Benzene

Carcinogenicity

Description of key information

Long term experimental carcinogenicity bioassays have shown that benzene is a carcinogen producing a variety of tumours in animals (including lymphomas and leukaemia).

Human epidemiological studies indicate that benzene levels of ≥10 ppm are associated with increased risk of acute myeloid leukaemia (AML). 

The mode of action (MOA) for AML development leading to mortality is anticipated to include multiple earlier key events, which can be observed in hematotoxicity and genetic toxicity in peripheral blood of exposed workers. Prevention of such initial events would lead to prevention of the key, adverse outcomes, the morbidity and mortality caused by the myelodysplastic syndrome (MDS) and AML. The leukemic risk modelling analysis by North et al., 2020b (under review) supports the conclusion that cancer risk is not expected at an OEL of 0.25 ppm, which was proposed by Schnatter et al 2020.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, near guideline study, published as NIH report, minor limitations in design but fully adequate for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: mean weights per group males 151-155 g ; females 112-115 g
- Housing: 5 per sex per cage in polycarbonate cages
- Diet: NIH 07 Rat and Mouse Ration (Ziegler Bros, Gardners, PA) ad libitum
- Water: ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23±3°C
- Humidity: 40-60%
- Air changes: 15 per h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 10 December 1979 To: 11 December 1981

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: A weighed amount of benzene was mixed with the appropriate amount of corn oil and mixed by inversion (21 times). Rats were dosed at a rate of 5 mL/kg bw. Benzene in corn oil was found to be stable at 25ºC for at least 7 days. Dose mixtures were used within 2 weeks of preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Benzene/corn oil mixtures were analyzed by gas chromatography. All analyzed samples were within ±10% of the target concentrations.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

once per day, 5 days/week

Post exposure period:

Up to 15 days

Remarks:

Doses / Concentrations:
Males: 0, 50, 100, 200 mg/kg bw; Females: 0, 25, 50, 100 mg/kg bw
Basis:
other: nominal in corn oil

No. of animals per sex per dose:

50/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 17 week study: No compound-related deaths occurred; Final mean body weights (relative to those of the vehicle controls) were depressed 14%-22% for male and female rats that received 200, 400, or 600 mg/kg benzene. A dose-related leukopenia was observed for both male and female rats. Lymphoid depletion in the B-cell of the spleen was observed in 3/5 male and 4/5 female rats that received 200 mg/kg benzene and 5/5 male and 5/5 female rats that received 600 mg/kg benzene for 60 days and in 10/10 male and 10/10 female rats that received 600 mg/kg for 120 days. Increased extramedullary haematopoeisis was observed in the spleen of 4/5 male and 315 female rats that received 600 mg/kg for 120 days. Based on these composite observations, the doses were selected for rats for the 2-year study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for initial 13 weeks, monthly thereafter

HAEMATOLOGY: Yes
- Time schedule and number of animals for collection of blood: Main study - 10/sex/group bled orbitally at 12, 15, 18, 21 and 24 (termination) months. 40/sex/group bled by cardiac puncture at 24 months.
- Anaesthetic used for blood collection: No anaesthesia for interim samples, pentobarbital used prior to cardiac puncture terminal samples.
- Animals fasted: No data
- Parameters examined: packed cell volume, red blood cell count, total and differential white blood cell count, haemoglobin, and mean corpuscular volume, reticulocyte count and prothrombin time.

CLINICAL CHEMISTRY: No

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Examinations for grossly visible lesions were performed on major tissues or organs.

HISTOPATHOLOGY: Yes. The following tissues were examined histologically: gross lesions and tissue masses, mandibular or mesenteric lymph
node, salivary glands, sternebrae, femur, or vertebrae including marrow, thyroid gland, thymus, parathyroids, liver, small intestine, colon, brain, prostate/testes or ovaries/uterus, skin, heart, oesophagus, stomach, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder, pituitary gland, spinal cord, eyes, mammary gland, lung/mainstem bronchi.

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier. Statistical analyses for a possible dose-related effect on survival used the method of Cox for testing two groups for equality and Tarone's life table test for a dose-related trend.
The incidence of neoplastic or non-neoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined.
Three statistical methods were used to analyze tumour incidence data - Life table analysis, incidental tumour analyses and unadjusted analyses. The two that adjust for intercurrent mortality employ the classical method for combining contingency tables developed by Mantel and Haenszel. Tests of significance included pairwise comparisons of high dose and low dose groups with vehicle controls and tests for overall dose-response trends. Haematology data was initially screened for outliers the same animals were examined across time, a repeated measures analysis of variance was considered to be an appropriate method to investigate temporal and dose-related variation.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: Survival decreased with increasing dose for both male and female rats. Survival of the high dose male rats and of the mid and high dose female rats was significantly lower than that of the vehicle controls. Most rats that died early had neoplasia (Table 1).

BODY WEIGHT AND WEIGHT GAIN: Dose related weight gain reductions occurred in mid and high dose males, and in high dose females. Differences in bodyweight at 103 weeks were 12% and 23% in 100 and 200 mg/kg males, respectively and 10% in 100 mg/kg females. The low dose group was comparable to vehicle controls throughout the study.

HISTOPATHOLOGY: For male rats, benzene caused increased incidences of Zymbal gland carcinomas, squamous cell papillomas and squamous cell carcinomas of the oral cavity, and squamous cell papillomas and squamous cell carcinomas of the skin. For female rats, benzene caused increased incidences of Zymbal gland carcinomas and squamous cell papillomas and squamous cell carcinomas of the oral cavity (Tables 2 and 3).

