Toluene

Endpoint summary

Description of key information

Toluene exposure can produce central nervous system pathology in animals after high oral doses. Repeated inhalation exposure can produce ototoxicity in the rat and high concentrations are associated with local toxicity (nasal erosion).  In humans neuropsychological effects and disturbances of auditory function and colour vision have been reported, particularly when exposures are not well controlled and/or associated with noisy environments. The NOAEC for subchronic oral toxicity in rats is 625 mg/kg/day based on neuropathology.  The NOAEC for inhalation toxicity in the rat is 300 ppm (1131mg/m3) based on effects on body weight and mortality, with a LOAEC for local effects (nasal errosion) of 2261 mg/m3.  SCOEL concluded there was a great deal of human data demonstrating no reliable evidence of neurological effects or mucosal irritation at or below 50 ppm (192 mg/m3), hence 50 ppm was an appropriate level for an 8 hr TWA.

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: Near guideline, GLP animal experimental study, available as published report, fully adequate for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Cited as Directive 87/302/EEC, part B, p. 8

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI., USA
- Age at study initiation: approx. 6-7 weeks
- Housing: 5 per cage in polycarbonate cages with hardwood bedding
- Diet: IH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA) ad libitum
- Water: ad libitum
- Acclimation period: 18days

ENVIRONMENTAL CONDITIONS:
- Temperature: mean temperature 72.4°F (~22.4°C), range 63-82°F (~17-28°C)
- Humidity: mean humidity 59.8%, range 44-82%
- Air changes (per hr): not reported
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: Not reported

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Toluene was dissolved in corn oil. Solutions at 20 mg/kg were stable for at least 2 weeks at room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 31.2, 62.5, 126, 250 and 500 mg/mL
- Amount of vehicle (if gavage): dose volume of 10 mL/kg
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose mixtures were analysed several times during the 13-week studies and found to be within 10% of the target concentrations

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 5 days/ week

Remarks:

Doses / Concentrations:
312, 625, 1250, 2500, 5000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (for dead and moribund animals)
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: beginning of the study and weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination
- Anaesthetic used for blood collection: no data
- Animals fasted: yes
- How many animals: all
- Parameters examined: eosinophils, haematocrit, haemoglobin, lymphocytes, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean cell volume, methaemoglobin, monocytes, platelets, erythrocytes, reticulocytes, segmented neutrophils, leukocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination
- Animals fasted: yes
- How many animals: all
- Parameters examined: blood urea nitrogen (BUN), albumin, calcium, lactate dehydrogenase, inorganic phosphorus, total protein.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight prior to termination
- Animals fasted: yes
- How many animals: all
- Metabolism cage used for collection: yes
- Parameters examined: No data

Sacrifice and pathology:

Necropsy was performed on all animals.
Tissues examined in control, 2500 and 5000 mg/kg/day groups: adrenal glands, aorta, brain, caecum, colon, duodenum, oesophagus, heart, ileum, jejunum, kidneys, liver, lungs, and bronchi, mammary gland, mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral glands, prostate/testes or ovaries/uterus, rectum, salivary glands, spinal cord, spleen, sternebrae, including marrow, stomach, thymus, thyroid gland, tissue masses, trachea, urinary bladder and gross lesions. Tissues examined in other groups were: brain, kidneys, liver, and urinary bladder.

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):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
All rats in the 5000 mg/kg group died within the first week. In the 2500 mg/kg group 8 males and one female died before termination of the study. Clinical signs included prostration, hypoactivity, ataxia, piloerection, lachrymation, and excessive salivation in the 5000 and 2500 mg/kg groups.

BODY WEIGHT AND WEIGHT GAIN
The final mean body weight of males that received 2500 mg/kg was 19% lower than that of vehicle controls.

HAEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS
A there were no treatment related effects in the haematological and serum chemical analyses or urinalyses.

