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Description of key information

Repeated dose toxicity data are not available for the high benzene naphtha streams. However, there are substantial data on the repeated dose toxicity of a number of marker constituents present in some streams: benzene, toluene, DCPD, cyclohexane, xylene, and ethylbenzene. A number of the marker constituents demonstrate significant target organ toxicity, therefore they drive the mammalian toxicity effects in the streams (benzene and styrene - when present at 1%; toluene/  ethylbenzene - when present at 10%; n-hexane - when present at 5%).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, near guideline study, published as NIH publication, some limitations in design but fully adequate for evaluation
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no clinical chemistry analysis, no ophthalmological examination, no neurobehaviour, no organ weights.
GLP compliance:
yes
Remarks:
assumed - audit of 2 year data documented
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI)
- Age at study initiation: 6 weeks
- Weight at study initiation: mean weights per group males 110-137 g ; females 75-101 g
- Housing: 5 per sex per cage in polycarbonate cages
- Diet: Purina Lab Chow 5001 - pellets (Ralston-Purina Co., St. Louis, NJ) ad libitum
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±1°C
- Humidity: 40-65%
- Air changes: 15 per h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 14 October 1978 To: 13 February 1979
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: A weighed amount of benzene was mixed with the appropriate amount of corn oil and stirred for 5 minutes. Rats were dosed at a rate of 5 mL/kg. Benzene in corn oil was found to be stable at 25º C 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:
All benzene/corn oil mixtures analyzed by gas chromatography and were within ±10% of the target concentrations.
Duration of treatment / exposure:
120 days (17 weeks). Sub-group of animals from 0, 200, and 600 mg/kg groups killed after 60 days (8-9 weeks).
Frequency of treatment:
Once per day, 5 days/week.
Remarks:
Doses / Concentrations:
0, 25, 50, 100, 200, 400, 600 mg/kg bw/day
Basis:
other: nominal in corn oil
No. of animals per sex per dose:
10/sex/group for 0, 25, 50, 100 and 400 mg/kg; 15/sex/group for 0, 200 and 600 mg/kg
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: once per week

FOOD CONSUMPTION: Yes
- Time schedule: once per week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule / number of animals for collection of blood: animals killed at day 0 and day 60 and on 5 animals/group at terminal kill and on any animals killed in a moribund condition.
- Method of collection: from the orbital sinus
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters examined: haemoglobin, haematocrit, white blood cell count, red blood cell count, mean corpuscular volume, reticulocyte count, coagulation time.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Examinations on all animals except those with excessive autolysis or cannibalized.

HISTOPATHOLOGY: Yes. The following tissues were examined histologically in the predosing vehicle control, study vehicle control, and interim-kill animals and the 600 mg/kg animals at terminal kill: mandibular lymph node, salivary glands, femur, thyroid gland, parathyroid, small intestine, colon, liver, prostate/testes or ovaries/uterus, lungs and mainstem bronchi, mammary gland, heart, oesophagus, stomach, brain, thymus, trachea, pancreas, spleen, kidneys, adrenal glands, urinary bladder, pituitary gland; in addition, spleens were examined in all dose groups. Special histology studies performed on animals killed at days 0 and 60 and on 5 animals/group at 0, 200, and 600 mg/kg at terminal kill and on any animals killed in a moribund condition.
Statistics:
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 and as the same animals were examined across time, a repeated measures analysis of variance (Winer, 1971) method was used to investigate temporal and dose-related variation.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN: Final mean bodyweights, relative to vehicle controls, were depressed: 4, 2, 7, 14, 20 and 22% for males and 6, 9, 6, 16, 15 and 20 % for females at dose levels of 25, 50, 100, 200, 400 and 600 mg/kg respectively.

HAEMATOLOGY: A dose-related leukopenia was observed for both male and female rats. Day 60 mean WBC counts were 6.4, 2.5 and 1.7 (males) and 4.3, 2.3 and 1.7 (females) at 0, 200 and 600 mg/kg respectively. Day 60 mean LYM counts were 5.8, 2.0 and 1.3 (males) and 3.8, 1.9 and 1.5 (females) for 0, 200 and 600 mg/kg respectively. Day 120 mean WBC counts were 5.0, 7.0, 5.7, 6.0, 5.2, 3.5 and 3.1 (males) and 8.1, 6.2, 4.9, 5.0, 4.8, 3.4 and 3.8 (females) for 0, 25, 50, 100, 200, 400 and 600 mg/kg respectively. Day 120 mean LYM counts were 4.1, 5.1, 3.9, 3.9, 3.4, 2.3, and 2.4 (males) and 6.6, 4.7, 3.9, 3.6, 3.7, 2.7 and 2.8 (females) for 0, 25, 50, 100, 200, 400 and 600 mg/kg respectively.

