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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is no in vitro genetic toxicity data available for Hydrocarbons, C16-C18, Isoalkanes, <2% Aromatics. However, data is available for structural analogues isohexadecane, hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics, and hydrodesulfurized kerosene. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

All read across genetic toxicity tests listed below had negative results for Hydrocarbons, C16-C18, Isoalkanes, <2% aromatics.

Genetic Toxicity in vitro – Bacterial reverse mutation assay (OECD 471)

Genetic Toxicity in vitro – mammalian chromosome aberration test (OECD TG 473)

Genetic Toxicity in vitro - Mammalian Cell Gene Mutation Test (OECD TG 476)

Link to relevant study records

Referenceopen allclose all

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: supporting information
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
Target gene:
Reversion Histidine auxotrophy
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.
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.
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium 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
Species / strain:
E. coli WP2
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
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:

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
10 August to 21 September 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Few details on test material (no certificate of analysis)
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: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No certificate of analysis
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A culture medium supplemented with 10% fetal calf serum, 1% L-glutamine, and 1% penicillin and streptomycin, at about 37°C, in an atmosphere of about 5% C02 in air.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 from male Sprague-Dawley rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Range finding assay: half-log series of concentrations of 0.0835 to 2500 µg/mL
Main experiment:
- without metabolic activation: 3.13, 6.26, 9.35 and 12.5 µg/mL with 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL with 20-h harvest
- with metabolic activation: 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test material was insoluble in water and dimethylsulfoxide. A clear and homogeneous stock solution of 201 mg/mL with ethanol could be maintained.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: without metabolic activation: 7.25 and 17 h for 10 and 20 h assay, respectively; with metabolic activation: 2 h
- Expression time (cells in growth medium): with metabolic activation: 7.75 and 17.75 h for 20 and 10 h assay, respectively;
- Time in 0.1 µg/mL Colcemid: without metabolic activation: 1 and 0.5 h for 20 and 10 h assay, respectively; with metabolic activation: 2.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 10 h and 20 h without and without metabolic activation

STAIN (for cytogenetic assays): 5% Giemsa solution and BrdUrd (5-bromodeoxyuridine) at 10 µM

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 cells for test substance; at least 25 cells for positive controls

CYTOTOXICITY: visual observations based on confluence of monolayer and floating dead cells
Evaluation criteria:
Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 were analyzed.
The following factors were taken into account in the evaluation of the chromosomal aberrations data: the overall chromosomal aberration frequencies, the percentage of cells with any aberrations, the percentage of cells with more than one aberration, any evidence for increasing amounts of damage with increasing dose.
Chromatid and isochromatid gaps were not considered as they may be due to toxicity.
Statistics:
Fisher's exact test with an adjustment of multiple comparisons
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Range-finding without metabolic activation:
A very unhealthy cell monolayer, -70% reduction in the cell monolayer confluence, floating dead cells, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 25.0 µg/mL. Slight reductions in the number of visible mitotic cells and -15% reduction in the cell monolayer confluence were observed in the cultures dosed with 2.50 and 8.35 µg/mL.
Range-finding with metabolic activation:
An unhealthy cell monolayer, -85% reduction in the cell monolayer confluence, floating dead cells and debris, and severe reduction in the number of visible mitotic cells were observed in the culture dosed with 835 µg/mL. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 25.0 and 83.5 µg/mL.

Chromosomal aberrations assay without metabolic activation (Table 1):
In the 10 h assay, no toxicity was observed in any of the test cultures. These cultures were not analyzed for chromosomal aberrations as four dose levels were available for analysis from the 20 h assay. In the 20 h assay, an unhealthy cell monolayer, -70% and -45 % reduction in the cell monolayer confluence, floating dead cells and debris, and a severe reduction in visible mitotic cells were observed at 75.0 and 50.0 µg/mL, respectively. Toxicity was evident on the slides prepared from these cultures by the very sparse numbers of metaphases available for analysis.

Chromosomal aberration assay with metabolic activation (Tables 2 and 3):
In the 10 h assay, slight reductions in the numbers of visible mitotic cells were observed in the cultures dosed at 563 and 751 µg/mL.
In the 20 h assay, severe toxicity was exhibited on the slides prepared from the cultures dosed with 562 and 750 µg/mL by the presence of many dead cells and the sparse numbers of metaphases available for analysis. Reductions of -15% in the cell monolayer confluence were observed in the cultures dosed with 99.7, 187, 281, 375, 562, and 750 µg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 without metabolic activation (results from pooled duplicate cultures)

 

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

Concentration (µg/mL) 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

7

1

 

 

0.0

Positive (Mitomycin C)

0.04

7

 

4

7

28.0*

Test article

25.0

15

2

 

 

0.0

37.5

7

3

1

1

0.5

50.0

8

1

 

1

0.5

75.0

19

2

4

 

0.5

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 2: Chromosome aberrations in CHO cells fixed 10 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