ORGAN WEIGHTS
Absolute and relative kidney weight was increased in males at 625 mg/kg and higher, and in females at 1,250 mg/kg and higher. The differences in absolute and relative liver weights for female rats that received 2500 or 1250 mg/kg (22-62% relative liver weight increase) and for males that received 1250 or 625 mg/kg (8-78% relative liver weight increase) were statistically significant. Statistically significant increases were also seen in absolute and relative heart weight for male and female rats at 2500 and females at 1250 mg/kg. Absolute, but not relative brain weight was reduced in both sexes at 2500 mg/kg.

HISTOPATHOLOGY
Neuropathological changes in the brain, consisting of neuronal cell necrosis in the dentate gyrus and Ammons horn of the hippocampus, was seen in male and female rats that received 2500 or 1250 mg/kg. In addition to the hippocampal lesions, necrosis and/or mineralisation were present in the granular layer of the cerebellar cortex. Haemorrhage was present in the mucosa, submucosa, or muscularis of the urinary bladder of males and females of the two highest dose groups. A dose of 312 mg/kg/kg did not cause any effects.

Dose descriptor:

NOAEL

Effect level:

625 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: neuropathology (neuronal cell necrosis in hippocampus)

Dose descriptor:

LOAEL

Effect level:

1 250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: adverse clinical signs, differences in brain, liver, kidney and heart weight and neuropathology (neuronal cell necrosis in hippocampus)

Critical effects observed:

not specified

Conclusions:

NOAEL considered to be 625 mg/kg/day. Increased relative weights of liver and kidney are interpreted as toxicologically insignificant differences in liver and kidney weight unaccompanied by histological findings.

Executive summary:

Groups of 10 male and 10 female F344N rats received 0, 312, 625, 1250, 2500, or 5000 mg toluene/kg in corn oil by gavage for 13 weeks. All rats in the 5000 mg/kg group died within the first week. In the 2500 mg/kg group 8 males and one female died before termination of the study. The final mean body weight of males that received 2500 mg/kg was 19% lower than that of vehicle controls. Clinical signs included prostration, hypoactivity, ataxia, piloerection, lachrymation, and excessive salivation in the 5000 and 2500 mg/kg groups. Absolute and relative kidney weight was increased in males at 625 mg/kg and higher, and in females at 1,250 mg/kg and higher.

The differences in absolute and relative liver weights for female rats that received 2500 or 1250 mg/kg (22-62% relative liver weight increase) and for males that received 1250 or 625 mg/kg (8-78% relative liver weight increase) were statistically significant. Statistically significant increases were also seen in absolute and relative heart weight for male and female rats at 2500 and females at 1250 mg/kg. Absolute, but not relative brain weight was reduced in both sexes at 2500 mg/kg. There were no treatment related effects in the haematological and serum chemical analyses or urinalyses. Neuropathological changes in the brain, consisting of neuronal cell necrosis in the dentate gyrus and Ammons horn of the hippocampus, was seen in male and female rats that received 2500 or 1250 mg/kg. In addition to the hippocampal lesions, necrosis and/or mineralisation were present in the granular layer of the cerebellar cortex. Haemorrhage was present in the mucosa, submucosa, or muscularis of the urinary bladder of males and females of the two highest dose groups. A dose of 312 mg/kg/kg did not cause any effects.

The NOAEL for repeat dose oral toxicity is considered to be 625 mg/kg. At this dose increased absolute and relative liver and kidney weight (up to 46% increase in relative kidney weight, up to 78% increase in relative liver weight) were unaccompanied by histopathological findings were present. Therefore, the increased relative weights of liver and kidney are interpreted as toxicologically non-significant signs of metabolic activity related to exposure.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

625 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

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: Non-guideline, non-GLP, animal experimental study, published in peer reviewed literature, notable limitations in design, but contributing to weight of evidence.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

