p-cymene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Repeated dose inhalation toxicity study of the test chemical

Author:

Lam et al

Year:

1996

Bibliographic source:

Pharmacology & Toxicology

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

Details on test material:

- Name of test material: p-Cymene
- Molecular formula: C10H14
- Molecular weight: 134.221 g/mol
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): <1%

Test animals

Species:

rat

Strain:

Long-Evans

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mellegaard Breeding Center Ltd., LI. Skensved, Denmark
- Age at study initiation: 3 weeks at purchase
- Weight at study initiation: No data
- Fasting period before study:
- Housing: The rats were housed two per cage in plastic cages (14X23X42 cm) on steam-cleaned pine wood bedding
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2˚C
- Humidity (%): 40±5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hr reversed day:night cyclus with light from fluorescent tubes from 3 p.m. to 3 a.m.

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

not specified

Vehicle:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless
steel wire cages
- Method of holding animals in test chamber: No data
- Source and rate of air: No data
- Method of conditioning air: No data
- System of generating particulates/aerosols: The test chemical was evaporated in the air inlet by pumping it onto a glass spiral heated by the circulation of warm water (40˚).
- Temperature, humidity, pressure in air chamber: Temperature: 23±2˚C, humidity: 40±5%
- Air flow rate: No data
- Air change rate: The air exchange in the chambers during exposure was 13 time/hr.
- Method of particle size determination: No data
- Treatment of exhaust air : No data

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of the test chemical in each chamber was monitored with an infrared gas cell spectrophotometer (MIRAN-I
A, Foxboro, England) every 10 min throughout exposure.
- Samples taken from breathing zone: No data

VEHICLE (if applicable)
- Justification for use and choice of vehicle: No data
- Composition of vehicle: No data
- Type and concentration of dispersant aid (if powder): No data
- Concentration of test material in vehicle: No data
- Lot/batch no. of vehicle (if required): No data
- Purity of vehicle: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test chemical in each chamber was monitored with an infrared gas cell spectrophotometer (MIRAN-I A, Foxboro, England) every 10 min throughout exposure.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hr/day, 5 day/week

Doses / concentrations

Remarks:

0, 50 or 250 ppm (approximately 0, 0.25 or 1.23 mg/L)

No. of animals per sex per dose:

0 mg/L: 7 male rats
50 mg/L: 11 male rats
250 mg/L: 12 male rats

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. No data

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: yes, eight weeks after the end of exposure the rats were decapitated in C02/02-narcosis (at the age of 29 weeks). The cerebellum was weighed and homogenized in 4 ml ice-cold 0.32 M sucrose. Whole brain minus cerebellum was quickly transferred to 10 ml of ice-cold 0.32 M sucrose and weighed.

HISTOPATHOLOGY: yes, A 10% (w/v %I) homogenate was made. Synaptosomes were prepared at 0-4˚ by 'conventional' homogenization, differential- and discontinuous sucrose density gradient centrifugation techniques. The synaptosomal fraction obtained after gradient centrifugation as the 0.8-1.2 M sucrose interphase-band was isolated, diluted by slow addition of ice-cold buffer (I+ I), and centrifuged at 1000 Xg for 15 min. at W. The sediment was washed by resuspension in 10 ml ice-cold buffer and subsequent centrifugation as above. The final sediment was re-suspended in 5 ml ice-cold buffer.

Other examinations:

Immediately after homogenization (whole brain minus cerebellum and cerebellum) and resuspension (synaptosomes), samples were taken and processed for neurotransmitter analyses: 4 vol. homogenate or resuspension were deproteinized by addition of 1 vol. ice-cold 0.5 M perchloric acid and subsequent centrifugation. Resulting supernatants were stored at -20" until analysis. Aliquots of homogenate (whole brain minus cerebellum and cerebellum) and resuspension (synaptosomes) were taken for protein analysis and the determination of enzyme activities. The samples were stored at -20˚ until analysis.

