Reaction mass of ethylbenzene and m-xylene

Endpoint summary

Description of key information

In repeated dose studies, the principle effects of xylenes were adaptive changes in the liver, organ weight and body weight changes. Inhalation studies in rodents have demonstrated a potential to cause ototoxicity.

The repeated exposure toxicity of ethylbenzene has been evaluated in animals in subchronic and chronic inhalation studies, subchronic oral toxicity studies and numerous specialized investigations. Overall, ethylbenzene poses a moderate repeated exposure toxicity hazard with consistent targeted effects to the liver, kidney and hearing.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP status not known, near guideline study, published in peer reviewed literature, limitations in design and/or reporting but otherwise adequate for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.32 (Carcinogenicity Test)

Principles of method if other than guideline:

Mixed xylene was administered by oral gavage to groups of 50 male and 50 female F344/N rats at doses of 0, 250 or 500 mg/kg bw/day for 103 weeks. Animals were observed for survival, clinical signs and body weight gain and subject to a full necropsy with tissue histopathology at termination. However, only two dose levels were tested and ophthalmoscopy, food consumption, haematology, blood clinical chemistry and urinalysis were not assessed.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY, USA
- Age at study initiation: 7 weeks
- Housing: 5 per sex /cage in Polycarbonate cages
- Diet: NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA); available ad libitum
- Water: ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23° ± 1°C
- Humidity: 40 - 60%
- Air changes: 15 air changes/hr
- Photoperiod: 12 hr/d light; 12 hr/d dark

IN-LIFE DATES: From: 30 June 1980 To: 2 July 1982

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Oral (gavage): 0, 250 or 500 mg/kg xylenes (mixed) in corn oil; 4 mL/kg

Preparation: Weighed portions of xylenes (mixed) were placed in a graduated cylinder and mixed with corn oil to achieve the proper volume. The mixtures were shaken vigorously for 10 seconds.

Maximum Storage Time: 2 wks

Storage Conditions: Approximately 24ºC, 46% humidity under fluorescent light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of xylenes in corn oil was analysed by gas chromatography with flame ionization detection following extraction with methanol.
During the 2-year studies, the dose preparations were analyzed once every 2 months, with concentrations varying from 94.6% to 106.9% (within 10% target concentrations).

Duration of treatment / exposure:

5 days per week for 103 weeks.

Frequency of treatment:

Once daily (5 days / week).

Remarks:

Doses / Concentrations:
0, 250 or 500 mg/kg
Basis:
other: nominal concentration

No. of animals per sex per dose:

50 male / 50 female per group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Based on weight gain depression at 1,000 mg/kg in both sexes in the 14-day studies and in males in the 13-week studies and on the clinical signs in the 14-day studies, doses selected for rats for the 2-year studies were 0, 250, and 500 mg/kg xylenes (mixed) in corn oil by gavage, administered 5 days per week.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All animals were observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical signs were recorded once per day for 16 months and then once per month.

BODY WEIGHT: Yes
- Body weights were recorded weekly for 12 weeks and monthly thereafter

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Data were recorded in the NTP Carcinogenesis Bioassay Data System. The data elements included descriptive information on the chemicals, animals, experimental design, survival, body weight, and individual pathologic results.

Sacrifice and pathology:

Necropsy and histopathological examination performed on all animals, where possible. During necropsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. The following tissues were examined: gross lesions and tissue masses, mandibular lymph nodes, salivary gland, femur, including marrow, thyroid gland, parathyroids, small intestine, colon, liver, prostate / testis or ovaries / uterus, heart, oesophagus, stomach, brain, thymus, trachea, pancreas, spleen, skin, lungs and mainstem bronchi, kidneys, adrenal glands, urinary bladder, pituitary gland, eyes (if grossly abnormal), and mammary gland.

Statistics:

Survival Analyses: Kaplan and Meier (1958); Cox (1972) and Tarone (1975). All reported P values for the survival analysis are two-sided. Calculation of Incidence for neoplastic and non-neoplastic lesions. Analysis of Tumour Incidence: Mantel and Haenszel (1959). Continuity-corrected tests were used in the analysis of tumour incidence, and reported P values are one-sided. Life Table Analyses-- Mantel-Haenszel (1959) method used to obtain an overall P value. Life table method of Cox (1972) and of Tarone (1975). The underlying variable considered by this analysis is time to death due to tumour. Incidental Tumour Analyses-- (Haseman, 1984) Unadjusted Analyses--Primarily, survival-adjusted methods are used to evaluate tumour incidence. The Fisher exact test for pairwise comparisons and the Cochran-Armitage linear trend test (Armitage, 1971; Gart et al., 1979).

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality - Although the mortality was dose related in male rats (final survival: vehicle control 36/50, low dose 26/50, high dose 20/50), many of the early deaths in the dosed males were gavage related. Survival of the high dose males was significantly lower than that of the vehicle control after week 103.

Bodyweight - Bodyweights of high dose male rats were 5%-8% lower than those of the vehicle controls after week 59.

Tumour findings - There were no significant changes in the incidences of neoplastic or non-neoplastic lesions which were considered to be related to the administration of xylenes (mixed).

Testis findings - Although the overall incidences of interstitial cell tumours were comparable in male rat groups (vehicle control, 43/50; low dose,38/50; high dose, 41/49), survival-adjusted analyses indicated an increased incidence in the high dose group relative to vehicle controls. This apparent effect was due primarily to animals dying between weeks 62 and 92, for which the incidence of interstitial cell tumours was 13/13 for the high dose group compared with 4/9 for vehicle controls. Tumour incidences were comparable during the other time intervals. It is doubtful that this marginal effect is compound related.

Haematopoietic System and Pituitary Gland - Dose-related decreases in the incidences of mononuclear cell leukaemia (vehicle control,22/50; low dose, 18/50; high dose, 11/50) and pituitary gland adenoma or carcinoma (combined) (vehicle control, 24/49; low dose, 22/50; high dose, 12/45) were observed in male rats. However, these differences were due primarily to decreased survival of the high dose group relative to that of the vehicle controls.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decreased body weights in males from week 59 at 500 mg/kg bw/day and no evidence of systemic toxicity of xylenes (mixed) for male or female F344/N rats given 250 or 500 mg/kg

Critical effects observed:

not specified

Conclusions:

There was no evidence of treatment-related systemic toxicity or carcinogenicity following gavage administration of mixed xylenes to male and female F344/N rats at doses of 0, 250 or 500 mg/kg body weight/day for up to 103 weeks.

Executive summary:

The toxicity of mixed xylene was investigated in male and female F344/N rats following oral (gavage) administration at doses of 0, 250 or 500 mg/kg bw/day for 103 weeks. Animals were observed for survival, clinical signs and body weight gain and subject to a full necropsy with tissue histopathology at sacrifice. There was no evidence of treatment-related systemic toxicity in either sex under these conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Results are available from chronic and sub-chronic studies that have investigated the repeated dose toxicity of mixed xylene, xylene isomers, and ethylbenzene in rodents.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP status not known, non-guideline study, published in peer reviewed literature, limitations in design and/or reporting but otherwise adequate for assessment.