- only 5/sex/group examined for chronic toxicity at 1 year

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: Fischer-344 rats [CDF(F-344)/CrIBr]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, MA., USA
- Age at study initiation: approx. 6-7 weeks
- Housing: group housed, sexes separately, 10/cage in stainless steel suspended cages
- Diet: standard laboratory diet (Wayne Lab-Blox F6, Allied Mills Inc., Chicago, IL., USA) ad libitum (except during exposure)
- Water: ad libitum
- Acclimation period: quarantined for at least 10 days

ENVIRONMENTAL CONDITIONS: No details reported

IN-LIFE DATES: Not reported

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: rectangular stainless steel and glass chambers (nominal volume 8.0 m3)
- Source and rate of air: absolute and charcoal filtered air separate from the room air supply
- Temperature, humidity, pressure in air chamber: temperature 22°C / relative humidity 50%. The inhalation chambers containing toluene were maintained at slight sub-atmospheric pressure (0.1 in - 0.5 in H2O). Control chambers were under slight positive pressure (0.1 in - 0.5 in H20).
- Air flow rate: approx. 2000 L/min
- Generation: toluene vapour was generated by bubbling clean dry air (dew point -40°C) through liquid toluene. The rate of vapour generation was regulated by controlling airflow into the generator flask.

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography
- Samples taken from breathing zone: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The time weighted average concentration of toluene for each exposure group was 0.0, 30.1, 99.7, 299.0 ppm for target concentrations of 0, 30, 100 and 300 ppm, respectively.

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

5 days/week, up to 24 months (interim kills at 6, 12 and 18 months)

Remarks:

Doses / Concentrations:
0, 30, 100, 300 ppm
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
0, 30.1, 99.7, 299.0 ppm
Basis:
other: time weighted average concentration

Remarks:

Doses / Concentrations:
0, 113, 377 or 1131 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

120

Control animals:

yes, sham-exposed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (for dead and moribund animals)
- Time schedule: once/day non-exposure days, twice/day exposure days

DETAILED CLINICAL OBSERVATIONS: Yes (including palpation and measurement of tissue masses)
- Time schedule: every 2 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: beginning of the study (week 0), weekly for the first 6 months, every other week from 6 to 24 months and immediately prior to sacrifice

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to sacrifice
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months (prior to termination)
- Anaesthetic used for blood collection: no data
- Animals fasted: yes
- How many animals: selected (no further detail reported)
- Parameters examined: haemoglobin concentrations, haematocrits, total erythrocyte counts and total and differential leukocyte count. Mean corpuscular volumes, mean corpuscular haemoglobins, and mean corpuscular haemoglobin concentrations were calculated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 (months prior to termination)
- Animals fasted: yes
- How many animals: selected (no further detail reported)
- Parameters examined: blood urea nitrogen (BUN), serum glutamic pyruvic transaminase (SGPT) activity, and serum alkaline phosphatase (SAP) activity

URINALYSIS: Yes
- Time schedule for collection of urine: 16 hour period prior to blood collection (at 6, 12, 18 and 24 months)
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes
- Parameters examined: appearance, specific gravity, protein, pH, ketones, glucose and microscopic particles

Sacrifice and pathology:

SACRIFICE AND GROSS PATHOLOGY: Animals killed at each interval, and all survivors at 24 months, were exsanguinated following carbon dioxide anaesthesia. Each was examined grossly and the brain, heart, kidneys, liver, lungs and gonads weighed.

HISTOPATHOLOGY: The following tissues were examined histopathologically: brain, spinal cord, peripheral nerve, pituitary, parathyroid, salivary gland, heart, lungs, spleen, liver, pancreas, oesophagus, adrenals, lymph nodes, kidneys, bladder, prostate, ovaries, uterus, fallopian tubes, stomach, small intestine, large intestine, skeletal muscle, thymus, skin, mammary gland, bone marrow, adipose tissue, aorta, ear canal, nasal turbinate, trachea, tibial and plantar nerves, lumbar, sacral and dorsal ganglia, proximal and distal hind limb, eyes and testes.

