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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All read across genetic toxicity tests listed below had negative results for Hydrocarbons, C14-C20 (even numbered), n-alkanes, isoalkanes, <2% aromatics.

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

Genetic Toxicity in vitro – Mammalian chromosome aberration test (OECD 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:
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:
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 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ames test
Principles of method if other than guideline:
Guideline principles
GLP compliance:
yes (incl. QA statement)
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.
Key result
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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: “Acceptable, well-documented study report equivalent or similar to OECD guideline 473: 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 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
N/A
Species / strain / cell type:
primary culture, other: human lymphocytes from two male and one female donor
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 without S9 (40.36, 57.66, 82.34 ug/ml);
Experiment 1 with S9 for 3 hours followed by 17 hour recovery (490, 700, 1000 ug/ml)
Experiment 2 without S9 20h treatment 0h recovery (22.52, 28.15, 35.18 ug/ml)
Experiment 2 with S9 for 3 hours followed by 17 hours recovery (640, 800, 1000 ug/ml)
Experiment 2 with S9 for 3 hours followed by 41 hours recovery (1000 ug/ml)
Experiment 3 without S9 for 20 hours treatment and 0 hours recovery (28.15, 35.19, 43.99 ug/ml)
Experiment 3 without S9 for 44 hours and 0 hours recovery (43.99 ug/ml)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline, cyclophosphamide
Evaluation criteria:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and 2) the proportion of cells with structural aberrations at such doses exceeded normal range, and 3) the results confirmed in the second experiment. A positive result only at delayed harvest in Experiment 2 was to be taken as evidence of clastogenicity provided criteria 1 and 2 were met. Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, were likely to be concluded as equivocal. Full assessment of the biological importance of such increases is likely to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one aberration were to be considered of particular biological significance.
Key result
Species / strain:
primary culture, other: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

The mammalian chromosomal aberration test to assess the genotoxicity of SHELLSOL D70 was negative. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

The potential of SHELLSOL D70 to cause chromosome aberration was investigated in cultured human lymphocytes with and without the metabolic activation S9 system. Negative and positive control substances were include in both experiments to confirm the activity and sensitivity of the test systems.  In the first experiment, the maximum dose levels selected for chromosome analysis were 82.34 ug/ml and 1000 ug/ml, in the absence and presence of S9 respectively.  These dose levels caused inhibitions of the mitotic index of 57% and 30% respectively.  In the second experiment, the highest concentration used for chromosome analysis were 35,18 ug/ml and 1000 ug/ml in the absence and presence of S9 respectively, these gave a reduction in the mitotic index of 52% and 12% respectively.  In both Experiments 1 and 2 in the presence of S9; and in Experiment 2 in the absence of S9 only there were no significant increases in the frequency of the cells with structural aberrations in cultures treated with SHELLSOL D70.  Following treatment in Experiment 2 in the absence of S9 there was a significant increase in the frequency of structural aberrations at the lowest dose analyzed (22.52ug/ml).  Additional doses from Experiment 1 were analyzed (19.79 and 28.25 ug/ml) to confirm whether this effect was only apparent at low concentrations.  No increase in the frequency of structural aberrations was apparent at these concentrations.  In order to further clarify the findings seen in the initial experiments, a third experiment was performed in which there were no significant increases in the frequency of cells with structural aberrations in all cultures treated with SHELLSOL D70.  Since the increase in structural aberrations seen at 22.52 ug/ml in Experiment 2 was not apparent in other experiments at similar or higher concentrations, the effect was considered to be non-reproducible and of no biological importance.  Based on these results, it is concluded that SHELLSOL D70 did not induce chromosome aberrations in cultured lymphocytes when tested to its limit of toxicity in both the absence and presence of S9.  This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

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:
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:
experimental study
Adequacy of study:
key study
Study period:
1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to sound scientific principles.
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)
Deviations:
no
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Chinese Hamster V79 cells in vitro (V79/HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Chinese Hamster V79 cells in vitro (V79/HPRT)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor
Test concentrations with justification for top dose:
without S9: 0.1, 0.3, 0.6, 1, 2, 3 ug/mL
with S9: 100, 500, 1000, 5000 ug/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation 0.6 ug/mL; with metabolic activation 5000 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative with and without metabolic activation

Exposure to test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane is not considered to be mutagenic in this test system.
Executive summary:

Exposure to test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane is not considered to be mutagenic in this test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to sound scientific principles.
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)
Deviations:
no
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK+/ phenotype
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
TK+/ phenotype of L5178Y mouse lymphoma cells from subline 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Aroclor
Test concentrations with justification for top dose:
up to was 1000 ug/mL in dimethylsulfoxide (maximum dose)
Vehicle / solvent:
dimethylsulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
This assay was performed with the TK+/ phenotype of L5178Y mouse lymphoma cells from subline 3.7.2C using a minimum of eight test compound doses with and without metabolic activation by an Aroclor induced rat liver microsomal fraction. Appropriate negative, solvent, and positive controls were included with each assay. The test compound dose levels were determined by a preliminary multidose ranging study with the highest dose targeted to give approximately fifty to ninety percent inhibition of suspension cell growth depending on the solubility of the compound. C10-C13 isoalkanes achieved a homogeneous mixture at approximately 100 mg/ml in dimethylsulfoxide. The maximum dose selected for the mutagenicity test was 1000 ug/ml because it represents the limits of solubility of the test material.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Exposure to eight graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative with and without metabolic activation

Exposure to eight graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.
Executive summary:

Exposure to eight graded doses of the test material in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.

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

Genetic toxicity in vivo

Description of key information

Read across genetic toxicity test listed below had negative results for Hydrocarbons, C14-C20 (even numbered), n-alkanes, isoalkanes, <2% aromatics.

 

Genetic Toxicity in vivo – micronucleus assay (OECD 474)

 

Genetic Toxicity in vivo – Rodent Dominant Lethal Test (OECD TG 478)

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:
1990/10/24 - 1990/11/30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to OECD Guideline 474. 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 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system 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: 7d

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 1.0 ml/100 g bw.
Details on exposure:
The test material and the carrier were administered by oral gavage as a single dose. The carrier was dosed at a volume equal to the test material dose volume. The individual animal dose volumes did not exceed 1.0 ml/100 g body weight; animals were administered 1.0, 2.5, 5.0 g test material/ kg body weight. The positive control, cyclophosphamide was administered as a single dose of 40 mg/kg using water as a carrier.
Duration of treatment / exposure:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
Frequency of treatment:
One dose was given at either 1.0, 2.5, 5.0 g test material/ kg body weight. Cyclophosphamide was dosed at 40 mg/kg.
Post exposure period:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
No. of animals per sex per dose:
Male (65), Female (65) ; 5 Males and 5 Females per treatment group
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. Additional criteria for scoring micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cell’s diameter. 1000 PCE from each animal were examined for the presence of micronuclei and the ratio of PCE to NCE was determined for each animal by counting 1000 erythrocytes (PCE and NCE).
Statistics:
Calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period (ANOVA). When ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test.

A standard regression analysis was performed to test for a dose response.
Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance). Therefore nonparametric analysis was not performed.

Sexes were analyzed separately.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The positive control (cyclophosphamide) induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes, indicating that the positive control was clastogenic and was responding in an appropriate manner. Carrier control values for the mean percent of polychromatic erythrocytes and for the mean number of micronucleated polychromatic erythrocytes are within the normal range for the corn oil control. MRD-90-874 did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes which is a measure of bone marrow toxicity. MRD-90-874 did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes. MRD-90-874 did not induce a significant increase in the mean number of micronucleated polychromatic erythrocytes. MRD-90-874 was not cytotoxic at doses up to 5.0 g/kg to mouse bone marrow under the conditions of this test.
Conclusions:
Interpretation of results: negative
These data indicate that MRD-90-874 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg of body weight.
Executive summary:

The test material, MRD-90-874 was tested in the mammalian bone marrow micronucleus assay using CD-1 mice.  MRD-90-874 was 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 MRD-90-874 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 474: 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 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
According to US EPA Guideline 84-2
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: ca. 8-9 weeks
- Weight at study initiation: 21-40 grams
- Assigned to test groups randomly: [no/yes, under following basis: computer generated, body weight sorting program
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 28 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): not to exceed 1ml/100 grams bw
- Purity: assumed to be 100% pure
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighted out and on the day of dosing, mixed with the carrier to provide stock solutions such that individual animal dose volumes did not exceed 1ml/100grams body weight. The mice were administered 1.25, 2.5, or 5.0 grams of test material/kg of body weight. Corn oil served as the carrier for the test material and was dosed at the same volume as the test material.