HISTOPATHOLOGY: NON-NEOPLASTIC: Lymphoid depletion in the B-cell of the spleen was observed in 3/5 male and 4/5 female rats at 200 mg/kg and 5/5 male and 5/5 female rats at 600 mg/kg at 60 days and in 1/10 male and 10/10 females at 600 mg/kg at 120 days. Increased extramedullary haematopoiesis was observed in the spleen of 4/5 male and 3/5 female rats that received 600 mg/kg for 120 days.

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: decreased final body weight, white blood cell and lymphocyte counts and lymphoid depletion of B-cells in the spleen at 200 mg/kg and above
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: decreased final bodyweight, white blood cell and lymphocyte counts and lymphoid depletion of B-cells in the spleen at 200 mg/kg and above
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: reduction in white blood cell and lymphocyte counts at 25 mg/kg (lowest dose tested)
Critical effects observed:
not specified
Conclusions:
Repeat oral administration of benzene to rats is associated with adverse effects in the haematopoietic system. NOAEL for males was 200 mg/kg. No NOAEL was established for females. LOAEL for females was 25 mg/kg/day (lowest dose tested).
Executive summary:

Groups of 10 or 15 rats/sex were administered 0, 25, 50, 100, 200, 400 or 600 mg/kg benzene in corn oil by gavage, 5 days/ week for 17 weeks. Five rats/sex were killed on days 0 and 60 from the 0, 200, and 600 mg/kg groups, remaining surviving animals were killed on day 120. Clinical observations, bodyweights, food consumption, haematological analyses and histopathological examinations were performed. 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 mg/kg benzene. A dose-related leukopenia and lymphocytopenia was observed in males at ≥200 mg/kg and in females at ≥25 mg/kg. In the spleen, lymphoid depletion of B-cells was observed in both sexes at ≥200 mg/kg benzene and increased extramedullary haematopoiesis was observed 600 mg/kg.

NOAEL for males was 100 mg/kg/day. LOAEL for males was 200 mg/kg and for females was 25 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate information is available on the component substances to characterise the repeated oral hazards of these streams.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status not known, guideline compliant, animal experimental study, published in peer reviewed literature, adequate for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at delivery: Approximately 6 weeks
- Housing: individually in stainless steel wire mesh cages
- Diet: Purina Certified Lab Chow (#5002 Ralston Purina, St. Louis, MO) ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: 40 days

ENVIRONMENTAL CONDITIONS
- Temperature: 71-79°C
- Humidity: 35-80%
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: no data
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 6 m3 glass and stainless-steel exposure chambers
- Method of holding animals in test chamber: no data
- System of generating test atmosphere: the test atmosphere was generated by forced evaporation of warmed liquid benzene under a stream of dried filtered air.
- The air flows through the generation system and exposure chamber were monitored.
- The airflow through the chambers was maintained at 1,200 L/minute.
- All generation flasks were seated in water baths agitated with air and maintained at room temperature (Groups 2-4) or heated at 30±2°C (Group 5) with tank-type heaters.

TEST ATMOSPHERE
- Brief description of analytical method used: The chamber concentrations of benzene were established using gas chromatography with infrared (IR) analyzer
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC samples were obtained and analyzed by peak height at least once a day from each exposure chamber.
Overall mean GC analyses (for Groups 2-5) varied from the target concentrations by 20.0, 18.0, 2.0, and 2.4% (respectively)
Duration of treatment / exposure:
10 rats/sex/group sacrificed after 7, 14, 28, 56, and 91 days of treatment
Frequency of treatment:
5 days/week for up to 13 weeks
Remarks:
Doses / Concentrations:
1, 10, 30 or 300 ppm (3.2, 32, 96 or 960 mg/m³)
Basis:
nominal conc.
No. of animals per sex per dose:
50
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (mortality and morbidity)
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to initiation and on days 7, 14, 28, 56 and 91
- Anaesthetic used for blood collection: sodium pentobarbital
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters examined: haematocrit, total haemoglobin, erythrocyte count, mean cell volume, mean cell haemoglobin, total and differential leukocyte count, platelet count, reticulocyte count (only on days 28, 56, and 91), myeloid/erythroid (M/E) ratio (from bone marrow smears), leukocyte alkaline phosphatase (from peripheral blood smears), total protein, alkaline phosphatase, total bilirubin, creatinine, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and erythrocyte glycerol lysis time

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: 10 rats/sex/group randomly selected, fasted (overnight), weighed, and killed by exsanguination under sodium pentobarbital anaesthesia on days 0, 7, 14, 28, 56, and 91. Complete necropsies were performed on all these animals and on all animals found dead or sacrificed in a moribund condition during the study.