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

No details reported

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: air

Details on inhalation exposure:

no details

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0, 180, 460 or 810 ppm
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0, 781, 1996 and 3515 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

25 males

Control animals:

yes

Details on study design:

Groups of 25 male rats were assigned randomly to each of three graded levels of mixed xylenes or to the solvent- free air-control. Three rats from each group were killed for histological examination after 3 and 7 week intervals. Ten rats per group that survived 13 weeks were used for a challenge exposure to determine whether they had become more or less sensitive as a result of the repeated inhalation of vapour. The remaining rats were terminated after 13 weeks (65 days of exposure).

Positive control:

No

Observations and examinations performed and frequency:

Measurements included body weight change and urine and blood analysis. Evaluations on blood included: haematocrit, total erythrocyte count, reticulocyte count, total and differential leucocyte counts, serum alkaline phosphatase (SAP), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxalacetic transaminase (SGOT) and blood urea nitrogen (BUN).

Sacrifice and pathology:

At termination, the following tissues were taken for microscopic examination: adrenal, brain, pituitary, trachea, thyroid, parathyroid, lung, heart, liver, kidney, spleen, stomach, duodenum, pancreas, ileum, jejunum, colon, skeletal muscle, sciatic nerve, and bone marrow impression smear. All tissues were examined at each time point for the high dose and control groups, but for the low and intermediate dose only lung, liver, kidney, heart, spleen, adrenal, thyroid, parathyroid, trachea, oesophagus, and bone marrow impression smears were examined.

Other examinations:

Groups of ten rats per group that survived exposure over 13 weeks, were subjected to a 4-hr challenge period during which time they inhaled 29.0 mg/L (6700 ppm) of mixed xylenes. Their response was compared to the similarly handled control group and to 20 naive rats that were randomized from the same production lot as those under test but not subjected to daily handling.

Statistics:

No details

Details on results:

Two rats (one control and one low dose) died of extraneous pneumonic infections. All other rats gained weight normally. Minor differences in blood clinical chemistry and haematology were considered not to be adverse effects. Urinalyses performed before rats were sacrificed at 3, 7, and 13 wk revealed no abnormalities. No lesions ascribed to inhalation of mixed xylenes were found at any time point.

No statistically significant differences in median time to death were apparent following the challenge at the end of the study. No protective adaptive mechanism could be claimed as a result of exposure to concentrations as high as 3.5 mg/L (810 ppm) for 6 hr/day x 65 days (5 days /week for 13 weeks.

Dose descriptor:

NOAEC

Effect level:

810 ppm

Sex:

male

Basis for effect level:

other: 3515 mg/m3. No effects at the highest dose tested.

Critical effects observed:

not specified

Conclusions:

The NOAEC of mixed xylenes for male rats exposed 6h/day for 5 days in each of 13 weeks was 3515 mg/m3 (3.5 mg/L).

Executive summary:

Male rats were exposed 6h/day for 5 days in each of 13 weeks to 0, 0.77, 2.0 or 3.5 mg/L (0, 180, 460 or 810 ppm) mixed xylenes. The NOAEC was 3515 mg/m³ (3.5 mg/L).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3 515 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

A NOAEC of 3515 mg/m3 was reported by Carpenter et al. (1975) for generalised systemic effects in male rats and male dogs for xylene. Other studies have shown that some xylene isomers adversely affect hearing in the rat, with a sub-chronic NOAEC of 1950 mg/m3 reported for p-xylene; the NOAEC for ototoxicity of m-xylene and o-xylene was greater than 7810 mg/m3 (Gagnaire et al., 2001). The ototoxicity of mixed xylenes appears to be dependent upon composition (Gagnaire et al., 2007), with a sub-chronic LOAEC of 1080 mg/m3 reported for one sample while another had a NOAEC of 2170 mg/m3.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The multi-constituent substances covered by this registration comprise individual xylene isomers (m-xylene, o-xylene, p-xylene) and ethyl benzene (>10% - <20%). The following information is available to characterise their repeated dose toxicity.

 

Repeated dose toxicity: oral

 

Data are available for laboratory animals exposed to high doses of mixed xylene, adverse effects have been observed in the kidney and liver (IARC, 1989).

 

A near-guideline (equivalent or similar to OECD 408) subchronic oral gavage study with mixed xylene (comprising 18% o-xylene, 62% m- and p-xylene, 20% ethyl benzene) was conducted by Condie et al. (1988). It includes the range of toxicological endpoints routinely investigated in regulatory subchronic studies and is a key study for identifying the effects of mixed xylene. In the study, groups of 10 male and 10 female rats were given 0, 150, 750, or 1500 mg/kg bw/day of mixed xylene in corn oil for 90 consecutive days. A decrease in body weight was observed in males only at 1500 mg/kg bw/day. Although increased relative liver weights were seen at all dose levels in males and in females at 750 and 1500 mg/kg bw/day there were no adverse histopathology findings in the liver. An increased relative kidney weight was observed in high dose males and females and in intermediate dose males. In males there was a dose-related increase in the incidence of slight to mild hyaline droplet formation in tubules at all dose levels. This finding is indicative of alpha-2u-globulin which is considered to be male rat-specific and is not relevant for humans. In females the incidence of minimal nephropathy in females was statistically significantly increased in the 750 and 1500 mg/kg bw/day groups. This finding described as scattered tubular dilation and atrophy, with occasional regeneration and is the chronic progressive nephropathy typically seen in ageing rats.

 

No NOAEL was established in this study for males based on liver weight increases. However increases in liver weight with no adverse histopathological findings are considered to be an adaptive response to administration of mixed xylene rather than an adverse toxicological effect. A dose of 750 mg/kg bw/day is the NOAEL based on effects on male body weight. In females a NOAEL of 150 mg/kg bw/day is based on liver weight increases and the kidney effects observed at dose levels of 750 mg/kg bw/day and higher.

 

Treatment levels between 150 and 750 mg/kg bw/day are covered in a carcinogenicity study in rats (NTP, 1986). The test sample comprised 60% m-xylene, 14% p-xylene, 9% o-xylene, and 17% ethyl benzene. Although this study did not include all of the end points included in chronic studies to current guidelines, the key parameters affected in the sub chronic study, i.e. body weights and detailed pathology and histopathology are included. Rats were dosed with mixed xylene at concentrations of 0, 250 or 500 mg/kg bw/day 5 days per week for 103 weeks. The main finding was a decrease in body weights in males receiving 500 mg/kg bw/day in the second year of the study. There was no other evidence of systemic toxicity including no treatment-related pathology findings. A dose of 250 mg/kg/day was a NOAEL for both sexes and this is considered to be the key study for determining the NOAEL for repeated dose exposure to mixed xylenes via the oral route.