Statistics:

Analysis of variance (Kruskal-Wallis test for ratios): absolute body weight, total body weight change (surviving animals), organ weights, organ weight/body weight and organ weight/brain weight ratios. Other techniques for multiple comparisons: Tukey's for equal-sized groups, Scheffe's for groups of unequal sizes. Tests for homogeneity of variance, transformed (natural logarithm) as needed, and analysis of variance were used for haematology data.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were 140 unscheduled deaths (14.6% of 960 animals) over the 2-year course of the study. The distribution of spontaneous deaths among the treatment groups was not significantly different from controls.

There were no differences between control and toluene exposed groups with regard to clinical observations, blood chemistry analyses, urinalysis, ophthalmology, gross or histopathological findings.

The males in the toluene treatment groups were significantly heavier than the control males throughout the study although there was no clear cut dose response relationship. A similar finding was noted for the females but the effect disappeared during the final weeks of the study. Neither observation was considered to be adverse.

Female rats exposed to 100 and 300 ppm of toluene for 24 months had slightly, but significantly, reduced haematocrits and for the 300 ppm group only the mean corpuscular haemoglobin concentration was slightly, but significantly, increased. Other haematology parameters were not significantly different from controls.

Dose descriptor:

NOAEC

Remarks:

chronic toxicity

Effect level:

300 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: no toxicologically significant effects at highest dose tested (300 ppm)

Dose descriptor:

NOAEC

Effect level:

1 131 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: no toxicologically significant effects at highest dose tested (1131 mg/m3)

Critical effects observed:

not specified

All statistically significant differences from control were considered to be of no toxicological significance.

Conclusions:

Toluene does not cause adverse effects in the rat following inhalation exposure to 300 ppm for up to 24 months. The NOAEC for chronic systemic or local toxicity in this study was 300 ppm (1131 mg/m3).

Executive summary:

Chronic toxicity of inhaled toluene was assessed in Fischer-344 rats exposed to 0, 30, 100 or 300 ppm 6 h/day, 5 days/week for 6, 12 or 18 months. There were no toxicologically significant effects on bodyweight, clinical signs, ophthalmoscopy, haematology, blood and urine clinical chemistry, organ weights or gross and microscopic pathology. The NOAEC for chronic systemic and local toxicity from this study was 300 ppm (1131 mg/m3) for an exposure duration of 18 months.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

98 mg/m³

Study duration:

chronic

Species:

other: human

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:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Near guideline, GLP, animal experimental study, available as published report, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Version / remarks:

Cited as Directive 87/302/EEC, part B, p. 20

GLP compliance:

yes

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, Portage, MI., USA
- Age at study initiation: approx. 6-7 weeks
- Housing: singly in stainless steel wire mesh cages
- Diet: IH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA) ad libitum (except during exposure)
- Water: ad libitum
- Acclimatisation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature: 74-80°F during exposures
- Humidity: 45-55 % during exposures
- Air changes (per hr): not reported
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: not reported

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: no data
- Vapour generation system: toluene vapour was generated by delivering liquid toluene to a heated Spraying Systems atomizer that was operated with nitrogen. Toluene vapour was diluted with chamber ventilation air to produce the desired concentrations in the chamber
- Temperature, humidity: 74-80°F, humidity 45-55 % during exposures

TEST ATMOSPHERE
- Brief description of analytical method used: gas-phase infrared spectrophotometry
- Samples taken from breathing zone: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No details reported for 15 week exposure.

Duration of treatment / exposure:

6.5 h/day

Frequency of treatment:

5 days/week for 15 weeks.

