Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro studies were identified for a structurally related material; there was no evidence of genotoxic activity in-vitro either the modified Ames test or mouse lymphoma assay.

Additionally, in response to the Draft Decision Letter (dated 26th November 2019) CCH-D-2114492604 -43 -01/D, new in vitro genetic toxicity studies have been planned to be conducted on the target substance (OECD 471 and 473 or 487). In the event both studies are considered to be negative, an OECD 476 or 490 study will be conducted, fulfilling all information requirements.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recognised method in GLP compliant laboratory
Qualifier:
according to
Guideline:
other: Modified Ames Test according to ASTM E 1687
Principles of method if other than guideline:
14 Gas oils were examined for mutagenic activity in one histidine dependent auxotroph of Salmonella typhimurium, strain TA98, using a modification of the pour-plate assay designed to detect mutagenicity mediated by polynuclear aromatic compounds derived from petroleum, based upon the principles of the ASTM Standard Test Method E 1687.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
His- (reverse mutation to histidine-independence)
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
S. typhimurium TA98 hisD3052 rfa uvrB pKM101
Metabolic activation:
with
Metabolic activation system:
Aroclor 1254-induced S9
Test concentrations with justification for top dose:
Test substance
0, 12, 24, 36, 48, 60 microlitres per plate

Positive control
0, 3, 6, 9, 12, 15 microlitres per plate
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
concurrent DMSO extract of the Reference oil No. 1, a straight-run naphthenic vacuum distillate of known mutagenic activity
Details on test system and experimental conditions:
Preparation of test substance extract
Extracts of the test substance in dimethyl sulphoxide (DMSO) were prepared as follows:
Three separate aliquots (1 mL) of the test substance, warmed to approximately 45°C, were mixed thoroughly with 5 mL of DMSO. The mixtures were vortexed intermittently (every five minutes) for a total of thirty minutes. The mixtures were then centrifuged at 200 g for ten minutes, and the extract (lower phase) was removed. Only one extract of Reference oil No. 1 was prepared. All extracts were prepared on the day of use, and stored in the dark until required.
Dosing solutions were prepared for each test substance extract and for the reference oil extract by dilution with DMSO (the reference oil extract was diluted 1:3 with DMSO before preparation of the dosing solutions).

Mutation test procedure
The following sequence of steps was used in the performance of this test:
The S9 mix was prepared and placed on ice (for not longer than 2 hours before use).
“Top agar", consisting of 0.4% bacteriological grade agar and 0.5% NaCl in purified water (prepared by reverse osmosis) was melted and placed in a 45°C water-bath.
Dosing solutions for the test substance and Reference oil No. 1 were prepared as described above. Two (for each of the test extracts) or three (for the reference extract) sterile glass test tubes were dosed with 60 micro-L of each of these solutions and were allowed to stand for at least 30 minutes.
To each of the dosing tubes were added in order 0.5 mL of S9 mix and 0.1 mL of bacterial suspension, taking care to introduce both the S9 mix and the bacteria into the bottom of the tube. The tube was gently swirled after each addition. When each set of tubes (one test article including the solvent control) was completed, it was transferred to the gyratory incubator at 150 rpm and incubated at 37°C for 30 minutes.
The tubes were removed from the gyratory incubator. To each tube, beginning with the solvent controls and proceeding to higher doses, 2.0 mL of the top-agar, cooled to ca 40°C and supplemented with 10 mL of 0.5 mM histidine/0.5 mM biotin solution per 100 mL, was added. Immediately after addition, the mixture was vortex-mixed and poured onto previously prepared Petri dishes containing 25 mL minimal agar. Care was taken to obtain an evenly distributed and level top-agar layer. When the top-agar had hardened, the plates were inverted and incubated at 37°C for 48 hours. The plates were then removed from the incubator and the number of colonies determined by either automated or manual counting. Each Petri dish was individually labelled with a unique code recorded in the study file, identifying the contents of the dish.
Evaluation criteria:
For a test to be considered valid, the mean revertant colony counts obtained for Reference Oil No. 1 (diluted 1:3) must reach, in a dose-responsive manner, at least a two-fold increase over the mean solvent control count, and no more than three doses should produce mean revertant counts more than 15% below the following representative values:

Dose (µL/plate) 0 3 6 9 12 15

Mean revertants 46 54 69 73 81 93


The mean revertant count for the solvent controls should be in the range 30 - 60. Excursions from this range were considered acceptable only if there was no significant change in the slopes of the curves. The curve should be linear over at least four doses.