 

A supporting subchronic oral gavage study was conducted by NTP (1986) using groups of 10 male and 10 female rats treated via gavage 5 days/week for 13 weeks with 0, 62.5, 125, 250, 500 or 1000 mg/kg/day. In the same study, groups of 10 male and 10 female mice were similarly dosed with 0, 125, 250, 500, 1000 or 2000 mg/kg/day of mixed xylene in corn oil. The composition of the sample was as described above.

 

Limited toxicological endpoints were evaluated.

 

Treatment-related findings in rats were limited to a reduction in overall body weight gain (15% for males and 8% for females) with a NOAEL of 500 mg/kg/day. High dose mice exhibited transient CNS effects 5-10 minutes after dosing that lasted 15-60 minutes. Other treatment-related findings were limited to a reduction in overall body weight gain (7% for males and 17% for females) with a NOAEL of 1000 mg/kg/day. Neither blood clinical chemistry nor organ weight data were collected for either species. No treatment-related macroscopic or microscopic lesions were observed in the tissues examined including the liver and kidney.

 

 

o-xylene

A guideline OECD 408 90-day oral rat study with o-xylene was conducted (Britton LD, 2020).

 

The test material, o-xylene (CAS95-47 -6), was administered to male and female rats by oral gavage in order to evaluate its sub chronic toxicity. Dose levels were 0, 100, 300, and 1000 mg/kg/day (Groups 1 through 4, respectively), with 10 animals/sex/group dosed daily for at least 90 days.The following were investigated: clinical observations, ophthalmoscopy, body weight, food intake, functional observation battery, haematology, blood chemistry, thyroid hormone analysis, organ weights, macroscopic and microscopic pathology.

 

There were no deaths during the study. Males and females given 1000 mg/kg/day showed decreased activity and unsteady gait from Days 19/18 of dosing onwards (persisting for up to 4 hours after dosing), respectively. The incidence of these clinical signs decreased with time and by Day 72 of dosing, only one male was showing decreased activity and abnormal gait. Over Days 84 to 92 of dosing at most eight out of ten females showed one or both of these signs after dosing. No effects on sensory reaction or grip strength were elicited by o-xylene. In open arena testing, males given 1000 mg/kg/day exhibited effects on arousal, unusual posture and abnormal gait. Twitches or extended rearing were also observed in a small number of individual males given 1000 mg/kg/day. Increased episodes of arousal in the arena and abnormal gait were observed between Days 21 to 70 and Days 21 to 56 of dosing, respectively. Excessive salivation in the arena was also noted between Days 14 and 70 of dosing for males given 300 or 1000 mg/kg/day, with incidence increasing with time. Females given 1000 mg/kg/day also exhibited effects on arousal in the arena, abnormal gait and salivation. Although incidences increased with time similarly to the males, these observations were not seen in either sex after Day 77 of dosing. Movement counts and distance travelled were reduced in males given 100, 300 or 1000 mg/kg/day and this was occasionally accompanied by an increased time at rest. Males given 300 or 1000 mg/kg/day gained less weight over the study than Controls with terminal body weights that were 6 % and 11 % respectively, lower than those of the Controls. There was no effect on body weight for females and no effect on food intake in either sex. There were slight increases in Thyroxine (T4) and no effect on thyroid stimulating hormone (TSH) or triiodothyronine (T3) levels at the end of the treatment period and no adverse effects on haematology, coagulation or blood chemistry parameters. Both sexes given 300 or 1000 mg/kg/day and females given 100 mg/kg/day had statistically significantly higher liver and kidney weights when compared with Controls. In addition, thyroid weights for females given 1000 mg/kg/day were also higher than Controls.

 

Histopathology findings considered to be related to o-xylene were seen in the liver (hepatocellular hypertrophy) and thyroid (follicular cell hypertrophy) in both sexes and, in the kidneys, eosinophilic inclusions (hyaline droplets) in the tubular epithelium (males only). Following α2μ-globulin staining of the kidneys, although positive staining was observed in 3/10 high dose males, minimal staining was also observed in 2/10 Controls. The precise content of the hyaline droplets observed at an increased incidence/severity of within the cortical tubules of male kidneys therefore remained unestablished. Given the physiological differences between the male rat kidney and human, these findings are considered unlikely to represent a hazard to man.

 

In conclusion, when administered by gavage to the Han Wistar rat, once daily for at least 90 days, o-xylene was generally well tolerated in-life at 100 and 300 mg/kg/day with, at the latter dose, only a mild retardation of body weight gain in males and clinical signs being limited to some transient excessive salivation after dosing. However, more profound (decreased activity, unsteady gait, effects on arousal, unusual posture), but transient, clinical signs were apparent at 1000 mg/kg/day and a more marked retardation of body weight gains in males. Target organ pathology was restricted to rat specific adaptive responses consisting of hepatic centrilobular hypertrophy and secondary thyroid follicular hypertrophy consistent with enzyme induction and adverse renal tubular changes in males only likely reflecting proteinuria and inclusion of non α2μ-globulin proteins. Therefore, the No Observed Adverse Effect Level (NOAEL) was defined as 300 mg/kg/day o-xylene in the female rat because of the severity of clinical signs at 1000 mg/kg/day and as 100 mg/kg/day in the male rat, because of the incidence and severity of the renal changes. However, given the physiological differences between the male rat kidney and human, the observed tubular changes are unlikely to represent a hazard to man. Therefore, if the findings in the kidney are disregarded as being human relevant, the NOAEL for the male would be 300 mg/kg/day.

 

m-xylene

A GLP 90-day oral rat study with m-xylene was conducted (Wolfe GW, 1988).

 

The test material, m-xylene (CAS 108-38-3), was administered to male and female rats by oral gavage in order to evaluate its sub chronic toxicity. Dose levels were 0, 100, 200, and 800 mg/kg/day (Groups 1 through 4, respectively), with 20 animals/sex/group dosed daily for at least 90 days. Criteria evaluated included mortality, clinical signs, body weights, food consumption, ophthalmologic examinations, haematology, clinical chemistry, organ weights, organ-to-body weight ratios, gross pathology, and histopathology.

 

Mortality was significantly increased in male Group 3 and female Groups 3 and 4; however, based on histopathology findings, these deaths were most likely the result of vehicle and/or compound aspiration. Salivation was frequently observed in both sexes of Group 4 and only rarely in the other treated groups. Total body weight gain was significantly decreased in male Groups 3 and 4, and female Group 4. Food consumption was significantly decreased from Week 1 to 5 in Group 4 males and from Week 6 to 9 in Groups 3 and 4 males. A few clinical pathology parameters were significantly increased or decreased and with one exception (female Group 3), all were in Group 4. A few significant changes were seen in absolute or relative organ weights, all of which were in male Group 4. The most commonly observed lesions at necropsy were in the lungs (mottled and failure to collapse). These lesions were only seen in the animals found dead. No apparent compound-related effects were present in the ophthalmology or histopathology findings.