 

Concentration (µg/mL)

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

2

 

 

0.0

Positive (Cyclophosphamide)

25.0

1

 

8

13

44.0*

Test article

282

7

1

 

 

0.0

375

3

1

0.5

563

4

 

1

0.5

751

3

3

 

1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Table 3: Chromosome aberrations in CHO cells fixed 20 h after exposure to MRD-90-843 with metabolic activation (results from pooled duplicate cultures)

 

Concentration (µg/mL)

Number and type of aberration

 

 

 

Not computed

Simple

Complex

% cells with aberrations

 

 

Chromatid gap

Chromosome gap

 

 

 

Negative (vehicle)

-

7

 1

0.0

Positive (Cyclophosphamide)

12.5

1

 

17

31

80.0*

Test article

281

15

2

 

1.0

375

16

6

1

1

1.0

562

3

1

1

1.0

750

10

1

1.0

* Significantly greater than the pooled negative and vehicle controls, p<0.01

Conclusions:
Interpretation of results:
negative

MRD-90-843 was found not to increase chromosome aberrations in CHO cells with and without metabolic activation.
Executive summary:

In an in vitro chromosome aberration test, Chinese Hamster Ovary cells were exposed to MRD-90-843 at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation.

Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, MRD-90-843 is not classified according to the criteria of Annex VI to Directive 67/548/EEC and the CLP Regulation (1272/2008).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Equivalent or similar to OECD Guideline 476.
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: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
na
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate
Test concentrations with justification for top dose:
Without activation: 6.25 nl/ml to 37.5 nl/ml
With activation: 3.91 nl/ml to 62.5 nl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [ethanol]
- Justification for choice of solvent/vehicle:The test material was miscible with ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 hours

SELECTION AGENT (mutation assays): BrdU

NUMBER OF REPLICATIONS: Variable with or without activation

NUMBER OF CELLS EVALUATED: 3x10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 40.8 x 10^-6
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material induced a good range of toxicities for evaluation of the test material (percent relative growths, 65.3% to 2.8%). The toxicities did show some variability between replicate samples. In the presence of metabolic activation, no indication of mutagenic activity was observed. The average cloning efficencies for the solvent and untreated negative controls varied from 119.1% without activation to 82.7% with activation which demonstrated very good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive compounds yielded normal mutant frequencies that were greatly in excess of the background.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

The test material was examined for mutagenic activity in the mouse lymphoma forward mutation assay in the absence and presence of a liver S9 fraction for metabolic activation. The test material did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to 37.5 nl/ml without activation and 62.5 nl/ml with activation were assayed and high toxicities were induced without inducing significant increases in the mutant frequency. It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is no in vivo genetic toxicity data available for Hydrocarbons, C16-C18, Isoalkanes, <2% aromatics. However, data is available for structural analogue hydrodesulfurized kerosene and is read across to based on a worst case basis. A discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Hydrodesulfurized kerosene was non-mutagenic when tested in an in vivo mammalian bone marrow chromosome aberration test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1984
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 475: GLP.
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: supporting information
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
intraperitoneal
Details on exposure:
A pilot study was carried out in 4 male and 4 female young adult Sprague Dawley rats. These animals were given a single intraperitoneal (i.p.) dose (3 g/kg) of API 81-07. During the following 48 hours observation, no animals died. The doses selected for the cytogenetics study were therefore 0.3, 1 and 3 g/kg. Three groups of 15 male and 15 female rats were given a single i.p. dose of either 0.3, 1 or 3 g API 81-07/kg. At six, 24 and 48 hours after dosing 5 males and 5 females were killed at each dose level. An additional 15 males and 15 females were untreated and served as negative controls. These animals were otherwise treated the same as the test animals. A positive control group of 5 males and 5 females was administered 0.8 mg/kg Triethylenemelamine (TEM) as a single i.p. dose. These positive control animals were killed 24 hours after administration of the positive control substance. Three hours prior to being killed with CO2, animals were injected i.p. with 4 mg/kg of colchicine. After the animal was killed, the adhering soft tissue and epiphyses of both tibiae were removed and the marrow was flushed from the bone and transferred to Hank's balanced salt solution. The marrow button was collected by centrifugation and was then re suspended in 0.075M KCl. The centrifugation was repeated and the pellet resuspended in fixative (methanol:acetic acid, 3:1). The fixative was changed once and left overnight. Cells in fixative were dropped onto glass slides which were then air dried and stained with Giemsa. Slides were coded and scored for chromosomal aberrations. 50 spreads were read for each animal where feasible. A mitotic index based on at least 500 counted cells was also recorded. The index was calculated by scoring the number of cells in mitosis per 500 cells on each read slide.
Duration of treatment / exposure:
Three groups of 15 male and 15 female rats were given a single i.p. dose of either 0.3, 1 or 3 g API 81-07/kg. At six, 24 and 48 hours after dosing 5 males and 5 females were killed at each dose level. An additional 15 males and 15 females were untreated and served as negative controls.
Frequency of treatment:
Single i.p. dose of either 0.3, 1 or 3 g API 81-07/kg
Remarks:
Doses / Concentrations:
0, 0.3, 1 or 3 g/kg.
Basis:
analytical conc.
i.p.
No. of animals per sex per dose:
15 male and 15 female rats
Control animals:
yes, concurrent no treatment
Positive control(s):
These animals were otherwise treated the same as the test animals. A positive control group of 5 males and 5 females was administered 0.8 mg/kg Triethylenemelamine (TEM) as a single i.p. dose. These positive control animals were killed 24 hours after administration of the positive control substance.
Details of tissue and slide preparation:
Three hours prior to being killed with CO 2 , animals were injected i.p. with 4 mg/kg of colchicine. After the animal was killed, the adhering soft tissue and epiphyses of both tibiae were removed and the marrow was flushed from the bone and transferred to Hank's balanced salt solution. The marrow button was collected by centrifugation and was then resuspended in 0.075M KCl. The centrifugation was repeated and the pellet re suspended in fixative (methanol:acetic acid, 3:1). The fixative was changed once and left overnight. Cells in fixative were dropped onto glass slides which were then air dried and stained with Giemsa. Slides were coded and scored for chromosomal aberrations. 50 spreads were read for each animal where feasible. A mitotic index based on at least 500 counted cells was also recorded. The index was calculated by scoring the number of cells in mitosis per 500 cells on each read slide.
Evaluation criteria:
Data interpretation and evaluation Gaps were not counted as significant aberrations. Open breaks were considered as indicators of genetic damage as were configurations resulting from the repair of breaks. The latter included translocations, multiradials, rings, multicentrics, etc. Reunion figures such as these were weighed slightly higher than breaks since they usually resulted from more than one break. Cells with more than one aberration were considered to indicate more genetic damage than those with evidence of single events. Consistent variations from the euploid number were also considered in the evaluation of mutagenic potential.