The Mutagenicity Index (MI), defined as the slope of the curve (revertants per µL DMSO extract), was calculated for all test samples.

Thresholds for interpretation of MI values defined in ASTM E 1687 are as follows:
Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.
Test substances with MI values >1 but <2 may or may not be non-carcinogenic in a mouse skin-painting bio-assay.
Test substances with MI values >2 are considered to have a high probability of being carcinogenic in a mouse skin-painting bio-assay.

A lower threshold value of 0.4 has been selected as the cut-off for Residual Aromatic Extracts (s), based on the results of skin-painting studies (Blackburn et al, 1996): s with a MI >0.4 demonstrated carcinogenic potential upon dermal application to mouse skin with chronic exposure, whereas s with a MI <0.4 did not demonstrate a carcinogenic potential.
Statistics:
The mean number of revertant colonies and standard deviations were calculated for all groups.
All valid data were plotted and analysed using a linear regression analysis programme.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
MI = 0.33
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix and test substance extracts.
The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.
The mean revertant colony counts obtained for Reference Oil No. 1 (diluted 1:3) reached, in a dose-responsive manner, at least a two-fold increase over the mean solvent control count, and mean revertant counts were within the acceptable range.

It was, therefore, confirmed that the tests were valid.
Remarks on result:
other:
Remarks:
negative with metabolic activation

 

Reference Oil No. 1

 

 

 

 

 

 

 

 

 

 

 

 

 

Dose (µL)

Counts

Mean

SD

 

 

 

 

 

 

 

 

 

 

 

 

0

41

44

45

43.3

2.1

 

 

 

3

56

55

51

54.0

2.6

 

 

 

6

66

70

68

68.0

2.0

 

 

 

9

77

74

76

75.7

1.5

 

 

 

12

79

83

79

80.3

2.3

 

 

 

15

87

91

94

90.7

3.5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Con#16(i)

 

 

 

 

 

 

 

 

 

 

 

 

 

Dose (µL)

Counts A

Counts B

Counts C

Mean

SD

 

 

 

 

 

 

 

 

 

0

42

44

41

46

44

43

43.3

1.8

12

54

55

52

55

54

50

53.3

2.0

24

55

61

61

59

55

57

58.0

2.8

36

62

62

59

59

61

58

60.2

1.7

48

63

59

64

61

61

64

62.0

2.0

60

67

63

63

66

61

62

63.7

2.3

 

 

 

 

 

 

 

 

 

 

Con#16(i) MI value: 0.33

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation

The test substance had a Mutagenicity Index (MI) of 0.33

Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.
Executive summary:

The test substance had a Mutagenicity Index (MI) of 0.33

Test substances with MI values <1 are considered to have a high probability of being non-carcinogenic in a mouse skin-painting bio-assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: no information on GLP. Study report lacks some details but nevertheless provides useful 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:
L5178Y mouse lymphoma cells
Test concentrations with justification for top dose:
0.125, 0.188, 0.25, 0.375, 0.5 uL/ml - non activation study
0.064, 0.125, 0.188, 0.25, 0.375, 0.5 uL/ml - activation study
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ACETONE
Positive controls:
yes
Positive control substance:
other: ethylmethane sulphonate and dimethyl nitrosamine
Details on test system and experimental conditions:
L5178Y MOUSE LYMPHOMA CELLS
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
both with and without activation
Cytotoxicity / choice of top concentrations:
not specified
Vehicle 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 (migrated information):
negative

Diesel fuel was not mutagenic in the mouse lymphoma assay
Executive summary:

The genotoxic activity of diesel fuel was evaluated in a mouse lymphoma assay using L5178Y cells. Assays both in the presence and absence of a mouse liver mirosome fraction were conducted. Dose levels ranged between 0.064 and 0.5 uL/ML.