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: increased incidences of Zymbal gland carcinomas, squamous cell papillomas and squamous cell carcinomas of the oral cavity, and squamous cell papillomas and squamous cell carcinomas of the skin ≥50 mg/kg

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

LOAEL

Effect level:

25 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: increased incidences of Zymbal gland carcinomas and squamous cell papillomas and squamous cell carcinomas of the oral cavity ≥25 mg/kg

Remarks on result:

other: Effect type: carcinogenicity

The high-dosed male and mid- and high-dosed female groups had significantly decreased survival rates when compared to the control groups.  The female control group, however, apparently had exceptionally good survival when compared with historic control data for the same strain and sex of animal.

 

Table 1: Survival

Week	Vehicle Control	Low Dose	Mid Dose	High Dose
Male
92	35/50 (70%)	38/50 (76%)	31/50 (62%)	30/50 (60%)
104	32/50 (64%)	29/50 (58%)	25/50 (50%)	16/50* (32%)
Female
92	49/50 (98%)	42/50 (84%)	40/50 (80%)	38/50 (76%)
104	46/50 (92%)	38/50 (76%)	34/50* (68%)	25/50* (50%)
* Decreased (P0.05) survival compared with vehicle controls

 

Zymbal gland carcinomas occurred in both the male and female treated rats with a significant positive trend, the incidences in the mid- and high-dosed males and all the dosed females being significantly greater than in the vehicle control group (incidences = 6, 13, 24 and 40% in males and 0, 13, 11 and 30% in females for the control and three dose groups respectively). A significant positive trend was seen for the number of benzene-treated rats with squamous cell papillomas or carcinomas of the palate, lip and tongue when considered either separately (in the case of the males) or combined (in both the males and females) (combined incidence = 2, 18, 32 and 46% in males and 2, 10, 24 and 20% in females for the four groups respectively). The individual or combined incidences of skin squamous cell papillomas and carcinomas were increased in the male rats with a significant positive trend, the incidences in the high-dosed animals being significantly greater than in the controls (incidence = 0, 4, 2 and 10% for squamous cell papillomas and 0, 10, 6 and 16% for squamous cell carcinomas in the four groups respectively). A significant positive trend was found for endometrial stromal polyps of the uterus, the high-dose incidence being significantly greater than in the controls (incidence = 14, 14, 14 and 28% for the four groups respectively).

 

Table 2: Incidence of selected neoplastic lesions

		Male				Female
Tissue and lesion		control	Low dose	Mid dose	High dose	control	Low dose	Mid dose	High dose
Zymbal gland	Carcinoma	2/32	6/46	10/42	17/42	0/45	5/40	5/44	14/46
Oral cavity	Squamous cell papilloma or carcinoma	1/50	9/50	16/50	19/50	1/50	5/50	12/50	9/50
Skin	Squamous cell papilloma or carcinoma	0/50	7/50	4/50	11/50
Liver	Adenoma Adenoma or carcinoma	2/50 2/50	2/48 2/48	4/49 5/49	1/49 1/49	0/50	3/49	1/50	0/50

 

Table 3: Primary neoplasms (percentage of neoplasm-bearing animals)

	Control	Low Dose	Mid Dose	High Dose
Male
Oral cavity	2a	18b	32b	38b
Zymbal gland	6a	15	24b	43b
Skin	2a	14c	10c	24b
Female
Oral cavity	2a	10c	24b	18b
Zymbal gland	0a	13b	14b	33b
Uterus	14a	14	14	28b
a = dose-related trend (P0.05) b = increased (P0.05) relative to vehicle controls c = marginal increase (P0.10) relative to vehicle controls

Conclusions:

Benzene is carcinogenic in rats following oral exposure. Increased tumour incidences were seen in the Zymbal gland, oral cavity and skin.

Executive summary:

The carcinogenic potential of benzene was investigated in rats using oral exposures of 0, 50, 100 or 200 mg/kg in males and 0, 25, 50 or 100 mg/kg in females 5 days/week for 103 weeks. Increased incidences of neoplasms were observed at multiple sites for male and female rats. There was clear evidence of carcinogenicity of benzene for male and female F344/N rats. For male rats, benzene caused increased incidences of Zymbal gland carcinomas, squamous cell papillomas and squamous cell carcinomas of the oral cavity, and squamous cell papillomas and squamous cell carcinomas of the skin. For female rats, benzene caused increased incidences of Zymbal gland carcinomas and squamous cell papillomas and squamous cell carcinomas of the oral cavity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

Adequate information is available to characterise the oral carcinogenicity of benzene in animals.

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline required

Species:

other: human exposure studies

Route of administration:

inhalation

Key result

Dose descriptor:

NOAEC

Effect level:

0.59 ppm

Based on:

test mat.

Basis for effect level:

haematology

Remarks on result:

other: arithmetic mean

Key result

Dose descriptor:

NOAEC

Effect level:

0.69 ppm

Based on:

test mat.

Basis for effect level:

other: genotoxicity - chromosomal aberration and micronucleus

Remarks on result:

other: aggregate NOAEC

Key result

Dose descriptor:

other: OEL

Effect level:

0.25 ppm

Based on:

test mat.

Basis for effect level:

haematology

other: genotoxicity

Remarks on result:

other: aggregate NOAEC adjusted for assessment factor 2

Results

 

Quality scoring results for genotoxic and haematological studies

Among the group of 31 haematology and 56 genotoxicity study populations each had a top score of 20 (of a possible 24), which in both cases was due to the (Qu et al., 2003) study. Both haematotoxicity and genotoxicity studies showed wide ranges (8–20 and 6–20, respectively) indicating marked differences in study quality for each body of literature.

