Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

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. Benzene has been shown to be mutagenic in vivo in both somatic cells and germ cells.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key 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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
- no positive control.
Principles of method if other than guideline:
Mice were exposed to benzene vapour (up to 200 ppm; 640 mg/m3) for up to 8 weeks and blood and bone marrow samples evaluated for the presence of micronuclei within 2 hours of end of exposure
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Nil, USA
- Age at study initiation: 12 weeks±2 days
- Assigned to test groups randomly: yes, by weight
- Housing: one per cage in 8 m3 stainless steel and glass inhalation chambers
- Diet: NIH-07 rodent chow (Ziegler Bros., Gardener, PA, USA) ad libitum
- Water: Filtered tap water ad libitum
- Acclimation period: 10-17 days
Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION: Benzene exposures conducted in 8 m3 stainless steel and glass Hinners-style whole body inhalation chambers. Air was conditioned to 72°F and 50% humidity and maintained at a flow of 2000 L/min through the chambers. Temperature, relative humidity, airflow and static pressure continuously monitored.
- System of generation: Atmospheres of 10, 100 and 200ppm benzene generated by vaporizing liquid benzene contained in a 10-gallon stainless steel pressure vessel. A controlled flow of nitrogen was bubbled through the liquid benzene and carried vapours out of the vessel into the chamber air inlet. The 1 ppm benzene atmosphere was also generated from liquid benzene, however, the pressure vessel was maintained at 10 p.s.i. gauge pressure with nitrogen. The flow of the benzene vapours and nitrogen into the chambers controlled by mass flow controllers placed on the outlet side of the pressure vessels.

TEST ATMOSPHERE
- Brief description of analytical method used: An infrared (IR) gas analyser used to monitor the 0, 10, 100 and 200 ppm benzene concentrations. The 1 ppm chamber was monitored by gas chromatography.
Duration of treatment / exposure:
1, 2, 4 and 8 weeks of exposure
Frequency of treatment:
6 h/day, 5 days/week
Post exposure period:
None. Blood and bone marrow samples collected within 2 h of end of final exposure.
Remarks:
Doses / Concentrations:
0, 1, 10, 100 or 200 ppm
Basis:
other: target concentrations
No. of animals per sex per dose:
7
Control animals:
yes, sham-exposed
Positive control(s):
none
Tissues and cell types examined:
Micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and blood and micronucleated normochromatic erythrocytes (MNCE) in the blood.
Statistics:
For each timepoint, the data from each benzene exposure group was compared to the controls using Dunnett's analysis of variance (ANOVA) with a 5% significance level. Linear and quadratic regression models were fit to the bone marrow NIPCE data. Evaluation of linear and quadratic curves with the data required a weighted analysis because of nonhomogeneous variability. A P value of <0.05 for lack of fit test meant that the curve was not appropriate for the data set.
Sex:
male
Genotoxicity:
positive
Remarks:
at exposure concentrations of 100 and 200 ppm
Toxicity:
yes
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
Atmosphere analysis: The analytical concentrations of benzene within the individual chambers were stable during each exposure period and over the time course of the studies. The actual concentrations of benzene averaged 0.95±0.09, 9.9±1.2, 98.5±1.5 and 198.5±4.8 ppm.

100 and 200 ppm benzene induced a statistically significant increased frequency of micronucleated erythrocytes in the bone marrow and blood. No such increase was observed at levels of 1 or 10ppm benzene. The increase was seen at weeks 1, 2, 4 and 8 of exposure, and the frequency of MPCE plateaued at week 2 with 43/1000 (100 ppm) and 86/1000 (200 ppm) in the bone marrow as compared with 10/1000 for controls. The frequency of MNCE in the blood progressively increased to 13.4/1000 (100 ppm) and 32.5/1000 (200 ppm) at week 8 as compared with 1.8/1000 for controls. Cytotoxicity was seen at 100 and 200 ppm with decreases in both PCE and total erythrocyte counts.