Remarks:

Doses / Concentrations:
100, 625, 1250, 2500 and 3000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
377, 2355, 4710, 9421 and 11306 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (for dead and moribund animals)
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: beginning of the study and weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination
- Anaesthetic used for blood collection: no data
- Animals fasted: no data
- How many animals: all
- Parameters examined: haemoglobin concentration, haematocrit, total erythrocyte count, platelet count, reticulocyte count and total and differential leukocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, methaemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination
- Animals fasted: no data
- How many animals: all
- Parameters examined: blood urea nitrogen (BUN), albumin, albumin/globulin ratio, calcium, chloride, cholinesterase, creatinine, gamma glutamyl transferase, inorganic phosphorus, potassium, glucose, sodium, total bilirubin, total protein

URINALYSIS: No

SPERM MORPHOLOGIC AND VAGINAL CYTOLOGICAL EVALUATIONS: Yes
- Time schedule for evaluation: sperm morphology; at termination (the right cauda epididymis was removed, gently chopped with a scalpel, and incubated for 5 minutes to release its contents) sperm viability and motility were evaluated: vaginal cytology; smears were taken between 7:00 a.m. and 9:00 a.m. from 12-14 consecutive days before the animals were killed and also on the morning of the kill
-
- How many animals: all surviving animals exposed at 0, 100, 625, or 1250 ppm toluene
- Parameters examined: sperm motility

Sacrifice and pathology:

NECROPSY: Yes
- How many animals: all

ORGAN WEIGHTS: Yes
- brain, liver, lung, right kidney, right testis, and thymus were weighed

HISTOLOGICAL EXAMINATIONS: Yes
- How many animals: all animals that died before the end of the study, vehicle controls, and animals in 2500 and 3000 ppm groups.
- Tissues examined: adrenal glands, aorta, brain, caecum, colon, duodenum, epididymis/prostate/testes or ovaries/uterus, oesophagus, femur, heart, ileum, jejunum, kidneys, liver, lungs and bronchi, mammary gland, mesenteric lymph nodes, nasal tissue, pancreas, parathyroid glands, pituitary gland, preputial gland, rectum, salivary glands, spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Sternum examined for the 3000 ppm group and animals dying before the end of the study. Selected tissues of lower dose animals were examined.

Statistics:

For all end points, dosed groups were compared with the control group using the nonparametric multiple comparison test of Dunn or Shirley. Jonckheere’s test was used to assess the significance of the dose response trends and to determine whether Dunn’s or Shirley’s test was more appropriate for pairwise 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):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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

Histopathological findings: neoplastic:

no effects observed

Details on results:

Sperm morphologic and vaginal cytologic evaluations: no effects

Dose descriptor:

NOAEC

Effect level:

625 ppm

Sex:

male/female

Basis for effect level:

other: decrease in leukocyte count in females and relative organ weight increases at 1250 ppm

Dose descriptor:

LOAEC

Effect level:

1 250 ppm

Sex:

male/female

Basis for effect level:

other: decrease in leukocyte count in females, increased relative liver weight in males and increased relative kidney weight in both sexes

Dose descriptor:

NOAEC

Effect level:

2 355 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: decrease in leukocyte count in females and relative organ weight increases at 4710 mg/m3

Dose descriptor:

LOAEC

Effect level:

4 710 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: decrease in leukocyte count in females, increased relative liver weight in males and increased relative kidney weight in both sexes

Critical effects observed:

not specified

Haematology and clinical chemistry

Lower leukocyte counts were seen in females at 1250 ppm and above. Plasma cholinesterase activity decreased with increasing exposure concentration.

		Concentration of toluene (ppm)
Parameter		0	100	625	1250	2500	3000
Leukocytes (10^3/mm^3)	M	8.19±0.441	8.12±0.409	7.43±0.367	7.19±0.443	7.86±0.623	7.70±0.095
	F	7.26±0.392	6.75±0.387	6.46±0.480	5.98±0.352*	5.64±0.342**	6.39±0.385*
Plasma cholinesterase (IU/L)	M	714±16	712±18	682±15	671±14	630±19	595±9**
	F	3701±83	3644±78	3166±209*	297±205	1798±162**	1702±163**

* P< 0.05, ** P<0.01; M = male, F= females; n=10 for all group except males at 3000ppm, n=2

Organ weights

There were increases in a number of relative organ weights at and above 1250 ppm.