Ties in scores for the haematotoxicity studies resulted in initial stratification of 11 studies in the top tertile (score range 14.5–20), 9 studies in the second tertile (score range 11–14), and 16 studies at or above the median score of 12.5. Similarly, for genotoxicity studies, 21 studies are in the first tertile (score range 13.5–20), 17 studies form the second tertile (score range 11–13), and 29 studies are at or above the median score of 12.5.

 

LOAECs and NOAECs for high quality studies

Haematology

Derivation of LOAECs

The highest quality studies (i.e. first tertile) that generated a more certain LOAEC were: Qu et al., 2003 (2.26 ppm, neutrophils), Schnatter et al., 2010, (7.8 ppm, neutrophils), Ward et al., 1996,(7.2 ppm, total leukocytes), Lan et al., 2004, (2.2 ppm, various cell types), Rothman et al., 1996, (7.6 ppm, lymphocytes), and Zhang et al., 2016 (2.1 ppm, leukocytes). From these values, a bimodal distribution results, in which there are two clusters of studies: three studies that suggest a LOAEC near 2 ppm and three studies that suggest a LOAEC near 7−8 ppm.

Sensitivity analyses supported a LOAEC around 2 ppm: Looking at the LOAECs in which all studies at or above the median quality score are considered as high quality, the LOAECs are similar, with only (Bogadi-Šare et al., 2003) at 8 ppm added to the above list from the first tertile. Thus, there are four studies suggesting a LOAEC of 7−8 ppm and three studies suggesting a LOAEC near 2 ppm. This alternative definition of high quality is a sensitivity analysis that supports the top tertile result. For the highest quality (top tertile) studies that generated a less certain LOAEC, values were: (Swaen et al., 2010) (0.75 ppm); and (Koh et al., 2015) (2.6 ppm). Inclusion of these studies is another sensitivity analysis that would lend more weight to a LOAEC in the range of 2 ppm, rather than the second cluster at 7−8 ppm. Various sensitivity analyses incorporating all less certain LOAECs above the median did not change this conclusion.

 

Derivation of NOAECs

For first tertile studies, the more certain NOAECs are 0.25 ppm (Swaen et al., 2010), 2.9 ppm (Schnatter et al., 2010), 2.2 ppm (Ward et al., 1996), 0.19 ppm (Collins et al., 1991), 0.21 ppm (Koh et al., 2015), and 1.7 ppm (Pesatori et al., 2009). Thus, there are three studies that suggest a NOAEC near 2−3 ppm, and three studies that suggest a NOAEC near 0.2−0.25 ppm. When studies that scored above the median and that show a more certain NOAEC are included, the NOAECs are 0.55 ppm (Collins et al., 1997), 0.81 ppm (Khuder et al., 1999), and 0.33 ppm (Tsai et al., 2004). Collectively, all studies above the median with more definitive NOAECs show four studies near 0.2−0.3 ppm, two studies near 0.6−0.8 ppm, and three studies near 2−3 ppm.

Examining studies above the median is justified and increases the number of studies although quality is somewhat lower (i.e. average quality is 16.25 versus 14.93 for NOAECs, and identical (16.67) for LOAECs).

Based upon frequencies, a LOAEC of 7−8 ppm, and a NOAEC of 2−3 ppm is one defensible conclusion from the analysis above. The NOAECs of 2−3 ppm are of a similar magnitude to three LOAECs from high quality studies, which introduces the problem of overlapping NOAECs with LOAECs. An alternative strategy would be to select the higher quality study(/ies) with more certain LOAECs that do not overlap NOAECs from high quality studies. Thus, there are three studies (Qu et al., 2003; Lan et al., 2004; Zhang et al., 2016) that show LOAECs near 2 ppm. If the LOAEC selected is near 2 ppm, a lower NOAEC should be selected. The two studies with the highest NOAEC yet still below 2 ppm are Collins et 1997 (0.55 ppm) and Khuder et al., 1999 (0.81 ppm). More studies show NOAECs of 0.2−0.3 ppm (Collins et al., 1991; Koh et al., 2015; Swaen et al., 2010; Tsai et al., 2004). Collins et al., 1991; Swaen et al., 2010; Tsai et al., 2004, all studied exposures < 0.5 ppm, so that a NOAEC was not achievable for those studies. Collectively, the results are not in conflict with a 0.5 ppm NOAEC, which is four times lower than the LOAEC (see Table 5). All sensitivity analyses (using top tertile studies, above median studies, and lower certainty LOAECs and NOAECs) in Table 5 result in LOAECs between 1.98 and 2.19 ppm, and NOAECs of 0.58 and 0.59 ppm. Thus, the result based on first tertile studies (a LOAEC of 2.19 ppm and a NOAEC of 0.59 ppm) is a conservative yet coherent interpretation of this information and is the preferred approach or base case.

 

Genotoxicity

Factory workers

Of the 21 studies in the top tertile, ten studies were among factory workers, five among fuel handlers and six among workers exposed to traffic and ambient air. In factory workers, the five studies with more certain LOAECs were (Qu et al., 2003) (LOAEC=3.07 ppm), (Xing et al., 2010)(LOAEC>1.6 ppm), (Zhang et al., 2012) (LOAEC>2.64 ppm), (Zhang et al., 2007) (LOAEC=13.6 ppm) and (Zhang et al., 2014) (LOAEC=2 ppm). The top tertile study generating a less certain LOAEC (>0.56 ppm) was (Kim et al., 2004a) due to the presence of PAH co-exposures.

 

Fuel workers

Three studies (Carere et al., 1995; Pandey et al., 2008 and Rekhadevi et al., 2010) in the top tertile were associated with a more certain LOAEC and none with a less certain LOAEC. The three studies showed similar LOAECs of 2 ppm, 2 ppm, and > 1 ppm, respectively. A NOAEC in the Carere study for micronuclei is 0.47 ppm and in the Pandey study∼0.9 ppm. The quality scores of the first tertile fuel studies (14.5) are lower than those from the factory

setting (17.25).