 

Based on the results of this study, with the assumption that the decrease in survival was the result of vehicle and/or compound aspiration, the no-observed-adverse-effect level (NOAEL) of m-Xylene was 200 mg/kg/day in the females. However, due to significantly decreased body weight gain in Groups 3 and 4, the NOAEL for the males was 100 mg/kg/day.

 

Data are available for laboratory animals exposed to high doses of mixed xylenes, adverse effects have been observed in the kidney and liver (IARC, 1989).

 

A near-guideline (equivalent or similar to OECD 408) subchronic oral gavage study with mixed xylenes (20% ethylbenzene) was conducted by Condie et al. (1988). It includes the range of toxicological endpoints routinely investigated in regulatory subchronic studies and is a key study for identifying the effects of mixed xylenes. In the study, groups of 10 male and 10 female rats were given 0, 150, 750, or 1500 mg/kg bw/day of mixed xylenes in corn oil for 90 consecutive days. A decrease in body weight was observed in males only at 1500 mg/kg bw/day. Although increased relative liver weights were seen at all dose levels in males and in females at 750 and 1500 mg/kg bw/day there were no adverse histopathology findings in the liver. An increased relative kidney weight was observed in high dose males and females and in intermediate dose males. In males there was a dose-related increase in the incidence of slight to mild hyaline droplet formation in tubules at all dose levels. This finding is indicative of alpha-2u-globulin which is considered to be male rat-specific and is not relevant for humans. In females the incidence of minimal nephropathy in females was statistically significantly increased in the 750 and 1500 mg/kg bw/day groups. This finding described as scattered tubular dilation and atrophy, with occasional regeneration and is the chronic progressive nephropathy typically seen in ageing rats.

 

No NOAEL was established in this study for males based on liver weight increases. However increases in liver weight with no adverse histopathological findings are considered to be an adaptive response to administration of mixed xylenes rather than an adverse toxicological effect. A dose of 750 mg/kg bw/day is the NOAEL based on effects on male body weight. In females a NOAEL of 150 mg/kg bw/day is based on liver weight increases and the kidney effects observed at dose levels of 750 mg/kg bw/day and higher.

 

Treatment levels between 150 and 750 mg/kg bw/day are covered in a carcinogenicity study in rats (NTP, 1986). Although this study did not include all of the end points included in chronic studies to current guidelines, the key parameters affected in the sub chronic study, i.e. body weights and detailed pathology and histopathology are included. Rats were dosed with mixed xylenes at concentrations of 0, 250 or 500 mg/kg bw /day 5 days per week for 103 weeks. The main finding was a decrease in body weights in males receiving 500 mg/kg bw/day in the second year of the study. There was no other evidence of systemic toxicity including no treatment-related pathology findings. A dose of 250 mg/kg/day was a NOAEL for both sexes and this is considered to be the key study for determining the NOAEL for repeated dose exposure to mixed xylenes via the oral route.

 

p-xylene

A GLP 90-day oral rat study with m-xylene was conducted (Wolfe GW, 1988).

 

The test material, p-Xylene (CAS 106-42-3), 99% pure, was administered to male and female Sprague-Dawley rats by oral gavage in order to evaluate its sub-chronic toxicity. Dose levels were 0, 100, 200, and 800 mg/kg/day (Groups 1 through 4, respectively), with 20 animals/sex/group dosed daily for at least 90 days. Criteria evaluated included mortality, clinical signs, body weights. food consumption, ophthalmologic examinations, haematology, clinical chemistry, organ weights, organ-to-body weight ratios, gross pathology, and histopathology.

 

At least one animal in each treated group died before termination of the study and there was a significant increase in mortality in Group 4 males. However, based on histopathology findings, nearly all of the unscheduled deaths were related to aspiration of the vehicle/test material. Salivation was frequently observed and exclusively in both sexes of Group 4. Mean body weights were slightly decreased in Group 4 males and females while total food consumption was slightly increased in these groups. There was one incidental finding in the haematology results. In the clinical chemistries, a few significant differences were present in the Group 4 females at Week 5; however, none were present at Week 13. No apparent compound-related effects were present in the ophthalmology, organ weight, gross pathology, or histopathology data.

 

Based on the results of this study, with the consideration of vehicle/test material aspiration producing the deaths in the treated groups, the no-observed-effect level (NOEL) of p-xylene was 200 mg/kg/day.  

 

Repeated dose toxicity: dermal

 

No studies are available for xylenes (including mixed xylene and the individual xylene isomers).

 

 

Repeated dose toxicity: inhalation

 

The available subchronic inhalation studies are designed primarily to address neurological endpoints (including ototoxicity) in male rats and dogs. These endpoints are discussed in section 5.10.1.1.

 

In a recent study (Gagnaire et al., 2007a) the potential ototoxicity of two samples of mixed xylene was investigated in groups of male rats exposed to 250, 500, 1000 and 2000 ppm for 6 h/day, 6 d/wk over 13 weeks, with a recovery period of 8 weeks. One sample contained 10% ethyl benzene, the other 20% ethyl benzene. There was no adverse effect on body weight at any of the dose levels.

 

In another investigation (Gagnaire et al., 2001), the potential ototoxicity of individual xylene isomers was evaluated using electrophysiological methods in male rats exposed by inhalation to three different concentrations 6 hours/day, 5 days/week for 13 weeks was evaluated. The highest exposure concentration of 1800 ppm had no significant effect on body weight or body weight gain.

 

In an older study by Carpenter (1975), male rats and male dogs were exposed 6h/day for 5 days in each of 13 weeks to 0, 180, 460 or 810 ppm mixed xylene. The highest exposure level was a NOAEC for both species.

 

Ethylbenzene

Oral route

Data obtained after oral exposure support the main findings of inhalation studies. The most comprehensive investigation is that of Mellert et al. (2006) (BASF, 2004) exposing rats by gavage at levels of 75, 250 and 750 mg/kg bw/d over 90 days. Body weight was significantly decreased in males at 750 mg/kg bw/ d. Liver and kidney weights were increased at 250 and 750 mg/kg bw/d accompanied by slight centrilobular hypertrophy, those in the kidney are characterised as chronic progressive nephropathy and accumulation of male specific protein α-2u-globulin. In addition at the two top dose levels there was a slight increase of serum alanine aminotransferase and gamma-glutamyltransferase in males and signs for a minimal regenerative anemia. Increases in total bilirubin and mild increase in hemosiderosis (observed for 1-phenylethanol only) might indicate on a mild hemolysis as possible cause of anemia. Overall, the NOAEL was 75 mg/kg bw/d.

