Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All Ames tests on C14-20 Aliphatics (≤2% aromatic) showed no mutagenic effect with and without metabolic activation. The chromosome aberration study in CHO cells on hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics,<2% aromatics also showed no signs of mutagenicity. A mouse lymphoma forward mutation assay performed with hydrodesulfurised kerosene also showed no mutagenic properties.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 20, 1993 to February 4, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Ames test (5 Salmonella strains), GLP. Substance identification: information available from supplier for commercial name Substance analytical certificate available
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ames test
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Reverse gene mutation assay
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Stock of S. typhimurium tester strains were obtained from B. N. Ames (University of California Berkeley, USA). Master stocks are held in liquid nitrogen and were aliquots of nutrient broth cultures then stored at -80°C. See below Table 7.6.1/1.
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix was prepared in laboratory from liver of a Sprague-Dawley male rat (IFFA CREDO, France) induced by Aroclor 1254 and stored at -80 °C as aliquots. See below Table 7.6.1/2
Test concentrations with justification for top dose:
HDF 200 was tested as an emulsion in 10% Pluronic F68 aqueous solution (1/1)( v/v) in preliminary test and both main tests.
Doses: 0 (5% Pluronic F68 solution as solvent), 2, 6, 20, 60, 200 µL/plate in Pluronic F68 aqueous solution for all S. typhimurium strains (see below Table 7.6.1/3)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 10% Pluronic aqueous solution
- Justification for choice of solvent/vehicle: the test substance (oil) was insoluble in water and other vehicles (DMSO, acetone)
Untreated negative controls:
yes
Remarks:
Sterile test: plates without the addition of bacteria are prepared in order to assess the sterility of HDF 200, the S9 mix and the medium.
Negative solvent / vehicle controls:
yes
Remarks:
Aqueous Pluronic F68 solution at 5%
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See below Table 7.6.1/4
Remarks:
6 plates for negative control (solvent). 3 plates for positive controls. 3 plates for controls of sterility (S9, solvent, medium).
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 and positive controls, controls for sterility) were tested in the same conditions.

DURATION
- Preincubation period: yes- S9 activation system preincubation at 37°C for 60 min before agar plate incorporation (when negative results in the main first test in the presence of S9 activation)
- Exposure duration: 48h at 37°C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 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: other: a preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate (test substance dilutions in Pluronic F68 solution at 10% (1/1)( v/v)).


OTHER: 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: the ratio between test substance revertants and solvent negative control revertants was performed at each dose-concentration
Evaluation criteria:
CRITERIA OF DECISION:
-Biological significance:
a reproducible 2-fold increase in the number of revertants (3 times in the case of TA 1535 and TA 1537 strains) compared with the vehicle controls, in any strain at any dose-level with some evidence of a dose-relationship (3 dose-concentrations) will be considered as a positive result. Reference to historical data may be taken into account in the evaluation of the data obtained.

-Statistical significance:
the test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.
-Reproductibility:
Positive results should be observed in two independently tests. Positive results observed in one test without reproducibility in two tests independently conducted should not be considered as significant (Brusick ,1980). Complementary test could be performed.

ACCEPTANCE CRITERIA:- The mean of the solvent control revertants for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the study director. The positive control compounds must induce an increase in mean revertants of at least twice (3 times in the case of strains TA 1535 and 1537) the concurrent solvent controls.The test substance must be sterile at the highest concentration after agar plate incubation 48h at 37°C.
Statistics:
The test data were subjected to analysis to determine the statistical significance of any increase in revertants according to the Dunnett method.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 102 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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no

RANGE-FINDING/SCREENING STUDIES: The substance was freely soluble in the vehicle Pluronic F68 . A preliminary toxicity assay was conducted in S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations between 2 and 200 µL/plate in 10% Pluronic F68 solution (1/1) (v/v). A slight cytotoxicity (62.4% to 102.2% of the control survival) at the highest test substance was observed in all strains. Considering the slight toxicity this concentration (200 µL/plate) was used in the main test both in the absence and the presence of S9 activation system.