 

Traffic/ambient air

There were only two studies (Leopardi et al., NOAEC=0.003 ppm; Maffei et al., LOAEC=0.008 ppm) in the top tertile which produced a more certain LOAEC or NOAEC. Violante et al. (15.5) has a less certain NOAEC of 0.005 ppm and Angelini (14.5) has a less certain LOAEC of 0.006 ppm. Since the exposure concentrations present in the traffic/ambient air studies are lower than other NOAECs based on fuel and factory studies, this group of studies does not add meaningful information to the NOAEC analysis.

Since the single top tertile study that showed a more certain LOAEC is of lower quality (13.5) than studies from the factory and fuel sectors (average=16.07), this group of studies also does not add meaningful information to the LOAEC analysis. Thus, these studies are not subsequently considered.

 

Derivation of LOAECs

The highest quality studies (i.e. first tertile) that generated a more certain LOAEC originated from the factory and fuel study scenarios. There were five such studies from the factory scenario: Qu et al. (LOAEC=3.07 ppm), Xing et al. (LOAEC>1.6 ppm), Zhang et al. (2012) (LOAEC>2.64 ppm), Zhang et al., 2007(LOAEC=13.6 ppm), and Zhang 2014 (LOAEC=2 ppm).

Zhang et al., 2007 studied mainly higher exposures, and can therefore be excluded. The four remaining high-quality factory studies result in an average LOAEC of 2.33 ppm. This is the best supported LOAEC (leading case) since it is a weighted average of the highest quality studies, with an average quality score of 17.25. When the three additional studies from the fuel scenario: Carere et al. (2 ppm), Rekhadavi et al. (1 ppm), and Pandey et al. (2 ppm) are added, the resulting LOAEC is 2.04 ppm, which can be regarded as the sensitivity analysis based on the next highest quality studies.

If high quality is defined more inclusively as studies above the median, adding the one additional study from the factory setting with a more certain LOAEC (Eastmond et al., 1.29 ppm) with the other first tertile more certain factory studies, results in an average LOAEC of 2.12 ppm. The average quality score in this sensitivity analysis decreases to 16.3 (from 17.25), but still supports a LOAEC of approximately 2 ppm. There were no additional studies from the fuel nor ambient scenarios which generated more certain LOAECs above the median score of 12.5. All high certainty LOAECs above the median score from the factory and fuel sector combined, result in a LOAEC of 1.95 ppm (average score – 14.85). Although average quality score has decreased, this also supports an aggregate LOAEC of∼2ppm.

Consideration of the Less certain LOAECs included Kim et al., 2004a, >0.56 ppm, potential confounding by PAH exposure; average LOAEC for all factory studies in the first tertile was 1.97 ppm, quality score of 17.10); Factory studies with a less certain LOAEC (Bogadi-Sare et al., 2003 LOAEC=13 ppm, Holz et al., 1995, LOAEC=0.6–1 ppm). The LOAECs from Bogardi-Sare and Holz differ by more than two orders of magnitude, thus sensitivity analyses are not warranted.

The leading case LOAEC of 2.33 ppm is supported by the leading sensitivity analyses which account for more studies with a lower quality score and suggest slightly lower LOAECs near 2 ppm. Interpreted with due regard to quality, in aggregate the literature supports a LOAEC of 2 ppm.

Derivation of NOAECs. Three studies from the factory scenario that suggest NOAECs: Bogadi-Sare et al. 1997a (8 ppm), Zhang et al., 2011 (4.95 ppm) and Basso et al., 2011

(0.029 ppm). These studies differ by more than two orders of magnitude and as such, do not offer a good “base case” on which to justify a NOAEC. We face the problem of a NOAEC that is higher than the LOAEC. Despite the difficulty in isolating an effect of benzene in impure fuel and (especially) ambient studies, they are the best avenue at present for estimating a NOAEC for genotoxicity. In the fuel scenario, two studies scored in the first tertile and were characterized by more certain NOAECs: Carere et al. (1995) (0.47 ppm) and Pandey et al. (2008) (0.9 ppm). Combining these gives an average NOAEC of 0.69 ppm for genotoxicity. There are three other studies: Fracasso et al. (2010) (0.012 ppm), Pitarque et al. (1996) (0.3 ppm) and Göethel et al. (2014) (0.6 ppm) from the fuel sector that score above the median with more certain NOAECs. Using this set of studies as a sensitivity analysis a NOAEC of 0.45 ppm results. These analyses suggest that a NOAEC of 0.5 ppm is justified.

 

OEL derivation

Based on haematology studies

Method 1: (Use of the LOAEC)

POINT OF DEPARTURE FOR HAEMATOLOGICAL EFFECTS:

2.19 ppm (Based on three studies with a more certain LOAEC that are high quality (top tertile quality score). This is a conservative interpretation that does not consider that four other high-quality studies showed LOAECs of 7−8 ppm. 

POTENTIAL ASSESSMENT FACTORS:

• Dose-response (LOAEC to NOAEC). 2.19 ppm is the lowest level of exposure among three high quality studies with more certain LOAECs. Most other high-quality studies show a higher LOAEC. In addition, there are other high-quality studies (viz. Schnatter et al., 2010; Ward et al., 1996; Pesatori et al., 2009) (Pesatori et al., 2009; Schnatter et al., 2010; Ward et al., 1996) which report NOAECs for exposure levels similar to 2 ppm. Given this degree of potential overlap in LOAECs and NOAECs and the conservative selection of 2.19 ppm, the factor should be lower than the usual value of 3. A value of 2 is recommended.