Relative organ weights:

		Concentration of toluene (ppm)
Parameter		0	100	625	1250	2500	3000
Brain	M	5.1±0.10	5.1±0.10	5.2±0.11	5.1±0.13	5.8±0.11**	6.7±0.64**
	F	8.3±0.12	8.3±0.12	8.2±0.17	8.4±0.12	9.1±0.14**	9.1±0.23**
Heart	M	2.7±0.04	2.8±0.04	2.7±0.03	2.7±0.04	3.0±0.05**	3.1±0.02**
	F	3.1±0.04	3.1±0.04	3.1±0.05	3.0±0.07	3.3±0.07**	3.4±0.05**
Kidney	M	3.3±0.04	3.4±0.05	3.3±0.07	3.4±0.05*	3.8±0.07**	4.0±0.06**
	F	3.4±0.06	3.4±0.07	3.4±0.07	3.6±0.05*	3.8±0.05**	3.8±0.05**
Liver	M	35.8±0.58	36.0±0.61	37.6±0.55	38.9±0.54**	41.0±0.60	47.6±0.79
	F	34.1±0.55	34.8±0.85	33.8±0.33	34.6±0.46	39.5±0.54**	41.1±0.54**
Lung	M	3.3±0.06	3.4±0.05	3.4±0.05	3.5±0.08	3.8±0.07**	3.9±0.16**
	F	4.5±0.07	4.6±0.07	4.5±0.08	4.7±0.08	4.9±0.10**	4.9±0.08**

* P< 0.05, ** P<0.01; M = male, F= females

Conclusions:

Toluene exposure at concentrations = 1250 ppm, 6h/day, 5 days per week for 15 weeks induced adverse clinical signs, lower bodyweight and changes in haematology and organ weights.

Executive summary:

The sub-chronic toxicity of toluene was evaluated in a 15 week inhalation study in rats. Groups of 10 male and 10 female F344/N rats were exposed via inhalation t0 0, 100, 625, 1,250, 2,500, or 3,000 ppm (0, 377, 2,355, 4,710, 9,421, or 11,306 mg/m3) toluene 6.5 hours/day for 5 days/week for 15 weeks. The rats were observed for clinical signs daily, and weighed once per week, blood was collected prior to termination for haematological and clinical chemistry investigations. All animals were necropsied, organs weighed and tissues at 0, 2,500 and 3,000 ppm and from all decedents were examined microscopically.

Eight male rats of the 3000 ppm group died during week 2. The mean body weights of rats exposed to 2500 or 3000 ppm were 15-25% lower than that of controls. Clinical signs included dyspnea in all exposed groups, except males exposed at 3000 ppm and females exposed at 1250 ppm, and ataxia in rats exposed at 3000 or 2500 ppm. The relative kidney weight was statistically significantly increased at 1250 ppm and higher in both sexes. Males at 1250 ppm and higher also showed a statistically significantly increased relative liver weight while females at 2500 ppm and higher showed this effect. Relative weight of the brain, heart, and lung were statistically significantly increased in both sexes at 2500 and 3000 ppm. In male rats, relative testes weight was statistically significantly increased at 2500 and 3000 ppm. Plasma cholinesterase activity decreased as exposure concentration increased, and the leukocyte count was decreased for female rats at 1250 ppm or higher. No compound-related effects were seen on sperm or on oestrous cycle.

A NOAEC of 625 ppm toluene (2,355 mg/m3) can be derived on the basis of a lower leukocyte count in females and increased relative liver and kidney weights at 1250 ppm and above.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

2 261 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

LOA is currently reviewing the human and animal data supporting Human Health for Toluene. It is expected to be completed by Q4 2020.

The toxicity of toluene following repeated exposures was reviewed and reported in the EU RAR (2003). No additional relevant animal data have been found in the updated literature review.