The type of aberration, its frequency and its correlation to dose in a given time was considered in evaluating the test material as being positive or negative.
Statistics:
Statistical evaluation Performed by Student's t-tests on four parameters:
1. Number of structural aberrations per animal
2. Number of numerical aberrations per animal
3. % cells with one or more structural aberrations per animal
4. % cells with 2 or more structural aberrations per animal.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The data are given in the report for males, females and as male and female pooled data. When the results for males were compared with those for controls and the females were compared to controls, no statistically significant differences were found. The data summarized below, are the pooled data for males and females. The structural aberration frequency did not differ significantly from the negative control at any tested dose. The percentage of cells showing one or more structural aberrations or 2 or more structural aberrations were also similar to the negative controls. A concurrent positive control group induced significant increases in aberrations.
Conclusions:
Interpretation of results: negative
The test material did not cause chromosome aberration in the test model.
Executive summary:

The test material did not cause chromosome aberration in the test model.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In Vitro

In vitro gene mutation study in bacteria

In a reverse gene mutation assay in bacteria (EC Erdolchemie, 1990) strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to isohexadecane 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, isohexadecane did not induce in vitro mutagenic activity in the bacterial test system in the presence and the absence of S9 activation system.

In Vitro Chromosome Aberration in Mammalian Cells

In an in vitro chromosome aberration test (ExxonMobil, 1991), Chinese Hamster Ovary cells were exposed to the test material ( Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics) at concentrations of 3.13, 6.26, 9.35 and 12.5 µg/mL for 10-h harvest and 12.5, 25, 37.5, 50 and 75 µg/mL for 20-h harvest, for 7 and 17 h, without metabolic activation and 37.5, 93.8, 188, 281, 375, 563 and 750 µg/mL for 10 and 20-h harvest, for 2 h, with metabolic activation.

Positive controls (mitomycin C without metabolic activation and cyclophosphamide with metabolic activation) induced the appropriate response. As there was no evidence of chromosome aberration induced over background, the test material was not classified according to the criteria of CLP Regulation (1272/2008).

In vitro Gene Mutation study in Mammalian Cells

In a key study (American Petroleum Institute, 1984), the test material (hydrodesulfurized kerosene) was examined for mutagenic activity in the mouse lymphoma forward mutation assay in the absence and presence of a liver S9 fraction for metabolic activation. The test material did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to 37.5 nl/ml without activation and 62.5 nl/ml with activation were assayed and high toxicities were induced without inducing significant increases in the mutant frequency. It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

In Vivo

In a key mouse lymphoma forward mutation assay, the test material (hydrodesulfurised kerosene) showed no mutagenic properties (American Petroleum Institute, 1984).

Justification for classification or non-classification

The negative results observed in read across in vitro and in vivo genotoxicity assays do not warrant the classification of Hydrocarbons, C16-C18, isoalkanes, <2% aromatics as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).