• Intraspecies. A factor lower than 3 is recommended when a reasonably large human study is used in which a range of sensitivities are already present and extrapolations from the study data are to other occupational populations. In aggregate, the LOAEC studies considered included >2700 benzene exposed individuals. In addition, it can be seen that the lowest LOAECs ((Qu et al., 2003, (Zhang et al., 2016)) are those based on Chinese workers, who may be a more sensitive population. Thus, a value of 2 is recommended, although a value of 1 would not be unreasonable since the aggregate value is from studies showing the lowest LOAECs and the studies cover diverse populations, already including potentially sensitive sub-populations.

OEL=2.19 ppm / 4 (=2×2)=0.55 ppm METHOD 1

 

Method 2: (Use of NOAECs)

Method 2 is derived from the NOAECs of four studies of high quality.

NOAECs that are near or above the LOAEC from above are not considered, thus the Schnatter et al., 2010 (Schnatter et al., 2010) study (NOAEC 2.9 ppm) Ward et al( 1996), ( NOAEC 2.2 ppm) are excluded. A NOAEC is usually preferred to a LOAEC, provided that the lack of effect can be observed in a clear and precise way.

POINT OF DEPARTURE FOR HAEMATOLOGICAL EFFECTS:

NOAECs from four high quality studies (i.e. top tertile) are used as the basis for a weighted NOAEC of 0.58 ppm. These studies are: Collins et al., 1991; Koh et al., 2015; Pesatori et al., 2009; and Swaen et al., 2010. (Collins et al., 1997 and 1991; Khuder et al., 1999; Koh et al., 2015; Pesatori et al., 2009; Swaen et al., 2010; Tsai et al., 2004), in aggregate >11,700 benzene exposed individuals. Three studies (Bogadi-Šare et al., 2003; Schnatter et al., 2010; Ward et al., 1996) that report NOAECs of 2−3 ppm are not included, because they overlap with the chosen LOAEC value. The arithmetic average of these NOAECs is 0.59 ppm

POTENTIAL ASSESSMENT FACTORS:

• Dose response. A factor of 1 is suggested because the point of departure is derived from a NOAEC. • Intra-species factor. A factor of 1 is suggested because a larger aggregate human population is used (>11,700 benzene exposed individuals) than in METHOD 1 (>2700 benzene exposed individuals). Given that Method 1 (based on LOAECs) provides an OEL of 0.55 ppm (8 h TWA) and that Method 2 (based on NOAECs) provides an OEL of 0.59 ppm (8 hTWA) both methods would support an OEL of 0.5 ppm (8 h TWA).

 

The data supporting this position however are derived from worker studies examining effects in peripheral blood, while the target organ for benzene toxicity is bone marrow. An additional factor of two is proposed for possible subclinical effects in the bone marrow. We adopted ECHA RAC’s view that the associated uncertainty is relatively small and that an assessment factor of 2 would be appropriate (ECHA, 2018a, b). Although there is limited scientific experimental information available on this topic, and only in the rodent, French et al. (2015) and Ferris et al (1996) show that the bone marrow may (French et al 2015) or may not (Ferris et al 1996) be more sensitive than peripheral blood. The mouse micronucleus test results however cannot be simply translated to humans because of large differences in splenic function, such as removal of MN erythrocytes (Schlegel and MacGregor, 1983).

For workers, measured events in T-cells afford global assessments of in vivo mutagenicity but are not specific for bone marrow effects, while MN detected in reticulocytes are produced in reticulocyte precursors in the bone marrow (Albertini and Kaden, 2020). In humans only a small difference between MN in peripheral blood lymphocytes and reticulocytes was observed for radioiodine therapy (Stopper et al., 2005).

This available information overall would imply a small assessment factor. Therefore, an additional factor of two is proposed for possible subclinical effects in the bone marrow until additional research clarifies the sensitivity of peripheral blood versus bone marrow effects. This additional factor would support an OEL of 0.25 ppm (8 h TWA).

 

Based on genotoxicity studies

Method 1: (Use of the LOAEC)

POINT OF DEPARTURE FOR GENOTOXIC EFFECTS: >2.33 ppm.

This preferred approach is based on four studies (Table 6) in the factory setting with a more certain LOAEC that are high quality (top tertile). A fifth study (Zhang et al., 2007) which showed a higher LOAEC of 13.6 ppm was not considered. This preferred derivation is supported by additional sensitivity analyses summarized previously which consider the fuel sector as well as the factory sector, and the alternative definition of “high quality” using studies above the median rather than the top tertile.

 

POTENTIAL ASSESSMENT FACTORS:

• Dose-response (LOAEC to NOAEC).>2.33 ppm is the lowest level of exposure among four high quality (top tertile). Subsequently, a NOAEC of 0.69 ppm was calculated (see below). Other NOAECs which were near or greater than the LOAEC were not considered. In addition, the preferred LOAEC is noted as greater than 2.33, thus 2.33 should be regarded as the minimum preferred value. Given the degree of potential overlap in LOAECs and NOAECs, and the fact that there is some uncertainty in the inequality >2.33 ppm, the factor should be lower than the usual value of 3. A value of 2 is recommended. 

• Intraspecies. A factor lower than 3 is recommended when a reasonably large human study is used in which a range of sensitivities are already present and extrapolations from the study data are to other occupational populations. In aggregate, the LOAEC studies considered included >2700 benzene exposed individuals. In addition, all the LOAECs are based on Chinese workers, who may be a more sensitive population. Thus, a value of 2 is recommended. A value of 1 could also be considered since a possibly more sensitive population generates the LOAEC, thus, sensitive sub-populations may have already been accounted for in the selection of this LOAEC.