Non-human information

Oral

Two key studies were identified, one in rats and one in mice (Huff, 1990). In both studies groups of 10 animals/sex received 0, 312, 625, 1250, 2500, or 5000 mg toluene/kg in corn oil by gavage for 13 weeks. Deaths were seen before termination of the study as follows: all rats and mice in the 5000 mg/kg groups within the first week; 8 male and 1 female rat, 4 male and 4 female mice at 2500 mg/kg; 1 female mouse at 1250 mg/kg. The final mean body weight of male rats and mice that received 2500 mg/kg was lower than that of vehicle controls. Clinical signs in both species included prostration, hypoactivity, ataxia, piloerection, lachrymation, and excessive salivation in the 5000 and 2500 mg/kg groups. There were no treatment related effects in the haematological and serum chemical analyses or urinalyses in either species.

In rats at 1250 and 2500 mg/kg there were differences in the weight of a number of organs and neuropathological changes in the brain, consisting of neuronal cell necrosis in the dentate gyrus and Ammons horn of the hippocampus. In addition to the hippocampal lesions, necrosis and/or mineralisation were present in the granular layer of the cerebellar cortex. Haemorrhage was present in the mucosa, sub-mucosa, or muscularis of the urinary bladder of males and females. At 625 mg/kg increases in absolute and relative liver and kidney weight, unaccompanied by histopathological findings, were interpreted as toxicologically non-significant effects. A dose of 312 mg/kg/kg did not cause any effects.

In mice, increases in absolute and/or relative liver weights in males at 1250 or 2500 mg/kg and in females at all doses were not accompanied by histopathological effect and, therefore, considered to be of no toxicological significance. Relative brain and testis weight, and absolute kidney weight was increased in male mice at 5000 mg/kg. Myocardial degeneration was found in 3 male and 2 female mice from this group.

The NOAEL for repeat dose oral toxicity is considered to be 625 mg/kg in rats and mice.

Dermal

No data have been identified.

Inhalation

Toluene toxicity via inhalation has been investigated in sub-chronic and chronic toxicity studies the rat and mouse. The key studies are considered to be the 15 week and chronic studies in rats (Gibson and Hardisty, 1983; Huff, 1990).

In a 15 week study in rats, rats were exposed via inhalation to 0, 100, 625, 1250, 2500, or 3000 ppm toluene 6.5 hours/day for 5 days/week (Huff, 1990). Eight male rats of the 3000 ppm group died during week 2. Treatment-related effects at 3000, 2500 and 1250 ppm included lower body weights, adverse clinical signs and differences in absolute or relative organ weights (particularly liver and kidney). Plasma cholinesterase activity decreased as exposure concentration increased, and the leukocyte count was decreased for female rats at 1250 ppm or higher. 625 ppm toluene was the NOAEC on the basis of a lower leukocyte count in females and increased relative liver and kidney weights at 1250 ppm and above.

In the chronic toxicity study using 600 or 1200 ppm toluene body weight was slightly lower than control at 1200 ppm during the second year of the study. No effects of toluene exposure were found on absolute and relative weight of kidney, liver or brain. The severity of nephropathy was slightly increased in exposed female rats. Results of haematological analyses did not indicate any substance-related effects. In the nasal cavity, mild to moderate degeneration of the olfactory and respiratory epithelium was more obvious in toluene-exposed rats and goblet cell hyperplasia somewhat increased. No other treatment-related lesions were observed. 600 ppm (2261 mg/m³) was the LOAEC for local toxicity and the NOAEC for systemic toxicity.

Supporting sub-chronic and chronic toxicity inhalation toxicity studies were conducted in B6C3F1 mice (Huff, 1990). Concentrations = 625 ppm, 6h/day, 5 days per week for 15 weeks caused mortality but no other evidence of significant target organ toxicity.