Conclusions:
Interpretation of results (migrated information): positive at exposure levels of 100 ppm and 200 ppm
Benzene produced an increased frequency of micronuclei following inhalation exposure in mice and is considered to be an in vivo clastogen.
Executive summary:

The frequencies of micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and blood and micronucleated normochromatic erythrocytes (MNCE) in the blood of male B6C3F1 mice were measured following inhalation of benzene at 0, 1, 10, 100 or 200 ppm for 1, 2, 4 or 8 weeks of exposure. 100 and 200 ppm benzene induced a statistically significant increased frequency of micronucleated polychromatic erythrocytes and/or micronucleated erythrocytes in the bone marrow and blood of exposed animals at all time points. The frequency of MPCE was not statistically raised in animals exposed to 1ppm or 10ppm benzene, indeed in a manual count the frequency of MPCE was lower than control levels. It is concluded that benzene is an in vivo clastogen in the mouse.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

in vitro data

Benzene has been extensively examined for the core endpoints of gene mutation in bacteria, gene mutation in mammalian cells and chromosomal damage in mammalian cells in a number of laboratories (EHC/IPCS, 1993). The results have been conflicting, with predominantly negative results being reported from earlier studies, especially with bacterial systems. However, a number of positive results have been reported with these core endpoints, including studies with enclosed systems together with auxiliary metabolic activation. Positive results have been reported for bacterial mutation (Glatt et al, 1989), mammalian cell gene mutation (Tsutsui et al, 1997) and mammalian cell chromosomal damage (Ishidate and Sofuni, 1985). A similar profile of mixed results has been reported for additional endpoints including DNA repair, DNA strand breaks and cell transformation (Ashby et al 1985, Tsutsui et al, 1997; EHC/ IPCS, 1993).  It is considered that containment of the material in contact with the target cells, together with an appropriate source of metabolism is required to allow the identification of a mutagenic response. Overall, it is concluded that benzene shows some evidence of mutagenic potential in vitro (EHC/IPCS 1993, EU RAR 2008).

in vivo data

The key studies in animal in vivo systems are considered to be cytogenetic studies in the bone marrow and germ cells Ciranni et al, 1991; Farris et al, 1996. A gene mutation study in somatic cells Mullin et al , 1995). has been reported  but this and other similar studies have been observed to have limitations.

Of the in vivo cytogenetic studies benzene was studied in a rodent bone marrow cytogenetic assay in which male Swiss SD1 mice were exposed to a single oral dose of 0, 88, 440 or 880 mg/kg and samples of bone marrow cells taken for cytogenetic analysis. Samples of differentiating spermatagonia were similarly taken after 0, 220, 440 or 880 mg/kg benzene exposure. Dose-related significant increases in chromosomal aberrations were found in both tissues. Benzene was mutagenic in this assay in both the bone marrow and germ cells (Ciranni et al, 1991).  Additionally in a bone marrow micronucleus assay, male B6C3F1 mice were exposed to benzene by the inhalation route at atmospheres of 0, 1, 10, 100, 200, 400 ppm over an 8 week exposure period. A dose-related increase in micronucleus incidence was reported (Farris et al, 1996).  A number of other studies have confirmed the clastogenicity of benzene in both rats and mice (EU RAR, 2008). Benzene was also active in a comet assay in mice measuring DNA strand breaks, but only showed marginal activity for sister chromatid exchange induction in rats and mice (EU RAR, 2008).