OEL=2.33 ppm / 4 (=2×2)=0.58 ppm METHOD 1

 

Method 2: (Use of NOAECs)

Method 2 is derived from the NOAECs of two studies of high quality in the fuel sector since studies in the factory sector showed higher NOAECs when compared to the preferred LOAEC. NOAECs that are

near or above the LOAEC from above are not considered, thus this could be considered a conservative approach.

POINT OF DEPARTURE FOR GENOTOXIC EFFECTS:

NOAECs from two high quality studies are used as the basis for a weighted NOAEC of 0.69 ppm. Studies of Zhang et al., 2011 (NOAEC=4.95) and Bogadi-Šare et al., 2003 (NOAEC=8) were not considered, thus the value of 0.69 may be conservative. On the other hand, only two studies are used to calculate the aggregate NOAEC, which could balance the conservative nature of the selection of studies that were included. Concordance with method 1, arguably based on stronger data (average quality score of LOAEC studies=17.25, average quality score of NOAEC studies=14.5) would also justify an intra-species factor of 1.

OEL=0.69 ppm. METHOD 2.

 

Given that the haematology data suggest an OEL of 0.5 ppm, the genotoxicity based OELs of 0.58 ppm (Method 1), and 0.69 ppm (Method 2) it can be agreed that both datasets would support an OEL of 0.5 ppm (8 h TWA). 

As was the case for haematotoxicity, the data supporting this position are mainly derived from worker studies examining effects in peripheral blood (except for (Xing et al., 2010). An additional factor of two is proposed for possible subclinical effects in the bone marrow until additional research clarifies the sensitivity of peripheral blood versus bone marrow effects. This additional factor would support an OEL of 0.25 ppm (8 h TWA) for both haematotoxicity and genotoxicity endpoints.

Conclusions:

The data presented by Schnatter et al 2020 define a benzene LOAEC of 2 ppm (8 h TWA) and a NOAEC of 0.5 ppm (8 h TWA). However, the use of peripheral blood measures of bone marrow effects introduces some scientific uncertainty, thus until the issue of bone marrow sensitivity compared to that of peripheral blood is resolved an extra assessment factor of two is applied. An OEL of 0.25 ppm (8 h TWA) for benzene is the best estimate based on available genotoxicity and haematotoxicity data. The carcinogenicity information decribed by North et al., 2020b supports the conclusion by Schnatter et al 2020 that there is no cancer risk at the OEL of 0.25 ppm.

Executive summary:

Schnatter et al 2020 derived an occupational exposure limit for benzene using quality assessed data. The epidemiological carcinogenicity data was assessed in the initial scoping phase of this work, however, the MOA described by North et al., 2020a indicated genetic toxicity and/or haematotoxicity were key events preceding carcinogenic outcomes, therefore the carcinogenicity endpoint was not used to inform on a point of departure in the OEL derivation.

Seventy-seven genotoxicity and thirty six haematotoxicity studies in workers were scored for study quality with an adapted tool based on that of Vlaanderen et al., 2008 (Environ Health. Perspect. 116 1700−5). Lowest and No- Adverse Effect Concentrations (LOAECs and NOAECs) were derived from the highest quality studies (i.e. those ranked in the top tertile or top half) and further assessed as being “more certain” or “less certain”. Several sensitivity analyses were conducted to assess whether alternative “high quality” constructs affected conclusions. The lowest haematotoxicity LOAECs showed effects near 2 ppm (8 h TWA), and no effects at 0.59 ppm. For genotoxicity, studies also showed effects near 2 ppm and showed no effects at about 0.69 ppm. Several sensitivity analyses supported these observations. These data define a benzene LOAEC of 2 ppm (8 h TWA) and a NOAEC of 0.5 ppm (8 h TWA). Allowing for possible subclinical effects in bone marrow not apparent in studies of peripheral blood endpoints, an OEL of 0.25 ppm (8 h TWA) is proposed.

In a paper on risk models for benzene leukaemia by North et al., 2020b, a number of publications related to characterisation of benzene leukaemogenic effects in humans have been outlined. This carcinogenicity information supports the conclusion by Schnatter et al 2020 that there is no cancer risk at the OEL of 0.25 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Species:

other: Human data

Quality of whole database:

Genotoxicity and haematotoxicity data were used to to select an inhalation NOAEC for benzene. These endpoints were selected as they are the most sensitive and relevant to the proposed mode of action (MOA) and protecting against these will protect against benzene carcinogenicity (Schnatter et al.).

System:

other: haematopoietic system and genetic toxicity

Organ:

bone marrow

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

It is concluded that benzene is carcinogenic in animals and humans and therefore is classified as follows: Carcinogenic Cat 1A, H350 under Regulation (EC) No 1272/2008 of the European Parliament.

Additional information

Non-human data

Oral

Oral cancer studies showed increased tumour rates in multiple organs, some of which were also tumour sites in the inhalation studies. The majority of tumour types at sites other than the haematopoietic system are of epithelial origin. In mice benzene produced increased tumour incidences in Zymbal gland, (Cronkite et al,1985; Farris et al, 1993; NTP, 1986; Maltoni et al, 1989), lung (Farris et al, 1993; NTP, 1986; Maltoni et al, 1989), Harderian gland (NTP, 1986), preputial gland (Farris et al,1993; NTP, 1986), forestomach (Farris et al, 1993; NTP, 1996), mammary gland (NTP, 1986; Maltoni et al, 1989), liver (Maltoni et al, 1989) and ovaries (Cronkite et al, 1985; NTP, 1986). In rats, benzene treatment was associated to increased tumour incidences in the Zymbal gland (NTP, 1986; Maltoni et al, 1989), oral cavity (NTP, 1986; Maltoni et al, 1989), forestomach (Maltoni et al, 1989), nasal cavity (Maltoni et al, 1989), and skin (NTP, 1986; Maltoni et al, 1989).

