Registration Dossier

Administrative data

Description of key information

By read-across to 1,2,4- trimethylbenzene, it is concluded that 1,3,5-trimethylbenzene has low systemic toxicity in rats when administered by inhalational or oral routes. No significant treatment-related effects were observed up to oral doses of 1,3,5-trimethylbenzene of 600 mg/kg/day or inhaled concentrations of up to 1800 mg/m3.   Mild pulmonary lesions occurred, possibly as a result of respiratory irritation.

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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
nerobehavioural assessment not carried out
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley CD (Crl:CD®BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA.
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: random sample of 10/sex, at approximately 6 weeks of age, weighed 133-158g
- Housing: Individually in stainless steel wire mesh cages (24.0 x 17.8 x 17.6 cm)
- Diet: Purina Certified Chow 5002 (PMI Feeds Inc., St. Louis, MO, USA) ad libitum (except for approximately 16-20 hours prior to scheduled blood collection and necropsy).
- Water: Reverse osmosis-purified water ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23°C
- Humidity: 21-79%
- Air changes (per hr): Not reported
- Photoperiod: 12hrs dark / 12 hrs light

IN-LIFE DATES: From: 30 August 1994 To: 27 December 1994
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Stock dosing solutions of test substance in corn oil were prepared weekly during the study. The stock solutions were stored at room temperature and dosing aliquots were dispensed daily.

VEHICLE
- Concentration in vehicle: 10, 40 and 120 mg/mL for 50, 200 and 600 mg/kg/day dose groups, respectively.
- Amount of vehicle (if gavage): All animals were dosed at a constant dosing volume of 5 mL/kg of body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations prepared for the first two weeks of dosing were analyzed for homogeneity and concentration at all dose levels. The low and high dose formulations prepared for the following three weeks of dosing were analyzed to confirm their concentrationfollowed by biweekly concentration analysis of the low and high dose formulations for the remainder of the 13 week dosing period. Stability analyses were performed on low and high concentration solutions prepared for the first week of dosing. Stability was determined at 24 hours, I and 2 weeks after formulation.
Similar purity and composition percentages were found upon completion of the treatment phase of the study, indicating the test substance was stable during the period of its use. Low and high concentration solutions stored at room temperature were stable for at least 14 days. All preparations were found to be homogeneous. Overall mean analyzed concentrations were 98.9%, 96.9% and 100.8% of the target concentration for the 10, 40 and 120 mg/mL dosing solutions respectively. All individual sample concentrations were within 10% of the target concentrations. No test substance was detected in any vehicle control sample.
Duration of treatment / exposure:
90-91 days
Frequency of treatment:
once daily, 5 days per week (a total of 65-66 doses)
Remarks:
Doses / Concentrations:
0, 50, 200 & 600 mg/kg bw/day
Basis:
other: nominal in corn oil
No. of animals per sex per dose:
10 (for controls , low and mid-dose groups), 20 for high dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 29 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once/day for morbidity and mortality during acclimatisation, once ot twice daily therafter.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to treatment and weekly thereafter
BODY WEIGHT: Yes
- Time schedule: Immediately prior to first dose, weekly thereafter and Fasted weight immediately prior to termination.
FOOD CONSUMPTION:
- Time schedule: weekly
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation and during last week of dosing.
- Dose groups that were examined: All
HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 30 days dosing and at termination
- Anaesthetic used for blood collection: Yes :70% CO2:30%O2 (prior to 30 day samples collected from the orbital sinus); sodium pentobarbitol (termination samples collected from abdominal aorta)
- Animals fasted: Yes
- How many animals: All
- Parameters examined: total erythrocyte count (RBC), haemoglobin (HGB), mean corpuscular volume (MCV), total white blood cell count (WBC), differential white blood cell count and platelet count (PLT). The following values were calculated from the data globulin (GLOB), albuminlglobulin ratio (A/G Ratio), haematocrit (HCT), mean corpuscular hemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 30 days dosing and at termination
- Anaesthetic used for blood collection: Yes :70% CO2:30%O2 (prior to 30 day samples collected from the orbital sinus); sodium pentobarbitol (termination samples collected from abdominal aorta)
- Animals fasted: Yes
- How many animals: All
- Parameters examined: glucose (GLU), gamma glutamyl transpeptidase (GGT), alaaine aminotransferase (ALT), aspartate aminot:aasferase (AST), alkaline phosphatase (ALK P), creatine kiaase (CK), urea nitrogen (BUN), creatinine (CREA), sodium (Na), potassium (K), calcium (Ca), chloride (Cl), phosphorus (PHOS), total protein (T PRO), albumin (ALB), total bilirubin (MIL) and cholesterol (CHOL).
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Full necropsy of all animals
ORGAN WEIGHTS: Yes. adrenal glands, brain, gonads, kidneys, liver and lungs of all rats were weighed at necropsy and the organ weights relative to body weights (fasted) were calculated.
HISTOPATHOLOGY: Yes. Following tissues from all animals collected and fixed: adrenals, brain, eyes, exorbital lacrimal glands, esophagus, gonads, heart, duodenum, jejunum, ileum, caecum, colon, rectum, kidneys, liver, lungs, mesenteric lymph nodes, pancreas, aorta, pituitary, prostate, epididymides and seminal vesicles, salivary glands, sciatic nerve, skeletal muscle, skin (with mammary gland), spinal cord (cervical, mid-thoracic and lumbar), spleen, sternum (bone and marrow), stomach, thymus, thyroid (with parathyroids), trachea, urinary bladder, uterus, Zymbal glands, nasal turbinate, femur, gross lesions and ear. The complete set of collected tissues (except skin and mammary gland, ear, skeletal muscle, femur, spinal cord, eyes, exorbital lacrimal glands, Zymbal glands, epididymides, prostate and seminal vesicles) from all vehicle control and 600 mg/kglday dose group animals sacrificed at the end of the 90-day dosing period was processed and examined by light microscopy. In addition, the lungs from the 50 and 200 mg/kg/day dose group animals were also processed and examined microscopically, along with gross lesions observed in all animals. The remaining tissues
from the 50 and 200 mg/kg/day dose group animals were collected, but were not processed and examined microscopically. After the 28-day recovery period, a complete set of tissues was collected from all of the recovery 600 mg/kg/day animals; however, no tissues from any of these animals were examined microscopically.

