reaction mass of ethylbenzene and 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.

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

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key 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

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 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:

dog

Strain:

Beagle

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, 0.77, 2.0 or 3.5 mg/L
Basis:
analytical conc.

No. of animals per sex per dose:

4 males

Control animals:

yes

Details on study design:

Groups of 4 male beagle dogs were assigned randomly to each of three graded levels of mixed xylenes or to the solvent- free air-control. They were exposed for 6h/day, 5 days/week for 13 weeks (65 days of exposure) and then terminated. .

Positive control:

No

Observations and examinations performed and frequency:

Measurements included body weight change, food consumption was followed 1 day of each week, initial and terminal electrocardiograms were made, and urine analyses were performed. 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), bilirubin and blood glucose.

Sacrifice and pathology:

Liver and kidney weights were recorded 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 section. Bifurcation of the trachea, pharynx, tonsil, nasal mucosa were also taken. 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 section were examined.

Statistics:

No details

Details on results:

There were no treatment-related differences for any of the endpoints evaluated.

Dose descriptor:

NOAEC

Effect level:

>= 810 ppm

Sex:

male

Basis for effect level:

other: 3515 mg/m3

Critical effects observed:

not specified

Conclusions:

The NOAEC of mixed xylenes for male dogs exposed 6h/day for 5 days in each of 13 weeks was >=3515 mg/m3.

Executive summary:

Male dogs 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3 515 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Several studies are available that report the systemic toxicity and ototoxicty of xylene isomers (including mixed xylenes) in the rat following inhalation exposure.

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.

No studies are available for the individual xylene isomers.

In an OECD Guideline 90-day oral study ethyl benzene was gavage dosed at 0, 75, 250 and 750 mg/kg in corn oil (Mellert et al. 2007). The NOAEL for this study was 75 mg/kg/day based on changes in haematology indicative of a mild regenerative anaemia and changes in clinical chemistry parameters. There was also an increase in liver weights with centrilobular hepatocellular hypertrophy indicative of hepatic microsomal enzyme induction.

Repeated dose toxicity: dermal

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

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.

Ethyl benzene has also been the subject of a recent study to assess potential ototoxicity (Gagnaire et al, 2007b). Groups of male rats were exposed to 0, 200, 400, 600 and 800 ppm ethyl benzene for 6 h/day, 6 d/wk over 13 weeks with a recovery period of 8 weeks. There was no adverse effect on body weight at any of the dose levels.

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).

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

Justification for classification or non-classification

No classification of mixed xylenes is warranted when ethylbenzene content is <10%

Where ethylbenzene is >=10%, mixed xylene streams warrant classification under DSD or CLP as Xn, R48/20 with the equivalent classification as STOT-RE Cat 2 H373 under CLP [see Specific Investigations: other studies (ototoxicity)].