 

 

Dermal route

 

No information is available for systemic toxicity after repeated dermal exposure.

Inhalation route

A number of conventional toxicology studies on rats, mice and rabbits with repeated inhalation exposures are available. Additional data but of deficient quality were reported on inhalation exposure in rhesus monkeys and guinea pigs.

Clinical signs indicative of irritation were reported in rats starting at about 400 ppm with a NOEC of 100 ppm (Cragg et al., 1989; Biodynamics, 1986) with similar signs of irritation in mice at 400 ppm (Biodynamics, 1986). Slight body weight depressions in rats started at about 750 ppm (Stott et al.,1999; 2003; NTP, 1992) in studies up to 3 months of duration, while after an exposure period of 2 years (NTP, 1999) such slight effects on body weights were already observed at 250 ppm in rats but not in mice. Mortality was increased after 4 days of exposure in rats at 2400 ppm and in mice at 1200 ppm (Biodynamics, 1986) while after a 2-year exposure there was a decreased survival of male rats at 750 ppm (but not in female rats or mice), most probably related to chronic progressive nephropathy (CPN) (NTP, 1999).

The most consistent effect is an increase in liver weight of rats and mice without histopathological alterations by standard procedures in studies with duration up to 3 months (Cragg et al., 1989; Biodynamics, 1986; Stott et al., 1999;2003; Wolf et al., 1956; NTP, 1992, WIL Research Laboratories, 2004; Faber et al, 2006). Such effects were also noted in studies dealing with fertility (cf section 4.1.2.9), e.g. in parental animals of a 1- generation (WIL Research Laboratories, Inc., 2003) and 2 -generation study (WIL Research Laboratories, Inc., 2005) and in a non guideline investigation on female fertility (Andrew et al., 1981; Hardin et al., 1981). Liver changes most probably are related to enzyme induction as has been demonstrated by several authors (Stott et al., 1999; 2003; Elovaara et al., 1985; Toftgard, Nilsen, 1982). This is supported by a proliferation of smooth endoplasmic reticulum (Elovaara et al., 1985). An increase in kidney weight was also found. A detailed histopathological reevaluation of the kidneys of rats exposed for 3 months in the NTP (1992) study revealed a dose-related increase of the severity of CPN in male rats at 750 and 1000 ppm, but not in females. In addition, an increase in incidence of hyaline droplets was observed at these dose levels in male rats (Hard, 2002). CPN in rats has no specific relevance for humans and liver enzyme induction is not to be considered as a toxicological relevant effect for human risk assessment. The NOEC for all of these findings is 100 ppm and the NOAEC in the most relevant 90 day NTP study (1992) for extrapolation to humans is 1000 ppm (4.74 mg/l).

Similar organ-related systemic toxicity was observed after 2 years of exposure (NTP, 1999). In rats a detailed histopathological reexamination again revealed chronic progressive nephropathy at 750 ppm markedly in male and modestly in female rats in all treated groups. There was a high incidence of severe CPN with kidney alterations that may lead to renal failure. Cystic degeneration of the liver was increased in 750 ppm males, but the biological significance in the absence of other hepatotoxic changes is unclear. In mice there was a spectrum of non neoplastic liver changes for both males and females. Histopathological findings related to lung tumor formation will be reported in the section on carcinogenicity. A detailed reevaluation of the lung and liver slices from mice basically confirmed the original NTP findings (Brown, 2000). Hyperplastic changes were also reported for the thyroid in males and females at 750 ppm and for the pituitary in females starting at 250 ppm. In summary, as chronic progressive nephropathy has no toxicological relevance for human risk assessment, the NOAEC for rats was 250 ppm in males and 750 ppm in females if small reductions (5-6%) in body weight (males at 250 ppm and females at all exposure levels) for females are not taken into account. In mice the NOAEC was 75 ppm for males and females based on liver, lung, thyroid and pituitary pathology.

 

Organ-specific toxicity (effects on the nervous system)

In experimental animals ethylbenzene exposure induced various effects on the central nervous system:

 

Effects on neurotransmitters

Two studies reported modulation of neuronal transmitters at 2000 ppm (Andersson et al., 1981) or 750 ppm (Romanelli et al., 1986; Mutti and Franchini, 1987; Mutti et al., 1988), respectively, over a few days. Furthermore, at 2000 ppm a decrease of prolactin in serum was observed(Andersson et al., 1981). However, the significance of these effects is unclear.

 

Depressive and narcotic effects

Although depressive or narcotic effects have been observed in humans by aromatic solvents in general, the animal data are less consistent showing transient effects for ethylbenzene only at very high acute exposures.

In a 90 day oral guideline study specifically designed for the detection of neurotoxic effects dose levels up to 500 mg/kg bw/d did not lead to findings indicative of neurotoxicity in rats (Li et al., 2010).

The results of in vitro tests indicate that ethylbenzene may affect the regulatory functions of astrocytes (Naskali et al., 1994;Vaalavirta and Tähti, 1995).

It may be suspected that ethylbenzene could lead to lesions in the central nervous system similar to other organic solvents. But no indications for such morphological alterations of the central nervous system have been reported in other animal experiments including the 2-year bioassay with exposures up to 750 ppm (NTP, 1999).But because of the limited reliability of standard H&E staining for detecting neurological disorders this is not sufficient proof for the absence of minor morphological abnormalities.

 

Ototoxicity

Vyskocil et al.(2008) reviewed the literature for ethylbenzene induced effects on the auditory system. In workers, they found no evidence on either ethylbenzene related hearing losses or ototoxic interaction after combined exposure to ethylbenzene and noise. But given the current evidence from animal studies they recommend that ethylbenzene should be considered also as an ototoxic agent for humans.

Ethylbenzene leads to ototoxicity in rats. Persistent hearing loss in the mid-frequency range was confirmed in a series of auditory tests in rats (sound-evoked electrical responses, otoacoustic emissions, behavioral auditory tests) corresponding to a concentration dependent death of sensory cells (outer hair cells – OHC, especially in the 3rdrow) in the upper basal and middle turns of the cochlea. Outer hair cell death determined by histopathology is the most sensitive endpoint for auditory effects (Cappaert et al., 2000; Gagnaire et al., 2007). For death of outer hair cells 200 ppm was a LOEC and the NOEC was calculated to be 114 ppm (95% confidence limit) (Gagnaire et al., 2007). With increasing ethylbenzene concentrations the other endpoints for ototoxicity become affected, too, and OHC death spreads over the frequency range in the cochlea and from row 3 to row 2 and 1 of the outer hair cells. OHC death as confirmed by histopathology indicates that hearing loss is irreversible. This is substantiated by Cappaert et al. (1999) since hearing loss did not change between 1 and 4 weeks post exposure to 800 ppm over 5 days. On the other hand, electrophysiological investigations did not show any further deterioriation of auditory function when exposure to 400 ppm was prolonged from 4 over 8 up to 13 weeks (Gagnaire et al., 2007). Auditory loss remained at the same level following additional 8 weeks of recovery. This might indicate that OHC loss at this concentration was already complete at week 4 and was irreversible.