Statistics:
Data collected electronically (i.e., body weights, body weight gains, food consumption, haematology) were analyzed using the statistical capabilities provided by the software, which consisted of analysis of variance (ANOVA) followed by Dunnett's multiple range comparison test. Other data (i.e, clinical chemistry, absolute and relative organ weights) were analyzed by ANOVA followed by Dunnett's multiple-range comparison using SYSTAT software (SYSTAT, SPSS, Inc, Chicago, IL, version 5.0). Analyses were performed on the two sexes independently. Analyses consisted of comparing the treated groups with the vehicle control at the 30-day interim and dose termination time points. Since no vehicle control animals were retained for recovery observation, statistical comparison of recovery animal parameters could not be performed. A minimum significance level of p ≥ 0.05 was used in all comparisons.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No test substance-related deaths occurred during the study. Clinical signs observed predominantly in the high dose animals consisted of discolored inguinal fur, wet inguinal fur and salivation. All treatment related effects appeared to be reversible, since these effects were no longer apparent in the recovery animals at the end of the 28-day recovery period.

BODY WEIGHT AND WEIGHT GAIN: No statistically significant effects on body weight, body weight gain (except for a significant decrease in high dose males after 5 weeks of treatment) were observed; however, cumulative body weight gain was decreased approximately 11% in the high dose males. All treatment related effects appeared to be reversible, since these effects were no longer apparent in the recovery animals at the end of the 28-day recovery period.

FOOD CONSUMPTION: No statistically significant effects.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related ophthalmic lesions were observed following the 90-day treatment period.

HAEMATOLOGY: No treatment-related changes.

CLINICAL CHEMISTRY: Increased phosphorus levels in high dose males and females. All treatment related effects appeared to be reversible, since these effects were no longer apparent in the recovery animals at the end of the 28-day recovery period.

ORGAN WEIGHTS: Absolute liver weight was significantly increased in high dose females and relative liver weights were significantly increased in high dose males and females at treatment termination. Relative kidney weight was also increased in high dose males at treatment termination.