Duration of treatment / exposure:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing.
Positive control animals were sacrificed 24 hours after injection
Frequency of treatment:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing
Post exposure period:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing
Remarks:
Doses / Concentrations:
5.0 g/kg/bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2.5g/kg/bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1.25 g/kg/bw
Basis:
nominal conc.
No. of animals per sex per dose:
30 animals (5 male; 5 female)/dose; 10/timepoint
Positive control(s):
cyclophosphamide;

- Route of administration: intraperitoneal injection
- Doses / concentrations:40 mg/kg using water as the carrier
Tissues and cell types examined:
Bone marrows were collected and extracted, smear preparations made and stained. Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored for each animal.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range finding study was performed using 5.0, 2.5, and 1.0 gram of test material per kg of body weight. Two males and two females were used for each dose group. All animals survived and were sacrificed 24 hours after dosing. bome marrow was removed and slides were prepared. Slides were evaluated for percent of polychromatic erythrocytes in 1000 erythrocytes and for number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes.




DETAILS OF SLIDE PREPARATION: After sacrifice, both femurs were removed. The bone marrow was then removed and suspended in fetal bovine serum. After the suspension was centrifuged, the pellet was resuspended and smears were prepared (two slides per animal). Slides were labeled with blind coding. Slides were stained using acridine orange. 1000 polychromatic erythrocytes from each animal were examined for the presence of micronuclei, and the ratio of PCE’s to NCE’s determined


METHOD OF ANALYSIS: staining color, and circular appearance and a diameter between 1/20 and 1/5 of the cell's diameter


Statistics:
Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period. When the ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test. A standard regression analysis was performed to test for a dose response. Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance) in more than 25% of the analyses. Therefore nonparametric analysis were not performed. Sexes were analyzed separately
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No deaths or clinical signs of toxicity were observed in animals dosed with the test material up to the maximum recommended dose of 5g/kg
Conclusions:
Interpretation of results: negative
The in vivo micronucleus assay of MRD-89-582 in mice was negative. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.
Executive summary:

MRD-89-582 was examined for its potential to induce chromosomal damage in bone marrow erythrocytes in mice dosed by oral gavage at concentrations of 5.0,2.5, and 1.25 g/kg. Vehicle and positive control animals received corn oil and cyclophosphamide, respectively.  Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing.  MRD-89-582 did not induce a statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control material (cyclophosphamide) produced a marked increase in the frequency of micronucleated PCE when compared to the concurrent vehicle control group The test material was considered to be non-genotoxic and non-clastogenic under the conditions of the test. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations guidelines.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 478.
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 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: Males (7-8 weeks); females pre-treatment mating period (8-9 weeks); females post treatment mating period (7-8 weeks)
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: males were house individiually during the treatment period and hosed with two females per week during the 2 week pretreatment mating period and the 6 week post-treatment mating period. Females were housed individually during the pre-mating and post-mating periods and housed with males in a 2:1 ratio during mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum


Route of administration:
inhalation: vapour
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
MRD-77-44 was transferred from a reservoir using a metering pump into a heated flask and flash evaporated. A stream of clean air was also passed through the flask and the vapor laden air transferred to a port in the chamber air inlet where it was diluted with normal chamber intake air to give the desired concentration.
- Exposure apparatus: inhalation chamber
- Rate of air: 125 liters/minute



- Air flow rate: 125 liters/minute
- Air change rate: 8 minutes
- Method of particle size determination:
- Treatment of exhaust air:


TEST ATMOSPHERE
- Brief description of analytical method used: Wilks Scientific Copr, Miran IA Ambient Air Analyzer (long path infrared)
- Samples taken from breathing zone: no
Duration of treatment / exposure:
Six hours /day
Frequency of treatment:
five days
Triethylenemelamine was administered by intraperitoneal injection (normal saline) as a single dose.
Post exposure period:
Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks. The females were sacrificed 12 days after the last day of cohabitation
Remarks:
Doses / Concentrations:
900 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Negative control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
Positive control: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
300ppm MRD-77-43: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
900ppm MRD-77-43: 10 males; 120 females during six week post-treatment mating period (two females/male/week)
Control animals:
yes
Positive control(s):
triethylenemelamine