Comparable ototoxic effects were observed in rats that were repeatedly receiving etyhlbenzene via the oral route (Gagnaire and Langlais, 2005), which is in line with the experience gained from other aromatic solvents (e.g., styrene). Since only one concentration of ethylbenzene was examined, (900 mg/kg, 5 d/wk/2 weeks), a N/LOAEL could not be estimated. Other oral tests, also those following OECD standard study designs, did not include specific auditory examinations. By comparison between behavioral and electrophysiological methods Cappaert et al. (1999)concluded that ethylbenzene primarily exerts its effects on the peripheral part of the auditory system.

In a comparative study with different aromatic solvents given orally by gavage ethylbenzene belonged to the most potent ototoxicants together with styrene by means of OHC death. The potency was higher than that of toluene and p-xylene. Gagnaire et al. (2007) found that ototoxicity in mixed exposure to xylenes and ethylbenzene mainly depends on the concentration of ethylbenzene and auditory loss was higher in combined exposure than after single exposure to same ethylbenzene concentrations.

Experiments with combined exposure to noise and ethylbenzene indicated to a synergistic effect of both (Cappaert et al., 2001). In the other experiment of Fechter et al. (2007) a very high exposure to the solvent mixture used (660 ppm ethylbenzene + 400 ppm toluene) (without noise) did not result in adverse hearing effects in contrast to all the other studies with ethylbenzene, while hearing loss was reported for the combination of this solvent mixture with noise exposure of 93-95 dB. Actually no explanation for this unexpected result could be given. As synergistic effects were also known from mixed exposure to other aromatic solvents, the outcome of this study appears questionable.

By comparison with rats, guinea pigs are very insensitive against ethylbenzene-induced ototoxicity (Cappaert et al., 2002). The low sensitivity of guinea pigs was attributed to the low blood concentration of ethylbenzene in comparison to that of rats.

The critical question is whether ototoxicity of ethylbenzene in humans is best comparable to that in the rat rather to that seen in the guinea pig. Until the exact position of humans within the inter-species ranking of susceptibility to ethylbenzene-induced ototoxicity is actually known, data from the rat are to be taken as relevant for humans. This assumption is supported by a number of reports on hearing deficits in humans occupational exposed to organic solvents or from people after solvent abuse (for review cf Risk Assessment Reports on toluene and styrene).

Ethylbenzene belongs to the most ototoxic aromatic solvents, its potency being comparable to that of styrene but higher than those of toluene or p-xylene. Comparing rat data on the lowest effective concentrations for ethylbenzene and toluene, the risk of ototoxicity is expected to be higher for ethylbenzene.

Irreversible damage of auditory function and of sensory cells of the cochlea is a serious health damage. After 13 weeks of exposure minimal effects were still observed at 200 ppm (0.88 mg/l) and the NOEC was extrapolated to 114 ppm (0.5 mg/l). Thus, the classification limit for R48/20 (0.25 mg/l) is formally not attained. Nevertheless, such a classification is proposed taking into account that the experimental ototoxicity of ethylbenzene is comparable to that of styrene and less than that of toluene and that both of these chemicals have been assigned R48/20.

 

 

With regard to DNEL derivation three aspects have to be critically evaluated:

 

1.    Influence of exposure duration on NOAEC

 

2.    Sensitivity of histopathological and (electro)physiological effects

 

3.    Anatomical/histological differences of target tissues between rats and humans.

 

1. Influence of exposure duration on NOAEC

Ototoxicity leading to an increase of audiometric threshold and specific histopathological alterations with destruction of the outer hair cells in the cochlea (predominantly in row 3) is not a specific effect only of ethylbenzene, but has been described for a variety of other aromatic solvents. In a comparative oral study such histopathological effects were observed for alpha-methylstyrene, trans-beta-methylstyrene, toluene, p-xylene, n‑propyl­benzene, styrene, and allylbenzene apart from ethylbenzene after oral application (Gagnaire and Langlais, 2005). After inhalation exposure qualitatively similar hearing impairments were found by reflex modification audiometry for styrene, toluene and mixed xylenes (Crofton et al., 1994). The inhalation of styrene and toluene again led to similar electrophysiological and histopathological findings (Loquet et al., 1999). Permanent hearing loss as determined by behavioral and electrophysiological methods was also described after inhalation exposure to toluene (Pryor et al., 1984), mixed xylenes, and styrene (Pryor et al., 1987).

The weight of evidence obtained from experiments with styrene and other aromatic solvents indicates that ototoxicity is exerted by the lipophilic aromatic parent chemical rather than by a metabolite:

-  Similar ototoxic effects were found for a range of lipophilic organic solvents. As all of these chemicals are biotransformed to different metabolites, indirect evidence is thereby obtained that the parent chemicals themselves most probably lead to ototoxicity

- When rats were exposed to toluene, pre-treatment with phenobarbital prior to toluene inhalation resulted in a marked reduction in blood toluene levels and prevented hearing loss (Pryor et al., 1991) showing induction of metabolism reduces ototoxicity.

-  A comparative study of ototoxicity in rats and guinea pigs showed much higher blood levels of styrene in the rat (i.e. 23 µg/g) leading to ototoxicity, while in the guinea pig the blood levels were about 4-fold lower (i.e. about 5 µg/g) with no evidence of ototoxicity (Lataye et al., 2003). This finding compares well with that of Cappaert et al. (2002) attributing the low sensitivity of guinea pigs for ototoxicity against ethylbenzene to the low blood concentration of the parent chemical in comparison to rats.

-  Inhalation to toluene induced auditory impairment as determined by auditory brainstem response in rats but not in chinchillas under similar exposure conditions. This was explained by species differences in hepatic microsomal metabolism of toluene being highest in chinchillas (Davis et al., 2002)

- No changes in auditory brainstem response were seen in rats treated with phenylglyoxylic acid, a major metabolite of styrene and ethylbenzene, in drinking water over 3 months at doses up to 5000 mg/l, corresponding to 293 mg/kg bw/d (Ladefoged et al., 1998)

- The higher sensitivity of young rats as compared to old ones for ototoxicity was explained by Lataye et al. (2004) by age related increases in styrene metabolism

In conclusion, the overall evidence indicates that the mechanism of aromatic solvent induced ototoxicity is very similar for these chemicals, notwithstanding different potencies, and driven by the parent compound and not by their hydrophilic metabolites. A read across approach is therefore indicated to fill specific data gaps for the risk assessment of ethylbenzene

There is strong evidence that the maximum of hearing impairment is already reached after one to a few weeks of exposure and that ototoxicity does not increase with prolongation of the exposure period. Campo P. (2001) exposed rats to 1000 ppm styrene (6h/d, 5d/week) for 1, 2, 3, or 4 consecutive weeks. Permanent hearing loss was observed using electrophysiological examinations 6 weeks after the end of each exposure period. An exposure duration of 1 week was enough to obtain the maximal hearing deficit without any further increase by prolongation of exposure. This was confirmed by histopathology of the cochlea carried out 6 weeks post-exposure: hair cell loss was virtually the same after an exposure duration of 1 or 3 weeks.