GROSS PATHOLOGY: No treatment-related effects.

HISTOPATHOLOGY: No treatment related microscopic lesions were observed in the liver or kidneys of the high dose animals, or in any other tissue or organ of any animal.
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Increases in phosphorus concentrations at 600 mg/kg bw/day not evident after recovery period and not associated with any histopathological findings. Increased liver and kidney weights at 600 mg/kg/day attributed to a-2u-globulin related nephropathy.
Critical effects observed:
not specified
Conclusions:
The NOAEL following 90 days oral gavage of 1,3,5-trimethylbenzene to rats is concluded to be 600 mg/kg bw/day.
Executive summary:

1,3,5-Trimethylbenzene was administered at dose levels of 0, 50, 200 and 600 mg/kg/day to 10 male and 10 female Sprague Dawley CD rats for 90 days. An additional 10 rats/sexwere dosed at 600 mg/kg/day group on the same dosing schedule but were subsequently retained without treatment for an additional 28 days to evaluate recovery from any toxic effects.

 

In the 600 mg/kg/day group an increase in serum phosphorus levels (males and females), liver weight increase (absolute and relative weight increase in females; relative increase only in males) and kidney weight increase in males were reported. In the recovery group, no apparent effects were reported at the end of the 28-day recovery period. 

 

Based on the recovery group data, the effects reported at the highest dose were considered reversible and were attributed to adaptive response (enzyme induction) to the test substance. In the liver this could be attributed to microsomal enzymes induction while in the male rat kidney, to a-2µ-globulin related nephropathy.  With respect to the elevated serum phosphorus levels observed, these levels werenot statistically different at the end of the 28 -day recovery period and no associated histopathological effects were reported whichcould be correlated with the increased serum phosphorus levels.  The NOAEL for this study can therefore be concluded to be 600 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available data provide information that is adequate for the purpose of hazard assessment

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Non GLP, near guideline study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
No ophthalmology, no detailed clinical observations, bodyweights weekly only for initial 4 weeks, no food consumption and efficiency, haematology only for controls and high dose groups, no clinical chemistry at 3 months, limited number of organs weighed.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall) Breeding Unit
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 150-300 g
- Fasting period before study: No
- Housing: Singly housed in the exposure chambers in hanging aluminum cages with stainless steel mesh bases during and between exposures. Recovery animals (after 12 months exposure) transferred to an adjacent animal room.
- Diet: ad libitum except during exposure
- Water: ad libitum

ENVIRONMENTAL CONDITIONS: Temperature and relative humidity constantly monitored (no results reported); 6 hours light / 18 hour dark cycle, air flow 3-6 m3/min.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with doors fitted with glass windows and a volume of at least 8 m3
- Method of holding animals in test chamber: individually housed in a hanging aluminium cage within the chamber.
- Source and rate of air: Laboratory air at a rate of 3-6 m3/min.
- Method of conditioning air: Filtered.
- Temperature, humidity, pressure in air chamber: Not reported.
- Atmosphere generation: Test atmospheres were generated by completely evaporating the solvent into part of the ventilating air entering each chamber using micrometering pumps and vapourizers.

TEST ATMOSPHERE
- Brief description of analytical method used: Total hydrocarbon analyzer for 10 minutes at intervals of 40 minutes throughout the exposure periods. Each test atmosphere was also analyzed consecutively for a period of two hours during each exposure period using a gas chromatograph fitted with a flame ionization detector.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No significant change in test substance composition over the period of the study. Atmospheric generation did not significantly change the composition of the solvent blend. The actual atmospheric concentrations expressed as the overall means of the daily atmosphere analyses were close to the
nominal concentrations Actual concentrations were 0, 470±29, 970±70 and 1830±130 mg/m3 for target concentrations of 0, 450, 900 and 1800 m3 respectively.
Duration of treatment / exposure:
12 months exposure
Frequency of treatment:
6h/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 450, 900, 1800 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 470±29, 970±70, 1830±130 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
50. 10/sex/group killed after 6 months exposure, 25/sex/group killed after 12 months exposure, 15/sex/group (recovery) killed 4 months after cessation of exposure.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on results from an earlier 13 week inhalation study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure, weekly for initial 4 weeks and monthly thereafter.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Schedule / numbers: weeks 1, 2, 3, 4, 6, 8, 12, 20, 24, 28 and 32 (tail vein blood) from 10/sex controls and high dose groups; 6 months, 12 months and end of recovery period (cardiac blood) from 10/sex/group.
- Parameters examined: Erythrocyte count (RBC), mean cell volume (MCV), haemoglobin concentration (Hb), leucocyte count (WBC), mean corpuscular hemoglobin (MCHC), haematocrit (Hct). Reticulocytes, differential leukocytes, prothrombin time, kaolincephalin time and erythrocyte osmotic fragility (Fragilograph) were only measured using the cardiac blood samples.