Protein analysis. Protein was quantitated by the method of Lowry et al. (1951) as modified by Hartree (1972) using bovine serum albumin as the standard.

Determination of enzyme activities. Samples were treated I hr at ambient temperature with 1% (v/v) Triton X-100 and centrifuged. The supernatant was used for determination of lactate dehydrogenase (LDH), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) activities at 37˚ on a COBAS-MIRA by use of commercially available kits: Boehringer Mannheim Art. No. 07-3658-9, 07-3646-5, and 14-4238-8, respectively.

Neurotransmitter analyses. One aliquot of supernatant was used directly for the determination of 5-HT. Another aliquot was used for NA and DA determinations after 'conventional' purification on aluminum oxide. These samples were concentrated 5-fold. N-o-methyl-5-HT (Sigma, M 1514) was the internal standard for 5-HT determinations. 3.4-Dihydroxybenzylamine was internal standard for NA and DA determinations.

NA, DA, and 5-HT were determined by high performance liquid chromatography with electrochemical detection. The instrumentation consisted of a Hewlett-Packard Ti-SERIES 1050 liquid chromatograph equipped with a 250x4 mm i.d. RP-18 Highbar, Supersphere LiChroCART (5 pm) analytical column protected by a 20x4 mm i.d. (5 pm) RP-18 guard column. Separation was achieved at ambient temperature by use of an acetonitrile-modified citrate/octenylsulfate buffer. The flow was 1.0 ml/min. The detector was a Waters M 464 electrochemical detector applying an oxidation potential of t0.8 V. A 75-pI sample was injected for the NA and DA analyses and a 200-µl sample for the 5-HT analysis. From the analyses of samples of homogenate of whole brain minus cerebellum and cerebellum the values in the reconstructed whole brain were calculated.

Statistics:

Data were entered into Quattro ProR (Borland) spreadsheet version 4.0 for calculations and transferred to the SAS PC-version software package (SAS Institute Inc. 1988) for statistical analyses. Data were analyzed by analysis of variance (PROC ANOVA/GLM) followed by Dunnett's two-tailed t-test when indicated. The general level of significance was set to P<0.05. Therefore, no individual P-values are given.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of overt toxicity during the exposure.

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no difference in body weight between the three groups of animals at any time during the investigation.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There was no test chemical -induced effect on the terminal weight of whole brain minus cerebellum, cerebellum, or whole brain

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

There was no treatment-induced effect on enzyme activities in whole brain minus cerebellum, cerebellum, or whole brain when expressed per g tissue or per mg protein.

There was no treatment-related effect on the protein concentration (mg protein/g tissue) in whole brain minus cerebellum, cerebellum, or whole brain.

The concentration of NA, DA, and 5-HT (nmol/g wet weight) in whole brain minus cerebellum, cerebellum, and the whole brain was unaffected by exposure. It was not possible to detect DA in the cerebellum. The detection limit was below 100 fmol/injection.

The relative yield of synaptosomal protein defined as the mg synaptosomal protein per g whole brain minus cerebellum was statistically significantly reduced at exposure to 50 and 250 ppm in a concentration-related manner.

Synaptosomal enzyme activities.
The relative activity of the cytoplasmatic marker enzyme LDH (U/mg synaptosomal protein) was statistically significantly increased at exposure to 50 and 250 ppm. The relative AChE and BuChE activities (mU/mg synaptosomal protein) were both statistically significantly increased at exposure to 50 and 250 ppm.

The total activity BuChE, AChE, and LDH (U in the synaptosomal fraction per whole brain minus cerebellum) was unaffected by treatment. When expressed in relation to the cytoplasmatic marker enzyme, LDH (BuChE ctivity/LDH activity and AChE activity/ LDH activity) the relative synaptosomal choline esterase activities were both unaffected by the treatment.

Synaptosornal neurotransmitter concentrations.
The relative concentrations of NA, DA, and 5-HT when expressed per unit of cytoplasmatic marker enzyme activity (pmol neurotransmitter/U LDH) were unaffected by the treatment.