The conclusion that maximal hearing impairment is already reached after a few weeks of exposure can also be indirectly inferred by comparison of the results of The Dow Chemical Company (1992) and Maekitie et al. (2003). The Dow Chemical Company (1992) exposed male Fisher-344 rats (6h/d, 5d/week) to 0, 50, 200 and 800 ppm styrene over 13 weeks. Effects on the auditory system were determined by electrophysiology and histopathology of the cochlea a few days after the end of exposure. Clear effects were noted at an exposure of 800 ppm and the NOAEC was 200 ppm. This compares well to the NOAEC of 300 ppm determined in male Wistar rats by Maekitie et al. (2003)after an exposure to 0, 100, 300 and 600 ppm (12h/d, 5d/week) over only 4 weeks.

The conclusion that solvent induced ototoxicity already appears after a relatively short exposure duration and that continued exposure does not enhance the intensity of the response or reduces the NOAEC is also supported by studies with toluene. In a review Johnson and Nylen (1995) described for toluene that a time-integrated concentration between 12000 and 16000 ppm x h/d over 3 days was sufficient to cause loss of auditory sensitivity, while 6000 ppm x h/d over 18 month failed to produce ototoxic effects. Pryor et al. (1984) showed that a 2 week exposure to 1000 ppm toluene (14h/d, 7d/week) produced ototoxicity in Fischer rats while no effects were seen for a similar exposure schedule over 16 weeks to 400 and 700 ppm. Even 3 days at 1500 ppm (14h/d) or at 2000 ppm (8h/d) were sufficient to induce hearing loss. Nylen et al. (1987) demonstrated that the ototoxicity of toluene is governed by intensity of exposure and not by duration or cumulative exposure. Impairment of auditory function as determined electrophysiologically was found in male Sprague Dawley rats after exposure to 1000 ppm (22h/d, 7d/week) over 1 month corresponding to 0.62 million ppm x h. On the other hand, no effects were found after a cumulative exposure of 2.46 million ppm x h achieved by a shorter weekly exposure regime to 1000 ppm (6h/d, 5d/week) over 19 month.

Direct evidence that maximal hearing impairment will already be reached after a few weeks of exposure is also obtained by the data of Gagnaire et al. (2007) with ethylbenzene itself. Hearing deficits recorded electrophysiologically 4, 8, and 13 weeks after start of exposure (6h/d, 6d/week) and after additional 8 weeks of exposure free observation were all essentially the same: there was no increase of hearing loss over time and the NOAEC of 200 ppm did not change. The same applied for the two different mixed xylenes tested, containing either 10% or 20% of ethylbenzene.

In summary, an exacerbation of hearing deficits is not to be expected by prolongation of exposure beyond a few weeks or from 13 weeks to life time.

 

2. Sensitivity of histopathological and (electro)physiological effects

 

In the overall assessment it has also be taken into consideration that the NOAEC for ototoxicity as defined by auditory dysfunction is clearly higher than that defined by histopathological effects on the outer hair cells of the cochlea. Numerous investigations have shown that audiometric hearing deficits occur at higher exposure concentrations than (small) losses of hair cells in the outer row 3 of the cochlea. This was described by Gagnaire et al. (2007) for ethylbenzene and two different mixed xylenes. The NOAECs/LOAECs were found at the following concentration: ethylbenzene: auditory threshold - NOAEC 200 ppm; histopathology - LOAEC 200 ppm; mixed xylene (1): auditory threshold - NOAEC 500 ppm; histopathology - LOAEC 250 ppm; mixed xylene (2): auditory threshold - NOAEC 1000 ppm; histopathology - NOAEC 500 ppm.

Similar findings were reported for styrene by Loquet et al.(1999), Lataye et al. (2005), Pouyatos et al. (2002), or Lataye et al.(2001).In this respect the findings of Lataye et al. (2001) are important. After exposure to styrene at 1000 and 1500 ppm there was a clear cell loss in the medium spiral ganglion, but not at 750 ppm corresponding better to the dose response relationship of hearing loss (by electrophysiology) than the outer hair cell loss in the cochlea. Thus, the functional results are in better agreement with the morphological results at the level of the spiral ganglion than with those obtained at the level of the cochlea.

The inconsistency in the dose response relationship of the degenerative process of hair cells along the cochlea on the one hand with electrophysiologically measured changes of the auditory threshold on the other hand can be explained by the anatomical situation. To begin with, the auditory message comes mainly from the inner hair cells which are much less prone to damage by organic solvents than the outer hair cells, especially those in row 3. Secondly, the functional results of hearing loss are in better agreement with histopathological changes in the spiral ganglion rather than with those at the level of the cochlea. In this respect Lataye et al. (2001) point to an investigation of Prosen et al. (1990) who showed that a massive loss of outer hair cells (up to 50% in the 30% apical region of the cochlea) did not cause hearing loss at any frequency. They conclude that the functional role played by the apical outer hair cells appears to be secondary in the rat. Furthermore after oral exposure of rats to styrene Chen et al. (2008) found that a loss of outer hair cells of <33% did not result in a significant shift of hearing threshold.

Thus, taking very small losses of outer hair cells in row 3 as the decisive endpoint is a very conservative approach for risk assessment based upon hearing impairment.

 

3. Anatomical/histological differences of target tissues between rats and humans.

 

The mechanism was clarified for styrene by Campo P. (1999), Loquet et al. (1999), and Campo P. et al. (2001): Styrene, transported by blood coming from the stria vascularis or the spiral prominence, diffuses through the outer sulcus to reach the lipid-rich Hensen’s cells. These cells are in close connection with the Deiters cells that are directly located under the outer hair cells. Thus, the target cells are reached by diffusion of styrene. This explains why hair cells are lost in a sequence from the outer row 3 to row 1 as diffusion continuous and why the inner hair cell are the least sensitive. Specifically, the involvement of Hensen’s and Deiters cells was supported by histopathology of semi-thick sections of the cochlea (Campo et al., 2001) and Chen et al. (2007) demonstrated that Deiters cells are an especially vulnerable target of styrene. The latter authors further reported that the styrene concentrations in the cochlea varied along with the basilar membrane with the lowest level in the basal turn being consistent with the lowest styrene induced threshold shift and hair cell loss in this region.