CLINICAL CHEMISTRY: Yes
- Schedule / numbers: 6 months, 12 months and end of recovery period (cardiac and tail vein blood) from 10/sex/group.
- Parameters examined: Total protein, urea nitrogen, alkaline phosphatase (AP), chloride, bilirubin, calcium, inorganic phosphate, uric acid, sodium, potassium, alanine amino transferase (ALT), aspartate amino transferase (AST), glucose and protein electrophoresis. (The six-month bloods were limited to measurement of protein, urea, AP, ALT, AST, chloride, sodium, potassium and protein electrophoresis).

URINALYSIS: Yes
- Schedule / numbers: prior to exposure, after 3, 6, 9 and 12 months' exposure and three months after exposure ended. Individual samples collected over 4 hours from 12/sex/group.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: Glucose, protein, ketones, bilirubin, blood pigments, pH, nitrite and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - all animals.
ORGAN WEIGHTS: Yes - all animals surviving to the scheduled necropsies (liver, kidneys, spleen, brain, heart and testes).
HISTOPATHOLOGY: Yes - all animals except recovery groups were examined (salivary gland, stomach, heart, brain, spinal cord, pituitary gland, lungs, prostate, seminal vesicles, testes, ovaries, uterus, skeletal muscle, adrenal glands, thyroids with oesophagus and trachea, caecum, spleen, thymus, lymph node, mammary gland, small and large intestine, pancreas, liver, kidneys, urinary bladder, eye and lachrymal glands, nasal cavity, spinal cord, tongue, knee joint and femur, sciatic and posterior tibial nerves and any gross lesion).

Statistics:
Body and organ weights were analyzed by covariance analysis using initial body weight as the covariate. Means were adjusted for initial body weight if a significant covariance relationship was found, otherwise unadjusted means were reported. In order to adjust for differences in terminal body weight, organ weights were also considered with terminal body weight as a covariate. Haematology and clinical chemical parameters were examined using analysis of variance. The significance of any differences between exposed and control group means was tested using the Williams 't'test. But if a monotonic dose response could not be assumed, Dunnett's test was used.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: 2 male and 1 female control and 2 males at 450 mg/m3 died during study. 7 animals were removed from the study during the 12-month exposure period, and a total of 30 rats was removed during the recovery period but none were related to exposure. A range of minor clinical signs was observed. None of the signs were treatment-related except for a possible increase in male "aggression" at the high exposure, when the animals were slightly more difficult to handle. Some "aggression" was also noted in three or four rats during the recovery phase.

BODY WEIGHT AND WEIGHT GAIN: Initial reduction in body weight gain occurred in both male and female rats at the higher exposures (1800 mg/m3 males, weeks 1-4 -2% compared to controls; 900 mg/m3 females, weeks 1-4 -2% and 1800 mg/m3 females, weeks 1-12 -3%). Thereafter, there were no differences between control and experimental animals for the duration of the study.

HAEMATOLOGY: Various statistically significant changes were transiently seen in males up to six months, but were considered not to be biologically significant.

CLINICAL CHEMISTRY: Various statistically significant changes were probably not of biological significance, since they were within the range of values obtained in normal rats of this strain and age and they were not supported by histopathological or any other evidence of organ damage.

ORGAN WEIGHTS: 1800 mg/m3 exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses.

HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathological examination of the animals revealed a variety of changes, none of which could be attributed to exposure.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): Only one tumor was found in the six-month exposed animals: a malignant mammary adenocarcinoma in a high-exposure female. After 12 months' exposure, several tumors were found. Pituitary adenomas in females were most common, several being found in each group. Of the remaining tumors, one high-exposure female had a leiomyoma on the left uterine horn, one low-exposure male had a malignant glioblastoma under the left lobe of the cerebellum, and one high-exposure male had a malignant lymphoma of the spleen. The tumors were mostly of a type frequently found in this strain and age of rat and were considered not to be treatment-related.
Dose descriptor:
NOAEC
Effect level:
1 800 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: There was no significant systemic toxicity following 12 months exposure in this study.
Critical effects observed:
not specified
Conclusions:
Chronic exposure to this high aromatic naphtha (a 50:50 blend of SHELLSOL A and SOLVESSO 100, which contains 9.35% 1,3,5-trimethylbenzene) iwas without systemic toxicity in rats under the conditions of this study.
Executive summary:

Rats were exposed to a 50:50 blend of SHELLSOL A and SOLVESSO 100 at exposure concentrations of 0, 450, 900 and 1800 mg/m3 for 12 months. Initial reduction in body weight gain occurred in both male and female rats at the higher exposures. Various statistically significant haematological changes were transiently seen in males up to six months, but were not considered biologically significant. High exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses. No treatment-related histopathological abnormalities were found. It is concluded that chronic exposure to this high aromatic naphtha is without systemic toxicity in rats under the conditions of this study with a NOAEC of 1800 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 800 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available data provide information that is adequate for the purpose of hazard assessment

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Non GLP, near guideline study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
No ophthalmology, no detailed clinical observations, bodyweights weekly only for initial 4 weeks, no food consumption and efficiency, haematology only for controls and high dose groups, no clinical chemistry at 3 months, limited number of organs weighed.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall) Breeding Unit
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 150-300 g
- Fasting period before study: No
- Housing: Singly housed in the exposure chambers in hanging aluminum cages with stainless steel mesh bases during and between exposures. Recovery animals (after 12 months exposure) transferred to an adjacent animal room.
- Diet: ad libitum except during exposure
- Water: ad libitum

ENVIRONMENTAL CONDITIONS: Temperature and relative humidity constantly monitored (no results reported); 6 hours light / 18 hour dark cycle, air flow 3-6 m3/min.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with doors fitted with glass windows and a volume of at least 8 m3
- Method of holding animals in test chamber: individually housed in a hanging aluminium cage within the chamber.
- Source and rate of air: Laboratory air at a rate of 3-6 m3/min.
- Method of conditioning air: Filtered.
- Temperature, humidity, pressure in air chamber: Not reported.
- Atmosphere generation: Test atmospheres were generated by completely evaporating the solvent into part of the ventilating air entering each chamber using micrometering pumps and vapourizers.

TEST ATMOSPHERE
- Brief description of analytical method used: Total hydrocarbon analyzer for 10 minutes at intervals of 40 minutes throughout the exposure periods. Each test atmosphere was also analyzed consecutively for a period of two hours during each exposure period using a gas chromatograph fitted with a flame ionization detector.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No significant change in test substance composition over the period of the study. Atmospheric generation did not significantly change the composition of the solvent blend. The actual atmospheric concentrations expressed as the overall means of the daily atmosphere analyses were close to the
nominal concentrations Actual concentrations were 0, 470±29, 970±70 and 1830±130 mg/m3 for target concentrations of 0, 450, 900 and 1800 m3 respectively.
Duration of treatment / exposure:
12 months exposure
Frequency of treatment:
6h/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 450, 900, 1800 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 470±29, 970±70, 1830±130 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
50. 10/sex/group killed after 6 months exposure, 25/sex/group killed after 12 months exposure, 15/sex/group (recovery) killed 4 months after cessation of exposure.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on results from an earlier 13 week inhalation study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure, weekly for initial 4 weeks and monthly thereafter.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Schedule / numbers: weeks 1, 2, 3, 4, 6, 8, 12, 20, 24, 28 and 32 (tail vein blood) from 10/sex controls and high dose groups; 6 months, 12 months and end of recovery period (cardiac blood) from 10/sex/group.
- Parameters examined: Erythrocyte count (RBC), mean cell volume (MCV), haemoglobin concentration (Hb), leucocyte count (WBC), mean corpuscular hemoglobin (MCHC), haematocrit (Hct). Reticulocytes, differential leukocytes, prothrombin time, kaolincephalin time and erythrocyte osmotic fragility (Fragilograph) were only measured using the cardiac blood samples.