Dermal

No published data are available.

Inhalation

From several animal studies with inhalation and oral exposure there is evidence that benzene is carcinogenic. Target organs were similar in several studies irrespective of the application route and include the haematopoietic system and tissues of epithelial origin. The predominant tumours induced in the inhalation studies were located in the haematopoietic system, particularly lymphomas in mice (Farris et al, 1993; NTP 1986; Cronkite, 1985). In rats, increased frequencies of leukaemia in comparison to controls were found in benzene-exposed Sprague-Dawley rats and Wistar rats (Maltoni et al, 1989) and one case (out of 40 animals) of chronic myelogenous leukaemia was reported in Sprague-Dawley rats exposed to benzene (Goldstein et al, 1982).

Human data

There are substantial uncertainties regarding the shape of the dose response for benzene carcinogenesis below 10 ppm, but in recommending a limit below 1 ppm there can be relatively greater confidence in protection against cancer, based on NOAECs for haematotoxicity and genotoxicity around 0.6 ppm. The analysis by North et al., 2020b using leukemic risk models, concludes that there is no cancer risk to be expected at the proposed OEL of 0.25 ppm.

References

Baan R, Grosse Y, Straif K, Secretan B, El Ghissassi F, Bouvard V, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L and Cogliano V (2009). A review of human carcinogens—Part F: Chemical agents and related occupations. The Lancet Oncology 10, 1143-1144.

Bloeman LJ, Youk A, Bradley TD, Bodner KM & Marsh G (2004). Lymphohaematopoietic cancer risk among chemical workers exposed to benzene. Occup. Environ. Med. 61, 270-274.

Cronkite EP, Drew RT, Inoue T and Bullis JE (1985). Benzene hematotoxicity and leukemogenesis. Am J Ind Med 7, 447-456.

Crump KS (1994). Risk of benzene-induced leukemia: a sensitivity analysis of the Pliofilm cohort with additional follow-up and new exposure estimates. J Toxicol Environ Health 42, 219-242.

Crump KS and Allen BC (1984). Quantitative estimates of risk of leukemia from occupational exposure to benzene. Prepared for the Occupational Safety and Health Administration by Science Research Systems, Inc., Ruston, LA. Unpublished

EC (1993). Occupational exposure limits: Criteria document for benzene. Report EUR 14491 en, ISSN 1018-5593, Commission of the European Communities, pp126.

EC (1999). Council Directive 1999/38/EC of 29 April 1999 amending for the second time Directive 90/394/EEC on the protection of workers from the risks related to exposure to carcinogens at work and extending it to mutagens. Official Journal of the European Communities, L138, 66-69, 1 June 1999.

ECHA (2008). Guidance on information requirements and chemical safety assessment. Chapter R.8: Characterisation of dose [concentration]-response for human health, pp150.

Farris GM, Everitt JI, Irons RD, Popp JA (1993). Carcinogenicity of inhaled benzene in CBA mice. Fund Appl Toxicol 20, 503-507.

Glass DC, Gray CN, Jolley DJ, Gibbons C, Sim MR (2005). Health Watch exposure estimates: do they underestimate benzene exposure? Chem Biol Interact 153-154, 23-32.

Glass DC, Gray CN, Jolley DJ, Gibbons C, Sim MR (2006). The health watch case-control study of leukemia and benzene: the story so far. Ann N Y Acad Sci 1076, 80-9.

Glass DC, Gray CN, Jolley DJ, Gibbons C, Sim MR, Fritschi L, Adams GG, Bisby JA, and Manuell R (2003). Leukemia risk associated with low-level benzene exposure. Epidemiology 14, 569–577. Epidemiology. 2004;15, 509; author reply 510-1.

Goldstein BD, Snyder CA, Laskin S, Bromberg I, Albert RE and Nelson N. (1982). Myelogenous leukaemia in rodents inhaling benzene. Toxicology Letters 13, 169-173.

Gun RT, Pratt N, Ryan P, Roder D (2006). Update of mortality and cancer incidence in the Australian petroleum industry cohort. Occup Environ Med 63, 476-81.

Hayes RB, Yin SN, Dosemeci M, Li GL, Wacholder S, Travis LB, Li CY, Rothman N, Hoover RN and Linet MS (1997). Benzene and the dose-related incidence of haematologic neoplasms in China. Natl Cancer Inst 89, 1065-1071

IARC (International Agency for Research on Cancer) (1982). IARC monographs on the evaluation of carcinogenic risks of chemicals to humans: some industrial chemicals and dyestuffs. Vol. 29, IARC, pp. 93-148.

Irons RD, Gross SA, Le A, Wang XQ, Chen Y, Ryder J, Schnatter AR. Integrating WHO 2001-2008 criteria for the diagnosis of Myelodysplastic Syndrome (MDS): a case-case analysis of benzene exposure. Chem Biol Interact. 2010;184:30-8.

Maltoni C, Ciliberti A, Cotti G, Conti B and Belpoggi F (1989). Benzene, an experimental multipotential carcinogen: results of the long-term bioassays performed at the Bologna Institute of Oncology. Environmental Health Perspective 82, 109-124.

North, C.M., Schnatter, A.R., Rooseboom, M., Kocabas, N.A., Dalzell, A., Williams, S.D., 2020b. Key event-informed risk models for benzene-induced acute myeloid leukemia. Toxicol. Lett (under review).