- Route of administration: Intraperitoneal injection
- Doses / concentrations: 0.5mg/kg/bw
Tissues and cell types examined:
Males: seminal vesicle, epididymides, prostate, and any abnormal lesion or tissue masses, testes.
Females: reproductive tissues examined (uterine horns preserved, implantation sites, resorption sites)
Statistics:
Comparisons were made during the treatment and post-treatment periods between negative control, positive control and test substance-treated groups by the chi-square method where applicable. Absolute data were compared using the F-test and Students t-test. When variances differed significantly, Students T-test was appropriately modified using Cochran’s approximation.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
When administered by vapor inhalation, MRD-77-43 is not mutagenic by the dominant lethal test. This finding does not warrant classification of (the test material as a genotoxin 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:

In a dominant lethal assay,  MRD-77-43 was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential.  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to MRD-77-43 produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, no treatment related effects were observed on the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resoprtions.  Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.  Based on these data, MRD-77-43 when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test.  This finding does not warrant the classification of MRD-77-43 as a genotoxin 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

There is no data available for Hydrocarbons, C14-C20 (even numbered), n-alkanes, isoalkanes, <2% aromatics. However, data is available for structural analogues Hydrocarbons, C10-C12, isoalkanes, <2% aromatics; Hydrocarbons, C10-C13, n-alkanes, <2% aromatics; Hydrocarbons, C10 -C13, isoalkanes, <2% aromatics; Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics; isododecane, Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics; Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics; Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics; and isohexadecane. 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.

In Vitro

In vitro gene mutation study in bacteria

Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics

The mutagenic potential of the test material (Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics) was assessed in a key Salmonella typhimurium microsomal assay according to the Ames test, and in compliance with Good Laboratory Practice (TOTAL Solvants, 1994). 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, the test material did not demonstrate anyin vitromutagenic activity in this bacterial test system.

 

A reverse gene mutation assay in bacteria was conducted according to OECD 471 in compliance with GLP (Carless Refining & Marketing Ltd., 1997a). Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 were exposed to the test material (Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics) at concentrations of 0 (as solvent control), 0.1, 0.33, 1.0, 3.33 and 10 mg/plate in the presence and absence of S9 metabolic activation. Cytotoxicity was not observed at any dose level for all strains. Under the test conditions, the test material did not induce a revertant increase in any strain, in the presence or the absence of S9, up to the highest dose (10.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains.

Under the test conditions, Hydrocarbons, C14-C18, n-alkanes, isoalkanes, cyclics, <2% aromatics was not mutagenic in S.typhimurium in the absence and the presence of S9 activation system.

Hydrocarbons, C14-C17, n-alkanes, <2% aromatics

The mutagenic potential of the test material (hydrocarbons, C14-C17, n-alkanes, <2% aromatics) was assessed in the Salmonella typhimurium microsomal assay according to the Ames test in compliance with Good Laboratory Practice (Petroquimica, 1985).

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 mutants 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 72 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 test substance was then tested in another experiment performed in the same way as the range-finding 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 dissolved in acetone. Dose levels used in the main assay were 0, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix. All determinations were made in triplicate (3 automatic scoring measurements / plate). Two independent main tests were performed. Simultaneous negative (solvent, triplicate) and positive controls (triplicate) were used in all experiments and compared.

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. No increase in the mean number of revertant colonies for any S. typhimurium strains with and without S9-mix in both tests (pre-test and main test). Positive controls gave the expected increases in the number of revertants, with and without S-9 mix. Under the conditions of this study, the test material did not demonstrate any in vitro mutagenic activity in this bacterial test system.