COMPARISON WITH HISTORICAL CONTROL DATA: yes


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

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

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

28

6.8

4

2.6

17

4.1

113

7.1

265

35

2**

21

4

5

0.6

13

3.2

119

10.3

263

46.8

6**

29

5.5

5

1.7

16

1.7

99

4.7

259

27

20**

24

5.5

4

1.7

16

5.1

110

6.9

251

45.4

60**

29

9.1

3

1.5

16

8.4

105

12.5

253

32.5

200**

31

5

3

0

16

3.5

116

9.3

205

16.5

Positive control***

461

37.2

543

159

340

19.7

701

88.9

1360

206.1

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

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

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

12

3.2

6

1.9

26

5

114

6

347

19.9

2**

11

2.5

7

2.9

21

3

127

5.7

355

35.1

6**

8

1.2

5

1.2

28

3.6

136

11.7

332

47.3

20**

12

3

5

2.3

24

5.6

141

1.5

331

4.6

60**

12

3.6

4

1

24

5.6

124

5.2

375

99.7

200**

9

0.6

7

3.2

29

5.6

137

8

375

19.2

Positive control***

892

104.6

94

10.5

1324

117.2

2653

361.7

1380

87

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/7: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

24

3.7

4

2.5

15

3.6

106

11.4

235

15.9

2**

28

5.3

4

2.9

15

4

113

10.6

273

33.1

6**

25

12.1

7

2.3

15

3

108

13.1

240

54.1

20**

25

4.7

4

1.2

15

6.4

108

11.7

225

28.3

60**

19

6

7

2.1

18

1.7

92

5

243

32.5

200**

21

4

5

1.2

18

4.6

105

3.2

225

31.9

Positive control***

825

67.1

133

21.4

318

29.4

1151

235.3

1213

41.6

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Table 7.6.1/8: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Second test)

Test substance concentration
(µL/plate)

TA 1535

TA 1537

TA 98

TA 100

TA 102

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

0*

9

5.1

4

1.7

18

6.3

138

12.6

395

45.1

2**

9

0.6

7

4.2

26

3.6

170

23

403

47.7

6**

10

2.6

7

0.6

29

10.7

183

19.2

373

25.7

20**

10

1.7

7

1.5

34

2.5

195

7

379

12.2

60**

9

1.7

7

0

33

11.6

213

9.3

384

41.8

200**

11

1

7

0.6

37

3.5

232

12.2

385

47.4

Positive control***

127

18.6

91

6

1457

193.5

1093

142.3

953

97.9

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Table 7.6.1/9: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation with pre-incubation (Third test)

Test substance concentration
(µL/plate)

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

0*

19

5.2

107

14.6

2**

26

2.6

100

8.5

6**

25

10.3

118

7.6

20**

31

0

153

5.7

60**

32

5.5

124

14.4

200**

30

1.5

135

17.6

Positive control***

1457

193.5

1305

51.4

* Solvent control = negative control: 5% Pluronic F68 aqueous solution

** Test substance diluted in 10% Pluronic aqueous solution (1/1) (v/v)

*** Mutagens positive controls: see Table 7.6.1/4

Red results: p 0.01

Conclusions:
Interpretation of results:
negative with and without metabolic activation

Under the test conditions, HDF 200 did not demonstrate any in vitro mutagenic activity in the Salmonella test system.
Executive summary:

The mutagenic potential of HDF 200 was assessed in the Salmonella typhimurium microsomal assay according to the Ames test, and in compliance with Good Laboratory Practice. The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 102, TA 98 and TA 100 were used in the presence and the absence of metabolic activation system from the liver fraction of Aroclor 1254-induced rats (S9-mix). Each strain was exposed to 5 dose levels according to the direct incorporating plate method. After 48 hours of incubation at 37°C, the revertant colonies were scored. A preliminary toxicity assay was performed according to the direct incorporating method to define the 5 dose levels to be used in the main test. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies. The test substance was tested in the main experiment according to two tests independently performed in the same way as the range-finding test. The test substance was diluted in 10% Pluronic F68 aqueous solution. Dose levels used in the main assay were 0 (solvent), 2, 6, 20, 60 and 200 µL/plate in the main test, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments. No toxicity was observed in any of the strains in the absence and in the presence of S-9 mix up to the highest dose tested in the main test (62.4% to 102.2% survival). No increase in revertant mean number was observed in any S. typhimurium strain with and without S9-mix in the preliminary test and in the first main test. However, positive results were observed in the second main test conducted with 60-minute S9 pre-incubation before plate incorporation.

Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Both statistically significant results and biologically significant results (two-fold increase by comparison with solvent) were observed at the highest test substance dose in S. typhimurium TA 98 but without a dose-effect relationship. Reduced but statistically significant positive results were also observed in S. typhimurium TA 100 with a dose-effect relationship. However, no biological significance was observed at any dose. A third test was performed in S. typhimurium TA 98 and TA 100 under the same conditions as the second main test. In this case the positive results obtained in the previous main test were not observed in either S. typhimurium TA 98 or TA 100. In the absence of reproducible results, the test substance was not considered as mutagenic in S. typhimurium according to the decision criteria of Brusick (1980).

Under the conditions of this study, HDF 200 did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
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:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
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. certificate)
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
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:
experimental study
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:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
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
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

Structurally analogous test materials, hydrodesulfurized kerosene and jet fuel A were non-mutagenic when tested in in vivo studies: mouse bone marrow micronucleus assay, mammalian bone marrow chromosome aberration test and a dominant lethal assay. 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 474.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: Charles River Breeding Laboratories, Inc.
Sex: Male (65), Female (65)
Age at study initiation: Approximately 9-10 weeks
Weight at study initiation: 23-39g
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent 5002 chow (pellets), ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 35d

ENVIRONMENTAL CONDITIONS
Temperature (°F): 68-76
Humidity (%): 40-70%
Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
Corn oil was used. Dose volume did not exceed 10 ml/kg bw.
Details on exposure:
The animals were randomly divided into 5 dose groups: 4 of the dose groups contained 30 mice (15 males and 15 females). The animals in the first 4 groups were treated with corn oil (the vehicle control) or with 1.0, 2.5, or 5.0 g/kg test material. Doses were administered by oral gavage: dosing volumes were 10 mL/kg. Five male and five female mice from each group were sacrificed 24, 48, or 72 hr after treatment, and the bone marrows were isolated and examined for the presence of micronuclei. The last test group contained 10 mice (5 males and 5 females), which were given 40 mg/kg cyclophosphamide by intraperitoneal injection. All of these mice were sacrificed 24 hr after test material administration.
Duration of treatment / exposure:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
Frequency of treatment:
One dose was given of either vehicle control or with 1.0, 2.5, or 5.0 g/kg test material.
Post exposure period:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
Remarks:
Doses / Concentrations:
0, 1.0, 2.5, or 5.0 g/kg
Basis:
actual ingested
oral gavage
No. of animals per sex per dose:
The animals were randomly divided into 5 dose groups: 4 of the dose groups contained 30 mice (15 males and 15 females). The animals in the first 4 groups were treated with corn oil (the vehicle control), or with 1.0, 2.5, or 5.0 g/kg test material. Marrows were isolated and examined for the presence of micronuclei. The last test group contained 10 mice (5 males and 5 females), which were given 40 mg/kg cyclophosphamide.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control, cyclophosphamide was administered as a single intraperitoneal injection (40 mg/kg) using water as a carrier.
Tissues and cell types examined:
Erythrocytes derived from femur bone marrow.
Details of tissue and slide preparation:
Immediately following the sacrifice of the animals, both femurs were removed and the bone marrow was removed and suspended in fetal bovine serum. After the suspension was centrifuged the pellet was resuspended and smears were prepared (two slides per animal).
Evaluation criteria:
Slides were stained using acridine orange; polychromatic erythrocytes (PCE) stained red/orange, nonchromatic erythrocytes (NCE) are unstained (dull green), and micronuclei stain bright yellow. Slides were evaluated at 400x by fluorescent microscopy. A total of 1000 erythrocytes were counted from each animal, and the numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. To determine micronucleus (MN) frequency, 1000 PCEs were examined and the number of MN per 1000 PCEs was reported.
Statistics:
Statistical analysis included calculation of means and standard deviations as well as a standard one way analysis of variance (ANOVA) at each time period. When the ANOVA was significant, comparisons of vehicle-treated to dosed group means were made according to Duncans Multiple Range test. A standard regression analysis was performed to test for dose-response relationships. Sexes were analyzed separately.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
None of the five test materials produced an increase in micronucleus frequency regardless of sex or sampling time. Additionally, there was no evidence of bone marrow depression. The positive control (cyclophosphamide) produced a significant increase in micronucleus formation, and the vehicle control values fell within the normal control limits.
Conclusions:
Interpretation of results: negative
These data indicate that kerosenes are not cytotoxic and are not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg of body weight. 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:

Kerosenes were tested in the mammalian bone marrow micronucleus assay using CD-1 mice.  The test materials were tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes. Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner.  These data indicate that kerosenes are not cytotoxic and are not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg of body weight.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:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
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:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
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.
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:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Equivalent or similar to OECD Guideline 483.
Justification for type of information:
The justification for read across is provided as an attachment in IUCLID Section 13.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Assigned to test groups randomly: [yes, under following basis: Litton Bionetics, Inc SOP ]
- Housing: Sanitary cages with bedding
- Diet: ad libitum
- Water: ad libitum
Route of administration:
inhalation
Details on exposure:
Following treatment, the males were sequentially mated to two females per week for 2 weeks. The number of females provided an adequate number of implantations per group per week. After the mating period, the females were removed and housed in other cages until killed.

The males were rested for 2 days at the end of each 5 day exposure period, and two new females were introduced into each cage. Conception took placed in more that 90% of females by the 5th day, and a 2-day rest was beneficial to the males with regard to subsequent weekly matings.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
5 days per week for eight weeks
Remarks:
Doses / Concentrations:
520 and 2080 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
12 males per dose
Control animals:
yes, historical
Positive control(s):
- triethylenemelamine
- Route of administration: acute IP injection
- Doses / concentrations: 0.3 mg/kg
Tissues and cell types examined:
Sperm cells
Details of tissue and slide preparation:
At necropsy, the uteri were examined, and the number of living and dead implantations were counted for each pregnant female.
Evaluation criteria:
Fertility Index, Total number of implantations, dead implantations, proportion of females with one or more dead implantations and the proportion of females with two or more dead implantations.
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The results show that the test article did not cause significant increases in either pre- or post-implantations loss of embryos when statistically compared with the negative control. It is therefore concluded that the test article did not induce dominant lethal mutations in mice at the two tested dose levels.
Conclusions:
Interpretation of results: negative
These data indicate that the test material did not induce dominant lethal mutations in CD-1 mouse sperm cells at concentrations of 100 and 400 ppm. 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 evaluated for its ability to to induce dominant lethal mutations in sperm cells of CD-1 male mice. The test material was administered at two exposure levels of 520 and 2080 mg/m^3 via inhalation exposure, 6 hours per day, 5 days per week for 8 weeks. The results show that the test article did not cause significant increases in either pre- or post-implantations loss of embryos when statistically compared with the negative control. It is therefore concluded that the test article did not induce dominant lethal mutations in mice at the two tested dose levels. 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)

Additional information

Five studies (Ames tests) were available on the mutagenic properties of different hydrocarbon substances. None of these studies showed any mutagenic effect of the different test materials without and with metabolic activation.

One chromosome aberration study in CHO cells on Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics,<2% aromatics also showed no signs of mutagenicity (Murli, 1991). In sister chromatid exchange, a structurally analogous test material hydrodesulfurized kerosene was determined to be non-clastogenic. 

A mouse lymphoma forward mutation assay performed with hydrodesulfurised kerosene also showed no mutagenic properties (American Petroleum Institute, 1984).

Structurally analogous test materials, hydrodesulfurized kerosene and jet fuel A were also non-mutagenic when tested in in vivo studies: mouse bone marrow micronucleus assay, mammalian bone marrow chromosome aberration test and a dominant lethal assay. 

Justification for classification or non-classification

All in vitro and in vivo tests on test substances and analogues were negative, the substances are considered not to be mutagenic and should not be classified for mutagenicity according to the criteria of Annex VI of Directive 67/548/EEC and CLP Regulation 1272/2008.