NTP (1986). Toxicology and carcinogenesis studies of benzene (CAS No. 71-43-2) in F344/N rats and B6C3F1 mice (gavage studies). NIH publication number 86-2545. Testing laboratory: Battelle Columbus Laboratories. Report no.: TR 289. Study number: NTP TR 289.

Paustenbach DJ, Price PS, Ollison W, Blank C, Jernigan JD, Bass RD, Peterson HD (1992). Reevalution of benzene exposure for the Pliofilm (rubberworker) cohort (1936-1976). J Toxicol Environ Health 36, 177-231.

Paxton MB, Chinchilli VM, Brett SM and Rodricks JV (1994a): Leukaemia risk associated with benzene exposure in the Pliofilm cohort: I. Mortality update and exposure distribution. Risk analysis 14: 147-54

Paxton MB, Chinchilli VM, Brett SM and Rodricks JV (1994b): Leukaemia risk associated with benzene exposure in the Pliofilm cohort: II. Risk estimates. Risk analysis 14: 155-61

Richardson DB (2008). Temporal variation in the association between benzene and leukemia mortality. Environ Health Perspect. 116(3):370-4.

Rinsky RA, Young RJ, and Smith AB (1981). Leukaemia in benzene workers. American Journal of Industrial Medicine 2, 217-45.

Rinsky RA, Smith AB, Hornung RW, Filloon TG, Young RJ, Okun AH, and Landrigan PJ (1987). Benzene and leukemia. An epidemiological risk assessment. New England Journal of Medicine 316: 1044-50

Rinsky RA, Hornung RW, Silver SR and Tseng CY (2002). Benzene exposure and haematopoietic mortality: a long-term epidemiologic risk assessment. Am J Ind Med, Vol 42, pp 474-480.

Rushton L and Romaniuk H (1997). A case-control study to investigate the risk of leukaemia associated with exposure to benzene in petroleum marketing and distribution workers in the United Kingdom. Occup Environ Med 54, 152-166.

Schnatter AR, Armstrong TW, Nicolich MJ, et al. Lymphohaematopoietic malignancies and quantitative estimates of exposure to benzene in Canadian petroleum distribution workers. Occup Environ Med. 1996;53:773–781.

Schnatter, AR, Glass DC, Tang G, Irons RD and Rushton L (2012).Myelodysplastic syndrome and benzene exposure among petroleum workers: an international pooled analysis. J Natl Cancer Inst, 104, 1724-37

Schnatter AR, Rooseboom M, Kocabas NA, North CM, Dalzell A, Twisk J, Faulhammer F, Rushton E, Boogaard PJ, Ostapenkaite V, Williams SD. Derivation of an occupational exposure limit for benzene using epidemiological study quality assessment tools. Toxicol Lett. 2020 Jun 1:S0378-4274(20)30226-5. doi: 10.1016/j.toxlet.2020.05.036. Epub ahead of print. PMID: 32497562.

Schnatter AR, Rosamilia K, Wojcik NC (2005). Review of the literature on benzene exposure and leukemia subtypes. Chem Biol Interact.153-154, 9-21.

Swaen GM, Scheffers T, de Cock J, Slangen J and Drooge H (2005). Leukemia risk in caprolactam workers exposed to benzene. Ann Epidemiol 15, 21-8.

Swaen GM, van Amelsvoort L, Twisk JJ, Verstraeten E, Slootweg R, Collins JJ, Burns CJ (2010). Low level occupational benzene exposure and hematological parameters. Chem Biol Interact 2010 Jan 14. [Epub ahead of print]

Texas Commission on Environmental Quality (TCEQ) (2007). Development Support Document. Benzene. Chief Engineer’s Office. Available: http://tceq.com/assets/public/implementation/tox/dsd/final/benzene_71-43-2_final_10-15-07.pdf

Utterback DF and Rinsky RA (1995). Benzene exposure assessment in rubber hydrochloride workers: a critical evaluation of previous estimates. Am J Indust Med 27, 661-76.

Vlaanderen J, Portengen L, Rothman N, Lan Q, Kromhout H, Vermeulen R. (2010) Flexible meta-regression to assess the shape of the benzene-leukemia exposure-response curve.

Environ Health Perspect. 118:526-32.

WHO (2000). Air quality guidelines for Europe, 2nd ed., World Health Organization Regional Office for 2000 (WHO Regional Publications, European Series No. 91).

Wong O, Harris F, Yiying W, Hua F. A hospital-based case-control study of acute myeloid leukemia in Shanghai: analysis of personal characteristics, lifestyle and environmental risk factors by subtypes of the WHO classification. Regul Toxicol Pharmacol 2009;55:340-52.

Justification for selection of carcinogenicity via oral route endpoint:
Rodent oral cancer studies showed increased tumour rates in multiple organs, some of which were also tumour sites after inhalation. The majority of tumour types at sites other than the haematopoietic system are of epithelial origin.

Justification for selection of carcinogenicity via inhalation route endpoint:

Rodent studies collectively indicate that benzene exposure can result in dose-related increases in various tumour types.The predominant tumours induced in animal inhalation studies were located in the haematopoietic system, particularly lymphomas in mice. In rats, increased frequencies of leukaemia in comparison to controls were found in benzene-exposed Sprague-Dawley rats and Wistar rats, with one report of chronic myelogenous leukaemia.Epidemiologic studies have indicated excesses myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML), acute non-lymphocytic leukemia (ANLL) (the data on other subtypes is significantly less consistent).

Carcinogenicity: via oral route (target organ): other: all gross lesions and masses

Carcinogenicity: via inhalation route (target organ): cardiovascular / hematological: bone marrow