Results showed no evidence of of genotoxic activity, either in the presence or absence of metabolic activation. The positive control materials both showed expected results.

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

Genetic toxicity in vivo

Description of key information

Three read-across in-vivo were identified for a structurally related material. In a dominant lethal assay in male mice there was no evidence of heritable genotoxic effects. In a well conducted bone marrow chromosomal aberration assay by the oral route there was no evidence of genotoxic activity. In a further bone marrow chromosomal aberration study by the intraperitoneal route weak activity was observed. Effects were only seen at high dose levels (2.0 and 6.0 ml/kg) and the magnitude of effect was small and restricted to chromosomal fragments.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because, though it does not follow a prescribed guideline, it is well conducted and reported.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
No specific GLP guideline was followed. The study is well conducted and reported.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Doses / Concentrations:
1.0, 2,5, 5,0 g/kg
Basis:
no data
No. of animals per sex per dose:
15 male 15 female / dose level
Tissues and cell types examined:
bone marrow erythrocytes
Details of tissue and slide preparation:
bone marrow smears harvested at 24, 48, 72 hrs post exposure
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
The study authors concluded that home heating oil did not exhibit a positive response when tested for micronucleus induction.
Executive summary:

In a micronucleus assay, fifteen male and female CD-1 mice were treated with 1.0, 2.5, or 5.0 g/kg of home heating oil dissolved in corn oil via oral gavage. A concurrent control group received only corn oil, while another group served as positive control and was treated with 0.04 g/kg cyclophosphamide.

Five male and female mice from each dose group were sacrificed 24, 48 or 72 hours after test material administration. Bone marrows were removed and examined for the presence of micronuclei. Mice treated with cyclophosphamide were sacrificed 24 hours after administration. The micronucleus assay was conducted using the Schmid method. Erythrocytes numbering 1,000, were counted for each animal bone marrow slide and the number of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. Frequency of micronucleus induction was determined by examining the number of micronuclei per 1,000 PCEs. 

There was no increase in the frequency of micronuclei for the test material. In addition, there was no evidence of bone marrow depression. Cyclophosphamide, the positive control, exhibited appropriate results and the vehicle control result was within the normal range. Based on these results the study authors concluded home heating oil did not exhibit a positive response. 

This study received a Klimisch rating of “reliable with restrictions” because, though it does not follow a prescribed guideline, it is well conducted and reported.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: other: dominant lethal assay
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No information on GLP Compliance, No guideline stated. Study report lacks some details but nevertheless provides useful supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
Male mice were exposed by inhalation to diesel fuel, 6 hrs/day, 5 days / week for 8 weeks prior to mating with untreated female mice. Dominant lethal outcome was assessed by monitoring successful uterine implants and also number of dead implants.
GLP compliance:
not specified
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
CD-1
Sex:
male
Route of administration:
inhalation
Vehicle:
filtered air
Duration of treatment / exposure:
8 weeks
Frequency of treatment:
6 hours / day, 5 days / week
Remarks:
Doses / Concentrations:
100 and 400 ppm
Basis:

No. of animals per sex per dose:
12 males / dose
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine (TEM)
Sex:
male
Genotoxicity:
negative
Remarks:
NO EFFECT ON MALE FERTILITY
Positive controls validity:
valid


The results were as follows:

                                Dose Group
                 100        400        Negative     Positive
  Parameter        ppm        ppm        control      control

 Fertility index  
    Week 1        0.727       0.750        0.708      0.458
    Week 2        0.955       0.792        0.833      0.792


Implants/female
    Week 1        11.06       10.72        11.18      8.36
    Week 2        10.81       11.68        10.95      7.42