ORGAN WEIGHTS: - Organs were weighed and organ/terminal body weight ratios determined on the following: brain, pituitary, adrenals, thyroids, lungs, heart, liver, spleen, kidneys and testes.

HISTOPATHOLOGY:
- The following tissues were taken and preserved in 10% neutral buffered formalin: heart, lungs (all lobes), bronchi, trachea, cervical lymph nodes, salivary gland, oesophagus, stomach, small intestine (three sections), caecum, colon, bone marrow (femur), bone marrow section and smear (fixed in alcohol), adrenals, testes or ovaries, prostate or uterus, mesenteric lymph nodes, urinary bladder, mammary gland, brain, pituitary, spinal cord (two sections), sciatic nerve/ muscle, thymus, spleen, liver, pancreas, kidneys, bone, eye (fixed in Bouin's solution), Harderian gland, nasopharynx, and Zymbal's gland.
- Sections from these tissues, from control and high-level groups at each sacrifice period were examined plus sections of lung, trachea, cervical lymph nodes, thymus, spleen, bone marrow (femur), mesenteric lymph nodes, and nasal turbinates from all animals at each interval.
Statistics:
Body weight data were analyzed by one-way classification analysis of covariance (ANCOVA) using pre-exposure (day 0) weights as the covariate. Clinical pathology data, organ weight and organ/body weight ratio data of the control group were analysed by Barlett's test for homogeneity of variance. If ANOVA of homogeneous data was significant, Scheffe's multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA of heterogeneous data was significant, Games and Howell's multiple pairwise comparison procedure was used to compare the group mean values. If ANOVA was not significant, no pairwise comparisons were made. The null hypothesis was rejected only if p <0.05 (one-tailed) in all cases (ie, the 5% level of significance was chosen).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Dose descriptor:
NOAEC
Effect level:
30 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: decreased white blood cell count, percentage of lymphocytes and femoral marrow cellularity at 300 ppm
Dose descriptor:
NOAEC
Effect level:
96 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: decreased white blood cell count, percentage of lymphocytes and femoral marrow cellularity at 960 mg/m3
Critical effects observed:
not specified

No exposure-related mortality or effects on mean body weight and clinical signs were seen. Female rats of the 30 ppm dose group showed lower thyroid weight on day 14 only. At 300 ppm statistically significant decreases in WBC counts were seen in males on day 14 and females on day 91; statistically significant decreases in percent lymphocytes were noted in both males and females on days 14, 28, 56 and 9 (no further detail provided). At this same dose level decreased femoral marrow cellularity was seen on day 7 only.

Conclusions:
Inhalation exposure of rats to 300 ppm (960 mg/m3) benzene for up to 90 days induced decreased lymphocyte counts, a relative increase in neutrophil percentages and slightly decreased femoral marrow cellularity. The NOAEC was 30 ppm (96 mg/m3) in males and females.
Executive summary:

A subchronic inhalation toxicity study of benzene was conducted in Sprague-Dawley rats. Four groups of animals consisting of 50 rats/sex each were exposed to concentrations of 0, 1, 10, 30, and 300 ppm (0, 3.2, 9.6, 960 mg/m3) benzene vapour, 6 h/day, 5 days/week, for 13 weeks. Ten rats/sex in each group were sacrificed after 7, 14, 28, 56, and 91 days of treatment. Criteria used to evaluate exposure-related effects included behaviour, bodyweights, organ weights, clinical pathology, gross pathology, and histopathology.

No consistent exposure-related trends were seen in the clinical observations and bodyweight data. Exposure-related clinical pathology changes were seen in the high-level (300 ppm) animals. Decreased lymphocyte counts and a relative increase in neutrophil percentages were the only exposure-related clinical pathology alterations. The only exposure-related lesion consisted of slightly decreased femoral marrow cellularity in the animals exposed to 300 ppm.

The NOAEC for toxicity at 28 and 90 days was 30 ppm (96 mg/m3) for both male and female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
11.2 mg/m³
Study duration:
chronic
Species:
other: human
Quality of whole database:
Adequate information is available on the component substances to characterise the repeated inhalation hazards of these streams.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
Adequate information is available on the component substances to characterise the repeated dermal hazards of these streams.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The available data on the marker consituents xylene and DCPD do not reveal any specific target organ toxicity of a severity that would warrant classification. Therefore, no classification or labelling is warranted for streams which only contain these components.

