

1.2

1.0

4

1.2

0.4

32

7.2

0.9

83

10.1

1.0

1000.0

7

3.5

1.2

6

2.1

0.6

31

6.0

0.9

81

9.2

0.9

5000.0

6

1.0

0.9

6

1.5

0.7

31

6.8

0.9

76

6.7

0.9

Positive control***

199

9.8

31.4

211

42.7

21.8

1250

102.1

36.8

1515

116.3

17.7

Negative control*: concurrent untreated control

Solvent control**: ethanol

Positive control***: see table 7.6.1/2

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative TA1535; TA1537; TA98 and TA 100

Under the conditions of the assay, Isohexadecan did not demonstrate in vitro mutagenic activity in the Salmonella test system with and without S9 mix activation system.
Executive summary:

In a reverse gene mutation assay in bacteria (Poth, 1990) and in compliance with Good Laboratory Practice, strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to Isohexadecan at concentrations of 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate in the presence and absence of mammalian metabolic activation. No cytotoxicity was observed with all the dose tested. Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used (+/- S9). The positive controls induced the appropriate responses in the corresponding strains. Under the test conditions, Isohexadecan did not induce in vitro mutagenic activity in the bacterial test system in the presence and the absence of S9 activation system.