Isohexadecane

In a supporting 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

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key study (Shell, 1998), the potential of the test material (Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics) to cause chromosome aberration was investigated in cultured human lymphocytes with and without the metabolic activation S9 system. Negative and positive control substances were include in both experiments to confirm the activity and sensitivity of the test systems. In the first experiment, the maximum dose levels selected for chromosome analysis were 82.34 ug/ml and 1000 ug/ml, in the absence and presence of S9 respectively. These dose levels caused inhibitions of the mitotic index of 57% and 30% respectively. In the second experiment, the highest concentration used for chromosome analysis were 35,18 ug/ml and 1000 ug/ml in the absence and presence of S9 respectively, these gave a reduction in the mitotic index of 52% and 12% respectively. In both Experiments 1 and 2 in the presence of S9; and in Experiment 2 in the absence of S9 only there were no significant increases in the frequency of the cells with structural aberrations in cultures treated with the test material. Following treatment in Experiment 2 in the absence of S9 there was a significant increase in the frequency of structural aberrations at the lowest dose analyzed (22.52ug/ml). Additional doses from Experiment 1 were analyzed (19.79 and 28.25 ug/ml) to confirm whether this effect was only apparent at low concentrations. No increase in the frequency of structural aberrations was apparent at these concentrations. In order to further clarify the findings seen in the initial experiments, a third experiment was performed in which there were no significant increases in the frequency of cells with structural aberrations in all cultures treated with the test material. Since the increase in structural aberrations seen at 22.52 ug/ml in Experiment 2 was not apparent in other experiments at similar or higher concentrations, the effect was considered to be non-reproducible and of no biological importance. Based on these results, it is concluded that hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, <2% aromatics did not induce chromosome aberrations in cultured lymphocytes when tested to its limit of toxicity in both the absence and presence of S9. This finding does not warrant the classification of this test material as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, <2% aromatics

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 is not classified according to the criteria of the CLP Regulation (1272/2008).

In vitro Gene Mutation study in Mammalian Cells

C10-C13 isoalkanes

In a key mammalian cell gene mutation assay (Chevron Phillips, 1982), exposure to eight graded doses of the test material (C10-C13 isoalkanes) in the presence of and in the absence of metabolic activation did not increase the induction of forward mutations in L5178Y mouse lymphoma cells at the T/K locus. Therefore C10-C13 isoalkanes are not considered to be mutagenic in this test system.

Isododecane

In a key mammalian cell gene mutation assay (INEOS, 1996), exposure to the test material in the presence of and in the absence of metabolic activation did not increase the induction of mutations. Therefore isododecane was not considered to be mutagenic in this test system.

In Vivo

Hydrocarbons, C10-C12, isoalkanes, <2% aromatics

In a key in vivo dominant lethal assay (ExxonMobil Corp., 1978), the test material (Hydrocarbons, C10-C12, isoalkanes, <2% aromatics) was administered by vapor inhalation for six hours/day for five consecutive days to male rats at dose levels of 300 and 900 ppm to test for mutagenic potential.  Included in the study was a negative (chamber exposed) control group and a positive control group.  The latter received 0.5mg/kg of triethylenemelamine administered intraperitoneally on a single day, two hours prior to mating.  Each group contained 10 proven fertile rats.  Following exposure, the males were mated with unexposed females (two female rats were mated with each male rat per week) for 6 consecutive weeks.  The females were sacrificed 12 days after the last day of cohabitation.  Exposure of males to the test material produced no adverse effects on mortality or body weight gain during the post-treatment mating period. Overall, no treatment related effects were observed on the number of pregnant females, number of implantations per litter, number of live fetuses, number of dead implantations, and the number of resoprtions. Exposures to male rats had no effect on their ability to mate and impregnate females, and to produce live fetuses.  Based on these data, the test material when administered by vapor inhalation to male rats is not considered mutagenic by the dominant lethal test. 

Hydrocarbons, C10-C13, n-alkanes, <2% aromatics

In a key in vivo study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, < 2% aromatics) was tested in the mammalian bone marrow micronucleus assay using CD-1 mice. The test material was 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 Hydrocarbons, C10-C13, n-alkanes, < 2% aromatics is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/Kg.

Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, <2% aromatics

In a key in vivo study (ExxonMobil, 1991), the test material (Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics) was examined for its potential to induce chromosomal damage in bone marrow erythrocytes in mice dosed by oral gavage at concentrations of 5.0,2.5, and 1.25 g/Kg. Vehicle and positive control animals received corn oil and cyclophosphamide, respectively. Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing. The test material did not induce a statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control material (cyclophosphamide) produced a marked increase in the frequency of micronucleated PCE when compared to the concurrent vehicle control group. The test material was considered to be non-genotoxic and non-clastogenic under the conditions of the test. This finding does not warrant the classification of Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics as a genotoxin under EU GHS guidelines.

Justification for classification or non-classification

The negative results observed in in vitro and in vivo genotoxicity assays do not warrant the classification of Hydrocarbons, C14-C20 (even numbered), n-alkanes, isoalkanes, <2% aromatics as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).