Resorptions/female
    Week 1        0.375       0.5         0.77        4.73
    Week 2        0.71        0.32        0.55        4.9


Proportion of females with 1 or more dead implants
    Week 1        0.32        0.39        0.41        1.0
    Week 2        0.52        0.26        0.55        1.0

Proportion of females with 2 or more dead implants
    Week 1        0.06        0.11        0.24        1.0
    Week 2        0.14        0.05        0           0.95


Dead implants/Total implants
    Week 1        0.03        0.04        0.07        0.57
    Week 2        0.07        0.03        0.05        0.66


Living implants/pregnant female
    Week 1        10.7        10.2        10.4        3.6
    Week 2        10.1        11.4        10.4        2.5

Conclusions:
Interpretation of results (migrated information): negative for effects on male fertility
Inhalation exposure of male mice to diesel fuel showed no evidence of an effect on male fertility.
Executive summary:

The effects of inhalation exposure to diesel fuel on male fertilty were investigated in mice exposed 6 hrs/day, 5 days /week for 8 weeks. Following exposure, mice were mated with untreated female mice. Levels of exposure were 100 and 400 ppm. Effect on male fertility was evalueted on the basis of number of succesful uterine implants and viability of the implanted embryos.

There was no effect on the number of succesful impants, pre or post implant loss or death of embryos. The positive control material (TEM ) gave the expected response.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 10-12 weeks
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ] yes
- Fasting period before study:
- Housing: 5/cage
- Diet (e.g. ad libitum): Purina lab chow
- Water (e.g. ad libitum):
- Acclimation period: 10 days

Route of administration:
intraperitoneal
Details on exposure:
animals dosed and 2 hrs before sacrifice administered 4mg/kg colchicine to arrest metaphase
Duration of treatment / exposure:
Up to 48 hours and 5 days
Frequency of treatment:
either single dose or 5 exposures 24 hrs apart
Post exposure period:
Single dose - 6 24 or 48 hrs post dose
Repeat dose - 6hrs after last treatment
Remarks:
Doses / Concentrations:
0.6, 2 and 6 ml/kg
Basis:

No. of animals per sex per dose:
Acute - 15 animals / dose
Repeat dose - 5 animals / dose
Control animals:
other: saline
Positive control(s):
triethylene melamine
Tissues and cell types examined:
bone marrow smears
Details of tissue and slide preparation:
femeur and tibia bone marrow cells were collected and tranferred to Hank's balanced salt solution. Cells were centrifuged and the cell pellet resuspended in potassium chloride solution prior to fixation in Carnoy's fixative.
Slides were prepared and satined with 5% Giesma solution. Slides were examined microscopically for metaphases.
Sex:
male
Genotoxicity:
positive
Remarks:
2.0 & 6.0 ml/kg gave weak positive responses (acute study)
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Similar aberrations were observed throughout the treatment and negative  

control groups, the only difference being the frequency of aberration.
The results of the evaluation are summarized in the following table.
Group   Time      No        Cells with      No. animals        MI**
        (hrs)     of        1 or more       without                
                cells       aberrations    aberrations*                

Saline   6       53         0 (0%)          2 (2)               0.7
        24       225        6 (2.6%)        2 (5)               3.2
        48       200        1 (0.5%)        3 (4)               3.6
        5 doses  250        2 (0.8%)        4 (5)               10.6        

TEM     24       144        68 (47.2%)      0 (5)               1.4

Diesel   6       189        5 (2.6%)        0 (5)               5.9
  0.6   24       48         1 (2.1%)        4 (5)               1.7
ml/kg   48       186        1 (0.5%)        4 (5)               2.3
     5 doses     250        4 (1.6%)        2 (5)               3.6


Diesel   6        228       8 (3.5%)        1 (5)               6.3
2.0     24        86        5 (5.8%)        2 (5)               1.7
ml/kg   48        216       2 (0.9%)        3 (5)               4.5
   5 doses        250        1 (0.4%)        4 (5)              7.4

Diesel   6        100       1 (1.0%)       1 (2)                4.0
6        24       227       5 (2.2%)       2 (5)                4.7
ml/kg    48       93        5 (5.4%)       2 (4)                3.8
        5 doses   200       10 (5.0%)      0 (4)                6.0

*         ( ) Number in parenthesis is No. of animals examined.
**        Mitotic Index

For each dose group, the total number of cells with aberrations were  

combined and the mean % aberrations was estimated. These data formed the  

basis for the report's conclusion, that at a dose level of 2 and 6 ml/kg  

diesel fuel was clastogenic.

