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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Near guideline, GLP, animal experimental study, available as published report, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Toluene
EC Number:
203-625-9
EC Name:
Toluene
Cas Number:
108-88-3
Molecular formula:
C7H8
IUPAC Name:
toluene
Details on test material:
Toluene was obtained in one lot (H-12-19-80) from Exxon Company, USA (Baytown, TX, USA) as a clear, colourless liquid. Purity was greater than 99% as determined by elemental analysis, Karl Fischer water analysis and gas chromatography (GC). GC detected three impurities with individual peak areas of less than 0.1% of the major peak area. Benzene content was determined to be 5.7 ppm (v/v). The calculated benzene concentrations were 0.82, 4.1 and 8.2 ppb for the toluene exposures at 120, 600 or 1200 ppm.

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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 (103/mm3)

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

Applicant's summary and conclusion

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.