The mode of action for hearing loss induced by styrene is well-defined and the weight of evidence indicates that it also applies to other aromatic solvents including ethylbenzene. As shown above ototoxicity is governed by direct impact of the (unmetabolised) parent compound on the hair cells of the cochlea. Passage to these target cells occurs via diffusion of the solvent from blood through Hensen’s and Deiters cells. Taking into account the identical target cells (outer hair cells, Hensen’s cells, Deiters cells) and the identical structure of the target organ (cochlea) and even its blood supply, the toxicodynamics for ototoxicity caused by aromatic solvents are very similar between species. This also applies to the comparison between humans and rats if as a conservative assumption humans are considered to be of comparable sensitivity as rats.

The following information is taken into account for any hazard/risk assessment:

 

The repeated exposure toxicity of ethylbenzene has been evaluated in animals in subchronic and chronic inhalation studies, subchronic oral toxicity studies and numerous specialized investigations. Overall, ethylbenzene poses a moderate repeated exposure toxicity hazard with consistent targeted effects to the liver, kidney and hearing.

 

Repeated dose toxicity (Oral):

 

Changes in hematology, indicative of a mild regenerative anemia, and clinical chemistry parameters, indicative of hepatic microsomal enzyme induction, decreases in prothrombin time, mild alimentary effects and kidney (males only) and liver pathology were observed in rats that received gavage doses of > 250 mg/kg bwt/day ethylbenzene for 90 days.

 

Repeated dose toxicity (Dermal):

 

No data

 

Repeated dose toxicity (Inhalation):

90 day inhalation study in rats concluded ototoxic effects at> 200 ppm. The study NOAEC was calculated to be 114 ppm (500 mg/m3).

 

Value used for CSA (via oral route - systemic effects):

(NOAEL: 75 mg/kg bw/day)

Value used for CSA (inhalation- systemic effects):

(NOAEC: 500 mg/m³)

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Results from oral repeated dose studies conducted on mixed xylene in the rat indicate a sub-chronic LOAEL of 750 mg/kg bw/d (Condie et al., 1988) and a chronic NOAEL of 250 mg/kg bw/d (NTP, 1986). A sub-chronic NOAEL of 75 mg/kg bw/d has been reported for ethyl benzene (Mellert et al., 2007). The chronic NOAEL of 250 mg/kg bw/d was used in this assessment.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A NOAEC of 3515 mg/m³ was reported by Carpenter et al. (1975) for generalised systemic effects in male rats and male dogs. Other studies have shown that some xylene isomers adversely affect hearing in the rat, with a sub-chronic NOAEC of 1950 mg/m³ reported for p-xylene; the NOAEC for ototoxicity of m-xylene and o-xylene was greater than 7810 mg/m³ (Gagnaire et al., 2001). The ototoxicity of mixed xylene appears to be dependent upon composition (Gagnaire et al., 2007), with a sub-chronic LOAEC of 1080 mg/m3 reported for one sample while another had a NOAEC of 2170 mg/m³. A LOAEC of 868 mg/m³ was reported for the ototoxicity of ethyl benzene in the rat (Gagnaire et al, 2007), equivalent to an extrapolated NOAEC of 500 mg/m³.

Repeated dose toxicity: via oral route - systemic effects (target organ) other: all gross lesions and masses

Repeated dose toxicity: inhalation - systemic effects (target organ) other: all gross lesions and masses

Substance selection for risk characterization:

The approach developed by the LOA Exposure Working Group on the selection of constituents for use in human health exposure assessments was implemented for the Reaction Mass of Ethylbenzene and m-Xylene. The primary constituents used are ethylbenzene and xylenes. A full description of this approach is available in Section 13 ("Selection of constituents for HH exposure" in section 13.2).

Justification for classification or non-classification

No classification of ethylbenzene and xylene reaction masses is warranted when ethylbenzene content is <10%

Where ethylbenzene is >=10%, mixed xylene streams warrant classification under as STOT-RE Cat 2 H373 under CLP [see Specific Investigations: other studies (ototoxicity)].

Ethylbenzene

According to EU CLP (Regulation (EC) No. 1272/2008), the NOAEL of 75 mg/kg bw/d falls within the range of 10-100 mg/kg bw/d for STOT-RE Cat. 2 classification. But the severity of the effects observed at the next dose level (250 mg/kg bw/d) does not justify classification according to section 3.9.2.8. Thus, a classification according to EU CLP (Regulation (EC) No. 1272/2008) is not warranted by this study.

 

Ototoxicity was not investigated by the above mentioned oral gavage study. Gagnaire and Langlais (2005) observed ototoxicity in rats at a single high dose level of 900 mg/kg bw/d over two weeks. This study is not sufficient for classification. But the inhalative NOAEC (500 mg/m³) may be extrapolated to an oral dose:

oral absorption rat: 84%

inhalative absorption rat: 45%

respiratory volume rat (6h): 0.29 m³/kg.

Thus, the NOAEC of 500 mg/m³ corresponds to

           500 x 0.29 x 0.45 : 0.84 = 78 mg/kg bw/d.

 

According to EU CLP (Regulation (EC) No. 1272/2008) Cat 2 is justified: H 373: May cause damage to the auditory system by prolonged or repeated oral exposure.

 

Dermal: No studies with repeated dermal exposure are available. Taking into account the dermal absorption of 4% (see DNEL derivation) a classification according to EU CLP (Regulation (EC) No. 1272/2008) is not warranted based on the same calculation as above:

           500 x 0.29 x 0.45 : 0.04 = 1630 mg/kg bw/d.

 

Inhalation: Irreversible damage of auditory function and of sensory cells of the cochlea is a serious health concern. After 13 weeks of exposure minimal effects were still observed at 200 ppm (0.88 mg/l) and the NOEC was extrapolated to 114 ppm (0.5 mg/l).

According to EU CLP (Regulation (EC) No. 1272/2008) and UN GHS the NOAEC of 0.5 mg/l is within the classification limit of 0.2-1 mg/l/6h/d for STOT-RE Cat. 2 leading to a classification with H 373: causes damage to the auditory system through prolonged or repeated inhalative exposure.

 

In summary, the following classifications are justified for repeated dose toxicity:EU CLP (Regulation (EC) No. 1272/2008): A harmonized classification exists for ethylbenzene, however this hazard category is not included. Recommendation is to add: STOT-RE Cat 2, H 373: causes damage to the auditory system through prolonged or repeated oral or inhalative exposure and also recommended for UN GHS. After reviewing this information, the Committee for Risk Assessment concluded that ethylbenzene had a potential to cause damage to organs (hearing) through prolonged or repeated exposure (RAC, 2012a).

RAC (2012) Opinion proposing harmonised classification and labelling at EU level of ethylbenzene. ECHA/RAC/CLH-O-0000001542-81-03/F. Committee for Risk Assessment, adopted 5 June 2012