The overall NOAEC for local effects and chronic toxicity can be derived from the chronic toxicity study of Gibson and Hardisty (1983). Fischer-344 rats were exposed to 0, 30, 100 or 300 ppm 6 h/day, 5 days/week for 6, 12, 18 or 24 months. There were no toxicologically significant effects on bodyweight, clinical signs, ophthalmoscopy, haematology, blood and urine clinical chemistry, organ weights or gross and microscopic pathology. The NOAEC for chronic systemic and local toxicity from this study was 300 ppm (1131 mg/m³) for an exposure duration of 18 months.

Special studies addressing neurotoxicity and ototoxicity (see CSR section 5.10) demonstrate that toluene is ototoxic in the rat and can produce neurochemical and pathological changes. However, the data are insufficient to determine NOAEC values. Therefore, the NOAEC of 300 ppm (1131 mg/m³) based on the study of Gibson and Hardisty (1983) will be taken forward to the risk characterisation.

Human information

Human data addressing a number of adverse effects associated with exposure to toluene are reviewed under the relevant section of the dossier (section 5.10). After repeated dose exposure via inhalation in humans toluene causes a number of adverse effects including neuropsychological effects, auditory dysfunction and disturbances of colour vision. Key data published since the EU RAR (2003) addressing these effects are those of Seeber et al (2004) and Schaper et al (2003, 2004). There was no evidence that long-term exposure to toluene at 26 ppm for 21 years had any effects on cognitive function (Seeber et al, 2004). There was no evidence of ototoxicity resulting from occupational exposure to toluene below 50 ppm (mean exposure 26 ppm / 98 mg/m³) in a longitudinal study over 5 years (Schaper et al, 2003). Similarly no effect of human occupational exposures to toluene on colour vision were found in a follow up study over 4 years with three repeated examinations (Schaper et al, 2004). These studies demonstrate that 26 ppm (98 mg/m³) is a NOAEC for human adverse effects.

When reviewing the effects of repeated human exposure to toluene vapour, SCOEL (2001) noted that concentrations in the range 90-150 ppm (345-575 mg/m3) for several hours lead to decrements in neurological test results although some of the findings were difficult to interpret due to uncertainties regarding the relationship between the effects monitored and contemporary / past levels of exposure. High concentrations of toluene vapour were also associated with symptoms of eye and upper respiratory tract irritation in human volunteers exposed to 75-100 ppm (286 – 383 mg/m3) for 4.5 – 6.5 hr, with the lower end of this range a threshold for mucous membrane irritation. Based on a great deal of human data indicating no reliable evidence of neurological effects or irritation at or below toluene concentrations of 50 ppm (192 mg/m3), SCOEL concluded that 50 ppm (8 hour TWA) was an appropriate level for an IOELV.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Two key sub-chronic oral studies were identified, one in rats and one in mice. The NOAEL (625 mg/kg) is based on neuropathological changes recorded in rats

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Key studies are available that report the sub-chronic and chronic inhalation toxicity of toluene in rats (chronic NOAEC = 1131 mg/m3, based on neuropathy). Other animal studies demonstrate that toluene is ototoxic in the rat and can produce neurochemical and pathological changes. Human data (reported under Epidemiological studies) demonstrate no evidence that long-term exposure to toluene (98 mg/m3 for 21 years) adversely effects human neurological or cognitive function.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Mild to moderate degeneration of the olfactory and respiratory epithelium was more obvious in toluene-exposed rats, and goblet cell hyperplasia somewhat increased, in rats exposed chronically to 2261 mg/m3 toluene vapour.

Repeated dose toxicity: via oral route - systemic effects (target organ) neurologic: other

Repeated dose toxicity: inhalation - systemic effects (target organ) neurologic: behaviour

Justification for classification or non-classification

After repeated dose exposure, toluene causes a number of adverse effects including impairment of auditory function and morphological evidence of cell loss in the rat cochlea, neuron loss in the central nervous system of animals and in humans neuropsychological effects, auditory dysfunction and effects on colour vision have been reported. Consequently, toluene is classified as Category 2 (H373), according to GHS / CLP.