Group             Total No. cells       Mean %
                with aberrations        aberrations
Control                 9                   1.0
TEM                     68                  47.2
Diesel 0.6 ml/kg        11                   1.7
Diesel 2.0 ml/kg        16                   2.7
Diesel 6.0 ml/kg        21                   3.4

Conclusions:
Interpretation of results (migrated information): positive weak positive response at 2.0 and 6.0 ml/kg
Diesel fuel was clastogenic in the rat following IP injection at levels of 2.0 and 6.0 mL / Kg bw. The % increase in abberration rate following a single exposure was small, ranging from 0.9 to 5.8%, and the predominant aberration seen was chromosomal fragements. Following repeated exposure small % increases were seen and the findings were not dose related. This suggests that the clastogenic activity of diesel fuel is at most weak.
Executive summary:

The genotoxic activity of diesel fuel was investigated in the rat following IP injection at 0.6, 2,0 or 6.0 ml/kg bw. Chromosomal aberrations in bone marrow cells were assessed following acute (single) or repeated exposure.

A small increase in the % of cells with aberrations (0.9 -5.8%), predominantly showing chromosomal fragements, was seen following acute exposure to diesel fuel at 2.0 and 6.0 mL/kg. The % increase at 0.6 mL/kg was within the range seen in control animals. Chromosomal fragements are not heritable changes and few 'rearrangement' aberrations were seen. Smaller % increases in cells with aberrations (0.4 -5.0%) were seen in the repeat dose study and the findings were not dose related.

These results suggest that diesel fuel may be at most weakly clastogenic in vivo but that changes observed are unlikley to be heritable.

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

Additional information

Additional information from genetic toxicity in vivo:

Based on read-across to data on structurally related materials, the substance is not considered to be genotoxic. There was no evidence of activity in two in-vitro assays and two out of three in-vivo studies. In one chromosomal aberration study, weak activity was seen following intraperitoneal treatment at high doses. The magnitude of effects was small and was restricted to small increases in chromosomal fragments.


Justification for selection of genetic toxicity endpoint
key in-vivo study, selected from 2 in-vitro and 3 in-vivo assays.

Justification for classification or non-classification

Some oil products containing relatively high concentrations of polycyclic aromatic compounds (PAC) are considered genotoxic carcinogens, and, consequently, are classified and labelled as carcinogenic, Cat. 1A or 1B (H350) or Cat. 2 (H351) according to the EU CLP Regulation (EC) 1272/2008. This classification as carcinogenic does not automatically imply that these substances need also to be classified as mutagenic as defined by the CLP Regulation. The EU legislation aims primarily to classify substances as mutagenic if there is evidence of producing heritable genetic damage, i.e. evidence of producing mutations that are transmitted to the progeny or evidence of producing somatic mutations in combination with evidence of the substance or relevant metabolite reaching the germ line cells in the reproductive organs. The PAC in oil products are poorly bioavailable due to their physico-chemical properties (low water solubility and high molecular weight), making it unlikely that the genotoxic constituents can reach and cause damage to germ cells (Roy, 2007; Potter, 1999). Considering their poor bioavailability, oil products which have been classified as carcinogenic do not need to be classified as mutagenic unless there is clear evidence that germ cells are affected by exposure, consistent with the CLP Regulation. For example, based on in vivo micronucleus tests on home heating oil as well as for read-across substances that were all negative for genotoxicity, vacuum distillate fuels are not classified as mutagens according to the EU CLP Regulation (EC) 1272/2008.