The predominance of clastogenic activity rather than gene mutation activity is suggestive of benzene not being a direct acting mutagen.  In cases where DNA adduct formation has been claimed this is likely to be associated with protein contamination of the examined DNA samples  32P post labelling studies have been suggested to show DNA adducts in mice however very high dose regimes much higher than the dose required to cause cancer in mice, , administration by the intraperitoneal route which is not relevant to human exposure and inconsistent dose responses point to this not being credible evidence of DNA adducts with relevance to human exposure. (Pathak et al 1995, Li et al 1996, Whysner et al 2004, DECOS 2013)

Whilst there are reports of findings of mutation in transgenic rodents exposed to benzene (Mullin et al 1995, Mullin et al 1998 and Provost et al 1996) these studies variously have design limitations ( eg sample size , species differences in organs affected by cancer compared to man, limitations in statistical analysis) marginal findings and inappropriate delays in sampling periods which have led to their outcome being discounted (Whysner et al 2004, DECOS 2013).

Overall the evidence points to the genetic toxicology of benzene being based on clastogenicity and benzene not being a direct acting mutagen.   The mechanisms of action for benzene genotoxicity have been attributed to events with a threshold of action (DECOS 2013)  

Human information

There are a number of reports showing that benzene exposure induces genotoxic effects in human lymphocytes in vivo. These are primarily cytogenetic investigations. Reliable conclusions, however cannot be drawn from many such studies due to poor exposure data and methodological deficiencies (EU RAR, 2008). However more recently significant efforts have been made to investigate cytogenetic aberrations in workers using more refined methods and with better definition of exposure. In particular work using Fluorescence In situ Hybridisation (FISH) methodology on chromosome 9 aberrations by Zhang et al 1996 (extended by Zhang et al 1998 and Smith et al 1998 to cover chromosomes 5,7 ,8 and 21) has shown that such aberrations are associated with contemporary 8 hour TWA exposures of >31 ppm.   There has been concern however that in studies which have taken substantial steps to define current exposure there may be some aberrations which persist and reflect previous historic exposures of greater magnitude, duration or intensity than current exposures.  In contradiction to this Smith et al 1998 found that the frequency of some aberrations to chromosomes 8 and 21 correlated better with current measured exposure rather than with cumulative exposure metrics (ppm years) which took account of historic exposure in earlier work.

It was noted by Smith et al 1998 that the significant translocation [t(8;21) ] associated with benzene exposure is also found with topisomerase inhibitor drugs. Coupled with the fact that some benzene metabolites (e.g. hydroquinone, etc.) are known poisons of topoisomerase II this provides a plausible mechanism for a threshold basis for benzene associated cytogenetic aberrations.

Rothman et al 1995 examined a group of Chinese workers (n=24) with a very high benzene exposure (median 8h TWA = 66ppm) and a mean duration of employment of 6.9 +/- 4.9 years (range 0.7-16,5 years). Median lifetime benzene exposure was 270ppm years. The study looked at Glycophorin A gene loss in individual heterogenous at the GPA locus and compared GPA gene mutation frequency with that in 23 matched non benzene exposed controls. The mutations were not attributed to point mutations, deletions or gene inactivating mutations but rather to gene duplicating mutations probably by mitotic recombination. 

Attempts by Qu et al 2003 to replicate the FISH methodology of Zhang et al 1996 in a large well organised study with measurement of current benzene exposure were not successful but Qu et al instead reported findings from traditional cytogenetic studies on these workers and matched non- exposed controls. Their findings whilst showing statistically significant exposure related trends for chromosome and chromatid aberrations and acentric fragments showed anomalous dose responses . The lowest dose group (>0-5ppm exposure) showed aberration frequencies higher than intermediate dose groups and verging on the levels achieved in the high dose group (>30ppm exposure) which led to the authors questioning if prior exposure rather than current measured  exposure might be linked to currently observed aberrations by virtue of long lasting stable aberrations. 