CLINICAL CHEMISTRY: Yes
- Schedule / numbers: 6 months, 12 months and end of recovery period (cardiac and tail vein blood) from 10/sex/group.
- Parameters examined: Total protein, urea nitrogen, alkaline phosphatase (AP), chloride, bilirubin, calcium, inorganic phosphate, uric acid, sodium, potassium, alanine amino transferase (ALT), aspartate amino transferase (AST), glucose and protein electrophoresis. (The six-month bloods were limited to measurement of protein, urea, AP, ALT, AST, chloride, sodium, potassium and protein electrophoresis).

URINALYSIS: Yes
- Schedule / numbers: prior to exposure, after 3, 6, 9 and 12 months' exposure and three months after exposure ended. Individual samples collected over 4 hours from 12/sex/group.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: Glucose, protein, ketones, bilirubin, blood pigments, pH, nitrite and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - all animals.
ORGAN WEIGHTS: Yes - all animals surviving to the scheduled necropsies (liver, kidneys, spleen, brain, heart and testes).
HISTOPATHOLOGY: Yes - all animals except recovery groups were examined (salivary gland, stomach, heart, brain, spinal cord, pituitary gland, lungs, prostate, seminal vesicles, testes, ovaries, uterus, skeletal muscle, adrenal glands, thyroids with oesophagus and trachea, caecum, spleen, thymus, lymph node, mammary gland, small and large intestine, pancreas, liver, kidneys, urinary bladder, eye and lachrymal glands, nasal cavity, spinal cord, tongue, knee joint and femur, sciatic and posterior tibial nerves and any gross lesion).

Statistics:
Body and organ weights were analyzed by covariance analysis using initial body weight as the covariate. Means were adjusted for initial body weight if a significant covariance relationship was found, otherwise unadjusted means were reported. In order to adjust for differences in terminal body weight, organ weights were also considered with terminal body weight as a covariate. Haematology and clinical chemical parameters were examined using analysis of variance. The significance of any differences between exposed and control group means was tested using the Williams 't'test. But if a monotonic dose response could not be assumed, Dunnett's test was used.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: 2 male and 1 female control and 2 males at 450 mg/m3 died during study. 7 animals were removed from the study during the 12-month exposure period, and a total of 30 rats was removed during the recovery period but none were related to exposure. A range of minor clinical signs was observed. None of the signs were treatment-related except for a possible increase in male "aggression" at the high exposure, when the animals were slightly more difficult to handle. Some "aggression" was also noted in three or four rats during the recovery phase.

BODY WEIGHT AND WEIGHT GAIN: Initial reduction in body weight gain occurred in both male and female rats at the higher exposures (1800 mg/m3 males, weeks 1-4 -2% compared to controls; 900 mg/m3 females, weeks 1-4 -2% and 1800 mg/m3 females, weeks 1-12 -3%). Thereafter, there were no differences between control and experimental animals for the duration of the study.

HAEMATOLOGY: Various statistically significant changes were transiently seen in males up to six months, but were considered not to be biologically significant.

CLINICAL CHEMISTRY: Various statistically significant changes were probably not of biological significance, since they were within the range of values obtained in normal rats of this strain and age and they were not supported by histopathological or any other evidence of organ damage.

ORGAN WEIGHTS: 1800 mg/m3 exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses.

HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathological examination of the animals revealed a variety of changes, none of which could be attributed to exposure.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): Only one tumor was found in the six-month exposed animals: a malignant mammary adenocarcinoma in a high-exposure female. After 12 months' exposure, several tumors were found. Pituitary adenomas in females were most common, several being found in each group. Of the remaining tumors, one high-exposure female had a leiomyoma on the left uterine horn, one low-exposure male had a malignant glioblastoma under the left lobe of the cerebellum, and one high-exposure male had a malignant lymphoma of the spleen. The tumors were mostly of a type frequently found in this strain and age of rat and were considered not to be treatment-related.
Dose descriptor:
NOAEC
Effect level:
1 800 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: There was no significant systemic toxicity following 12 months exposure in this study.
Critical effects observed:
not specified
Conclusions:
Chronic exposure to this high aromatic naphtha (a 50:50 blend of SHELLSOL A and SOLVESSO 100, which contains 9.35% 1,3,5-trimethylbenzene) iwas without systemic toxicity in rats under the conditions of this study.
Executive summary:

Rats were exposed to a 50:50 blend of SHELLSOL A and SOLVESSO 100 at exposure concentrations of 0, 450, 900 and 1800 mg/m3 for 12 months. Initial reduction in body weight gain occurred in both male and female rats at the higher exposures. Various statistically significant haematological changes were transiently seen in males up to six months, but were not considered biologically significant. High exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses. No treatment-related histopathological abnormalities were found. It is concluded that chronic exposure to this high aromatic naphtha is without systemic toxicity in rats under the conditions of this study with a NOAEC of 1800 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 800 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
The available data provide information that is adequate for the purpose of hazard assessment

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

Several repeated dose studies have been conducted to evaluate both oral and inhalation exposures to trimethylbenzenes. Typically liver and kidney effects are reported at high doses together with some acute central nervous system (CNS) effects. The focus in this evaluation is on 1,3,5-trimethylbenzene where data is available, but read across from other isomers is considered where specific data is unavailable. The trimethylbenzene isomers have similar toxicological profiles (Firth 2008). There are no repeat dose toxicity data available in humans.

Oral

A 28-day study in rats was conducted on 1,2,4-trimethylbenzene (Biosafety Research Center,1996). Administered doses were 0, 30, 100, 300 and 1000 mg/kg/day with primary effects reported at the top two doses including increased liver and kidney weights and increased salivation. Weight gain was suppressed at the highest dose level. No haematological changes or abnormalities at necropsy were reported. The NOAEL was 100 mg/kg/day. 

A 90-day oral study is not available for 1,2,4-trimethylbenzene, but a study is available on the isomer 1,3,5- trimethylbenzene. In this oral gavage study, rats were dosed at 0, 50, 200, and 600 mg/kg/day, with a recovery group being dosed at 600 mg/kg/day and observed for 28 days post-treatment. In the 600 mg/kg/day group an increase in serum phosphorus levels (males and females), liver weight increases (absolute and relative weight increase in females; relative increase only in males) and kidney weight increases in males were reported. In the recovery group, no apparent effects were reported at the end of the 28-day recovery period.    

Based on the recovery group data, the effects reported at the highest dose were considered reversible and were attributed to an adaptive response (enzyme induction) to the test substance. Liver growth is likely to be due to microsomal enzyme induction whilst the increased kidney weights in male rats are likely to be due to alpha-2µ-globulin related nephropathy. With respect to the elevated serum phosphorus levels observed, these levels were not statistically different at the end of the 28-day recovery period and no associated histopathological effects were reported which could be correlated with the increased serum phosphorus levels. The NOAEL for this study was therefore concluded to be 600 mg/kg/day.

Dermal

There are no repeat dose dermal studies available but testing via the dermal route is not required as human skin contact is unlikely and in accordance with column 2 of REACH Annex VIII, testing shall be performed using the most appropriate route of administration.

Inhalation

The key study is considered to be a 12 month chronic inhalation toxicity study with high flash aromatic naptha (a 50:50 blend of Shellsol A and Solvesso 100, which contains 44.81% total trimethylbenzene, all isomers) in rats (Clark et al, 1989). Rats were exposed to the aromatic solvent blend at concentrations of 0, 450, 900 or 1800 mg/m3 6 h/day, 5 days/week for 52 weeks. An initial reduction in body weight gain occurred in both male and female rats at the higher exposures. Various statistically significant haematological changes were transiently seen in males up to six months, but were not considered biologically significant. High exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses. Some rats in all exposure groups and the control group showed mild pulmonary macrophage infiltration and alveolar wall thickening however there was no appreciable difference between the groups and no evidence that exposure had influenced the incidence or severity of the changes. Overall, no treatment-related histopathological abnormalities were found. It was concluded that chronic exposure to this high aromatic naphtha was without systemic toxicity in rats under the conditions of this study with a NOAEC of 1800 mg/m3. The test material contained a mixture of di- and tri-alkyl benzenes (including methyl-ethylbenzene isomers and other trimethylbenzene isomers) possessing presumably similar physico-chemical and toxicological properties therefore no adjustment was made to the NOAEC to account for the specific trimethylbenzene content..

