Distillates (petroleum), heavy steam-cracked

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Genotoxicity data on streams within this category are limited and variable with both positive and negative in vivo and in vitro study results. However, there are substantial data on the genotoxicity of a number of specific components present in some streams. Of these, benzene has been shown to be mutagenic and when present in streams at concentrations equal or greater than 0.1% labelling for mutagenicity will be required.

Link to relevant study records

Reference

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 based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not GLP, but key cytogenetic parameters measured comparable to guideline study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 3-4 animals/group

Principles of method if other than guideline:

Mice given single oral gavage dose of benzene (1 mL/kg bw) and chromosomal aberrations in bone marrow and spermatogonal cells assessed at time points up to 48 h post-treatment.

GLP compliance:

not specified

Type of assay:

other: bone marrow chromosome aberration assay and mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Valco Co., Italy
- Age at study initiation: 2 months
- Weight at study initiation: 30-35 g
- no further details

ENVIRONMENTAL CONDITIONS
- no data

IN-LIFE DATES:
- no data

Route of administration:

oral: gavage

Vehicle:

- Olive oil

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

Single oral dose

Post exposure period:

Up to 48 h

Remarks:

Doses / Concentrations:
1 mL/kg
Basis:
actual ingested

No. of animals per sex per dose:

3-4 male mice

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Bone marrow and spermatogonal cells

Statistics:

The binomial dispersion test was applied to test homogeneity of results from control animals. Statistical differences between treated and solvent control groups were determined by Fisher's exact test. To compare the sensitivity of the two cell types the doubling doses were calculated. The doubling dose (DD) is defined as the dose that induces as many aberrations as occur spontaneously per cell generation. Based on the linear dose-response, Y = a + b D, where Y is the yield of aberrant cells, a the spontaneous frequency and b the linear regression coefficient, the doubling dose is calculated as the ratio of a to b (DD = a/b).

Sex:

male

Genotoxicity:

positive

Remarks:

Chromosomal aberration test; mouse (bone marrow)

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not applicable

Sex:

male

Genotoxicity:

positive

Remarks:

Germ cell chromosome aberration test; mouse (spermatogonia)

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Bone Marrow:

Benzene showed high clastogenic activity in bone marrow cells at all sampling times with a peak at 24 and 30 hours (approx. 20% aberrant cells (excl. gaps) versus 1% in controls). The dose-response was determined 24 h after treatment with 0.1, 0.5 or 1.0 mL/kg benzene (equivalent to 88, 440 and 880 mg/kg bw). All three doses were clearly positive and a dose-dependency was established.

 

Chromatid aberrations in bone marrow cells of mice treated with 1 mL/kg (880 mg/kg) of benzene: time-response

Time (h)	No. cells scored a	No. of aberrations			Highly damaged cells (n) b	Aberrant cells (%±SE)
		gaps	breaks	exchanges		including gaps	excluding gaps
Control	1000	24	11	-	-	3.2 ± 0.6	1.1 ± 0.1
6	600	47	29	-	-	11.3 ± 0.1	4.3 ± 0.6**
12	600	89	98	7	-	24.6 ± 2.4	16.6 ± 0.4**
18	600	59	112	7	3	22.6 ± 4.5	15.3 ± 3.0**
24	600	53	206	6	13	25.1 ± 2.7	20.8 ± 3.3**
30	600	117	237	15	13	28.7 ± 7.4	19.8 ± 0.5**
36	600	10	24	-	-	4.8 ± 0.4	3.5 ± 0.5**
42	600	26	31	-	-	8.6 ± 1.4	5.3 ± 0.4**
48	600	41	44	-	-	12.0 ±1.7	6.3 ± 1.1**

**P <0.01 (Fisher's exact test).

a  200 cells scored per animal.

b  Cells with more than 10 aberrations.

 

Spermatogonia:

After administration of 1 mL/kg (880 mg/kg bw) the maximum response was obtained 24 h after treatment (6.3% aberrant cells versus 1.2% in negative controls). In the dose-response study, all doses tested (0.25, 0.5 and 1.0 mL/kg bw, equivalent to 220, 440 and 880 mg/kg bw) increased the aberration frequency in a dose-dependent manner; at 880 mg/kg again 6.3% of the spermatogonia were aberrant. Since bone marrow clastogenicity was investigated in parallel, it can be concluded that clastogenicity in bone marrow cells and spermatogonia was induced in the same dose range, although effects were less pronounced in spermatogonia.

 

Chromatid aberrations in differentiating spermatogonia of mice treated with 1 mL/kg (880 mg/kg) of benzene: time-response

Time (h)	No. cells scored a	No. of aberrations			Aberrant cells (%±SE)
		gaps	breaks	exchanges	including gaps	excluding gaps
Control	1000	49	12	-	5.5 ± 0.9	1.12 ± 0.2
6	600	32	6	-	6.3 ± 0.1	1.0 ± 0.3
12	600	63	18	1	12.3 ± 0.3	3.3 ± 0.4**
18	600	67	24	-	13.3 ± 1.2	4.0 ± 0.5**
24	600	54	36	3	14.8 ± 2.6	6.3 ± 1.6**
30	600	33	16	1	7.8 ± 1.6	2.6 ± 0.5*
36	600	31	23	1	8.3 ± 1.0	3.5 ± 0.5**
42	600	21	14	-	5.2 ± 1.3	2.0 ± 0.5
48	600	19	23	-	18.2 ±2.0	3.5 ± 0.7**

**P <0.01 (Fisher's exact test).

a  200 cells scored per animal.