A review of some individuals who suffered significant haematotoxicity from high benzene exposure and showed cytogenetic aberrations at the time was carried out by Forni, 1996. It was noted that approximately 20 years later after recovery from benzene induced haematotoxicity there were still higher levels of chromosome aberrations in surviving cases (n=4) compared to controls (n=7), although this was not statistically significant.  When complex aberrations with more than one break were considered the difference between control and exposed was greater. Hyperdiploid cells were more frequent in surviving cases than contols (0.036> p 0.021). This would suggest that elevated aberrations can persist long after high levels of exposure ceased

Given these uncertainties it is not clear what the NOAEL is for cytogenetic aberrations in workers is but the work of Zhang et al 1996 and Smith et al 1998 would suggest that it is > 31ppm as an 8 hour TWA.

REFERENCES

Ashby et al (1985). Assays to measure the induction of unscheduled DNA synthesis in cultured hepatocytes, (Eds) Progress in Mutation Research, Vol 5, pp 371-373

Ciranni R et al (1991). Dose related clastogenic effects induced by benzene in bone marrow cells and in differentiating spermatogonia of Swiss CD1 mice, Mutagenesis6(5) 417-421

DECOS (2014). Benzene- Health-based recommended occupational exposure limit. Health Council of the Netherlands, Publication no.2014 /3 The Hague

IPCS (1993). Environmental Health Criteria No. 150. Benzene.

EU RAR (2008). Risk Assessment. Benzene.

Farris GM et al (1996). Benzene-induced micronuclei in erythrocytes: an inhalation concentration -response study in B6C3F1 mice, Mutagenesis. 11(5) 455-462

Forni A (1996). Benzene induced chromosome aberrations: a follow up study, Environ. Health Perspect.104 suppl. 6 1309-1312 

Glatt et al (1989). Multiple activation pathways of benzene leading to products with varying genotoxic characteristics, Environmental Health Perspectives. 82, 81-89

Li G et al (1996). Tissue distribution of DNA adducts and their persistence in blood of mice exposed to benzene, Environ. Health Perspect.104 suppl. 6 1337-8

Mullin AH et al (1995). Inhalation of benzene leads to an increase in the mutant frequencies of alacItransgene in lung and spleen tissues of mice, Mutat Res. 327 121-129

Mullin AH et al (1998). Inhaled benzene increases the frequency and length oflacIdeletion mutations in lung tissues of mice, Carcinogenesis. 19(10) 1723-1733

Pathak DN et al (1995). DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene, Carcinogenesis. 16(8) 1803-8

Provost GS et al (1996). Mutagenic response to benzene and tris(2.3-dibromopropyl)-phosphate in the lambda lacI transgenic mouse mutation assay: a standardized approach to in vivo mutation analysis, Environ Mol Mutagen. 28(4) 342-7

Qu et al (2003). Validation and evaluation of biomarkers in workers exposed to benzene in China,  Res Rep Health Eff Inst. (115) 1-72

Rothman N et al (1995). Benzene induces gene-duplicating but not gene-inactivating mutations at the glycophorin A locus in exposed humans, Proc. Natl. Acad. Sci. 92 4069-4073 

Smith MT et al (1998). Increased translocations and aneusomy in chromosomes 8 and 21 among workers exposed to benzene, Cancer Research. 58 2176-2181

Tsutsui T et al (1997). Benzene, catechol, hydroquinone and phenol induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduleded DNA synthesis in Syrian Hamster embryo cells, Mutat Res. 373 (1) 113-23

Whysner J et al (2004). Genetic toxicology of benzene and its metabolites, Mutat Res. 566(2) 99-130

Zhang L et al (1996). Interphase cytogenetics of workers exposed to benzene  Environ. Health Perspect. 104 suppl. 6 1325-1329

Zhang et al (1998). Increased Translocations and Aneusomy in Chromosomes 8 and 21 Among workers exposed to Benzene, Cancer Research. 58 2176-2181

Justification for classification or non-classification

Benzene is an in vivo mutagen in mammals and Humans, inducing chromosomal aberrations and micronuclei. Benzene is therefore classified as Mutagenic Cat 1B (H340) under Regulation (EC) No 1272/2008 of the European Parliament.