These data are supported by the studies of Korsak et al (1997, 2000 a & b). In these studies, rats were exposed to nominal vapour concentrations of 0, 123, 492 or 1230 mg/m3 of 1,2,4- trimethylbenzene or 1,3,5- trimethylbenzene for 90 days (6h/day, 5 days/week). In all 3 studies, all animals survived and there were no effects on body weight and no clinical signs of toxicity.  The 90 day studies (Korsak 2000 a & b), resulted in an overall low degree of systemic toxicity. Adaptive effects of exposure to 1,2,4- trimethylbenzene and 1,2,3 - trimethylbenzene included slightly increased sorbitol dehydrogenase activity and some blood changes (decreased red and increased white blood cell counts), observed at 1230 mg/m3.  Pulmonary lesions, comprising an increase in peribronchial, lung parenchymal and perivascular lymphocytic infiltration, were described by the authors at 492 and 1230 mg/m3 for both isomers, however the pathology was poorly reported. Since no comparable findings were reported by Clark et al. (1989) in a study of longer duration it is concluded that these findings are of limited relevance to hazard identification. There were no significant histopathological changes in the upper respiratory tract or in any other organs examined, demonstrating the lack of systemic effects. The overall systemic NOAEC from these investigations by Korsak et al. is therefore considered to be 1230 mg/m3 for both isomers.

In the supporting respiratory irritation study (Korsak et al, 1997), bronchiolar lavage fluid (BAL) was collected at termination (24 h after the last exposure) and BAL cell smears were stained and examined by light microscopy. Differential cell counts were made and cell viability was assessed using the trypan blue test. There was an increased number of cell macrophages, polymorphonuclear leukocytes and lymphocytes at all three test concentrations compared with the controls. Total protein lactate dehydrogenasee (LDH) and acid phosphatase activity in BAL were significantly increased in all exposed groups. However, the observed changes were not concentration-dependent, showing no progression of effects even with an exposure 10 times higherTherefore, although respiratory irritation effects were observed in the BAL even at the lowest concentration of 123 mg/m3, there were no toxicologically significant clinical effects at concentrations up to and including 1230 mg/m3.

When examining information on the health effects of trimethylbenzenes, the Scientific Expert Group (SEG, 1994) noted that only quite limited human data were available, with results from long-term animal toxicity testing (Clarke et al., 1989) therefore used as the basis of the IOELV. A single limit value of 20 ppm was recommended, and subsequently implemented (European Commission, 2000), for all trimethylbenzene isomers.

Conclusions

1,3,5- Trimethylbenzene has low systemic toxicity in rats when administered by inhalational or oral routes. An oral NOAEL of 600 mg/kg day was established from a 90 day study on 1,3,5-trimethylbenzene and an inhalational NOAEC of 1800 mg/m3 has been established from a 12 month study on high flash aromatic naphtha. The EU Scientific Expert Group concluded that no adverse effects on health were likely at an IOELV of 20 ppm (100 mg/m3).

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

Results from a sub-chronic oral toxicity study performed using 1,3,5-trimethylbenzene showed no toxicologically relevant effects at the highest dose tested.

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

Results from a chronic inhalation toxicity study conducted on a trimethylbenzene-containing stream showed no toxicologically relevant local effects at the highest exposure level tested.

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

Results from a chronic inhalation toxicity study conducted on a trimethylbenzene-containing stream showed no toxicologically relevant local effects at the highest exposure level tested

Justification for classification or non-classification

1,3,5-Trimethylbenzene has low oral and inhalational sub-chronic toxicity and dermal exposure is unlikely. It does therefore not warrant classification under Regulation (EC) No 1272/2008.