 

Conclusions:

Interpretation of results (migrated information): positive Chromosomal aberration test (mouse bone marrow) and Germ cell chromosome aberration test (mouse spermatogonia)
Benzene was positive in the chromosomal aberration test (mouse bone marrow) and germ cell chromosome aberration test (mouse spermatogonia), following a single oral dose of 1 mL/kg to male mice.

Executive summary:

The ability of benzene to induce chromosome damage in vivo was assessed by determining the frequencies of chromosomal aberrations in bone marrow and spermatogonial cells of male Swiss CDI mice. Initially a single dose of 1 mL benzene/kg (880 mg/kg) was assessed using a wide range of times (6, 12, 18, 24, 30, 36, 42 and 48 hours) to determine the time of maximum response. Benzene showed high clastogenic activity with a peak between 24 and 30 hours in bone marrow cells or 24 hours in differentiating spermatogonia. The effect in bone marrow cells was greater than in spermatogonia. Secondly, the dose response 24 hours after treatment was determined. Additional doses of benzene used were: 0.1 mL/kg (88 mg/kg) and 0.5 mL/kg (440 mg/kg) for bone marrow cells; 0.25 mL/kg (220 mg/kg) and 0.5 mL/kg (440 mg/kg) for differentiating spermatogonia.

Benzene was positive in this test with dose dependent clastogenic effects in both cell types. All dose levels showing a statistically significant increase in the incidence of aberrant cells.

It is concluded that benzene is a clastogen in male germ cells and the bone marrow of mice.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vivo:

Genotoxicity information for specific streams identified for this category are variable with both positive and negative results in vitro and in vivo. Further information is included in the Category Summary for Fuel Oils Category (ACC, 2005).

Aromatic Pyrolysis Oil (CAS 64742 -90 -1): Positive mutagenic effects were reported in the in vitro CHO/HGPRT test for point mutations. A significant increase in mutation frequency was seen at concentrations of 500 µg/mL and above when tested with metabolic activation (Gulf Life Sciences, 1984b). In addition positive results were reported in further in vitro and in vivo studies and Aromatic Pyrolysis Oil was determined to be mutagenic and clastogenic (ACC, 2005).

Light Pyrolysis Fuel Oil (CAS 68527-18-4): Mixed results were seen in both in vitro and in vivo tests. In a cell transformation test using mouse embryo cells toxicity was seen after 2 days exposure beginning at 32 µg/mL and 100% toxic at >128 µg/mL. There was no increase in the frequency of transformation foci compared to control at any dose level (Gulf Life Sciences, 1984a). Light Pyrolysis Fuel Oil caused dose-related unscheduled DNA synthesis (43.3% at 8 μg/mL and 96% at 32 μg/mL compared with 2.7% in controls) at all nontoxic levels. (ACC, 2005). Following oral dosing at up to 1 mg/kg for 1-2 days mice did not show any significant change in micronucleus formation and there was no significant change in the ratio of polychromatic to normochromatic erythrocytes in bone marrow (Gulf Life Sciences, 1984c).

Rohnaphthalin-gemisch (CAS 85117-10-8): In an in vitro bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 in the presence and absence or Aroclor-induced rat liver S9 toxicity was observed with some conditions at > 200 µg/plate. There was not a significant increase in revertant colonies in Salmonella strains with or without rat liver metabolic activation at any dose level and Rohnaphthalin-gemisch (CAS 85117-10-8) was considered not to be a mutagen in this test system (BASF, 1985).

The specific component benzene which has been identified as present in some streams at up to 30% has been shown to be mutagenic in vivo:

Benzene (Classification: EU –Toxic T Mutagen Cat 2 R46; GHS/CLP - Category 1B, H340): Benzene has been extensively examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It has shown mixed results for mutagenicity in vitro although in mammalian cells there is overall evidence for potential mutagenic activity (EU, 2008a). Benzene has been shown to be mutagenic in vivo in both somatic cells and germ cells (Ciranni et al, 1991).

Justification for selection of genetic toxicity endpoint
Although only limited experimental data are available for members of this category, the presence of the component substance benzene indicates that they should be considered as potentially genotoxic.

Justification for classification or non-classification

Adequate data are available from in vitro and in vivo rodent studies to characterise the genotoxic potential of Fuel Oils streams. The results of a diverse array of mutagenicity, transformation and clastogenicity assays indicate positive responses in some assays and negative responses in others. However, Fuel Oils streams are also known to contain up to 30% benzene which is mutagenic in vivo and when present at ≥ 0.1% triggers labelling. Overall it is it is concluded that these streams are likely to be genotoxic.

It is proposed that Fuel Oils streams are classified as Mutagenic and “May cause genetic defects” Cat 1B, H340 under Reg (EC) 1272/2008.