Other specific components which have been identified as present in some streams are benzene and toluene. These are all identified as producing serious target organ toxicity following repeated oral, dermal or inhalation exposures in animals and man:

Benzene (Classification: STOT-RE Category 1, H372): After repeated dose exposure via oral or inhalation routes, benzene causes adverse effects on the haematopoietic system of animals and man. The oral LOAEL was 25 mg/kg bw/day for male and female mice (NTP, 1986) and the inhalation LOAEC for haematotoxicity in mice is 10 ppm (32 mg/m3) (Ward et al, 1985). In humans, a recent study of petroleum distribution workers exposed to relatively low levels of benzene (Schnatter et al., 2012) reported associations between myelodysplastic syndrome (MDS) and benzene exposure, however while the association appeared to be reasonably robust, it was difficult to ascribe a precise dose/response relationship. For humans, therefore, a NOAEC of 3.5 ppm (11.2 mg/m3) is obtained based on the 95% LCL for the threshold level of neutrophils, the most sensitive endpoint reported by Schnatter et al (2010).

Toluene (Classification: STOT-RE Category 2, H373): Toluene exposure can produce central nervous system pathology in animals after high oral doses. Repeated inhalation exposure can produce ototoxicity in the rat and high concentrations are associated with local toxicity (nasal erosion). In humans neuropsychological effects and disturbances of auditory function and colour vision have been reported, particularly when exposures are not well controlled and/or associated with noisy environments. The NOAEC for subchronic oral toxicity in rats is 625 mg/kg/day based on neuropathology (NTP, 1990). The NOAEC for inhalation toxicity in the rat is 300 ppm (1131 mg/m3) based on effects on body weight, mortality and adverse local effects (nasal erosion) (Gibson and Hardisty, 1983). The NOAEC for neuropsychological effects, auditory dysfunction and disturbances of colour vision in humans is 26 ppm (98 mg/m3) (Seeber et al, 2004); Schaper et al, 2003, 2004).

Ethylbenzene (Classification: STOT-RE Category 2, H373): Ethylbenzene is classified in Annex VI of the CLP regulation as being harmful to hearing organs following repeated dose exposure.

Styrene (Classification: STOT-RE Category 2, H373): Styrene is classified in Annex VI of the CLP regulation as being harmful to hearing organs following repeated dose exposure.

n-hexane (Classification: STOT-RE Category 2, H373): n-hexane is classified in Annex VI of the CLP regulation as being harmful following repeated dose exposure.

References

Spencer PS and Schaumburg HH (1985). Organic solvent neurotoxicity - facts and research needs. Scand J Work Environ Health 11, 53 -60.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Several of the marker substances present are associated with serious target organ effects e.g. benzene, toluene. Results obtained for the key component benzene are considered indicative of the overall potential hazard of these streams after repeated exposure.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Several of the marker substances present are associated with serious target organ effects e.g. benzene, toluene. Results obtained for the key component benzene are considered indicative of the overall potential hazard of these streams after repeated exposure. Human data show haematological changes in neutraphil counts with a NOAEC of 3.5 ppm (11.2 mg/m3).

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

Sub-acute data available for representative streams indicate no potential to affect health following repeated skin contact, however several of the marker substances present are associated with serious target organ effects e.g. benzene, toluene. Results obtained for the key component benzene are considered indicative of the overall potential hazard of these streams after repeated exposure.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: bone marrow

Repeated dose toxicity: inhalation - systemic effects (target organ) cardiovascular / hematological: bone marrow

Repeated dose toxicity: dermal - systemic effects (target organ) cardiovascular / hematological: bone marrow

Justification for classification or non-classification

There are sufficient data available to conclude that the high benzene naphtha streams, which contain less than 1% benzene / styrene, less than 5% n-hexane, and less than 10% toluene/ethylbenzene, do not require classification for this endpoint.

Streams which contain ≥1% but less than 10% benzene or styrene will require to be classified as follows: Cat 2, H373 according to Reg (EC) 1272/2008; streams containing ≥10% benzene or styrene should be classified Cat 1, H372 according to Reg (EC) 1272/2008.

Streams which contain 5% n-hexane should be classified as Cat 2, H373 according to Reg (EC) 1272/2008.

Streams which contain ≥10% toluene/ ethylbenzene should be classified as Cat 2, H373 according to Reg (EC) 1272/2008.