The relative NA and DA concentrations (pmol neurotransmitter per mg synaptosomal protein) were statistically significantly increased at 50 and 250 ppm. The total amount of NA and DA in the synaptosomal fraction (pmol per whole brain minus cerebellum) was unaffected by the treatment.

The relative 5-HT concentration (pmol neurotransmitter per mg synaptosomal protein) was unaffected by the treatment, whereas the total amount of 5-HT in the synaptosomal fraction (pmol from whole brain minus cerebellum) was statistically significantly decreased by exposure to 250 ppm

Effect levels

Dose descriptor:

NOAEL

Effect level:

0.25 other: mg/L

Based on:

test mat.

Sex:

male

Basis for effect level:

other: The relative 5-HT concentration (pmol neurotransmitter/mg synaptosomal protein) was unaffected by the treatment, whereas the total amount of 5-HT in the synaptosomal fraction was statistically significantly decreased by exposure to 250 ppm

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Regional concentration of noradrenaline (nmol/g wet weight) in brains of rats exposed to 0, 50, or 250 p.p.m. (v/v) test chemical 6hr/ day, 5 dayslweek for 4 weeks, followed by an exposure-free period of 8 weeks.

	Control	50 mg/L	250 mg/L
Whole brain cerebellum	3.37±0.33 (7)	3.52±0.39 (10)	3.47±0.39 (10)
Cerebellum	0.32±0.03 (7)	0.31±0.03 (11)	0.29±0.05 (12)
Whole brain	2.94±0.26 (7)	3.09±0.36 (10)	3.05±0.36 (10)

Results are mean±S.D., with number of rats in parentheses

Table: Regional concentration of dopamine (nmol/g wet weight) in brains of rats exposed to 0, 50, or 250 p.p.m. (v/v) test chemical 6hr/day, 5 days/week for 4 weeks, followed by an exposure-free period of 8 weeks.

	Control	50 mg/L	250 mg/L
Whole brain cerebellum	5.38±0.58 (7)	5.06±0.40 (10)	5.11±0.39 (10)
Cerebellum	ND (7)	ND (11)	ND (12)
Whole brain	4.63±0.50 (7)	4.36±0.33 (10)	4.43±0.34 (10)

Results are mean±S.D., with number of rats in parentheses. ND: Not detectable.

Table: Regional concentration of 5-hydroxytryptamine (nmol/g wet weight) in brains of rats exposed to 0, 50, or 250 p.p.m. (v/v) test chemical 6hr/day, 5 days/week for 4 weeks, followed by an exposure free period of 8 weeks.

	Control	50 mg/L	250 mg/L
Whole brain cerebellum	3.55±1.25 (7)	3.65±0.50 (10)	3.73±0.63 (10)
Cerebellum	0.30±0.08 (7)	0.28±0.03 ( I 1)	0.30±0.05 (12)
Whole brain	3.10±1.03 (7)	3.20±0.43 (10)	3.28±0.55 (10)

Results are mean±S.D., with number of rats in parentheses

Table: Yield of protein in the synaptosomal fraction of whole brain minus cerebellum prepared from rats exposed to 0, 50, or 250 p.p.m. (v/v) test chemical 6hr/day, 5 day/week for 4 weeks, followed by an exposure-free period of 8 weeks.

	Control	50 mg/L	250 mg/L
Relative yield (mg protein/g whole brain-cerebellum)	16.4±3.1 (7)	9.20±2.11* (11)	8.62±1.71* (12)
Total amount (mg protein per whole brain-cerebellum)	0.30±0.08 (7)	0.28±0.03 (11)	0.30±0.05 (12)

Results are mean±S.D., with number of rats in parentheses.

* P<0.05 between values from control and exposed rats.

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) for the test chemical is considered to be 1.23 mg/L.

Executive summary:

Long-lasting effects of 4 week inhalation exposure to the test chemical on regional and subcellular brain neurochemistry were studied. The study was performed using Long Evans male rats. The rats were exposed to 0, 50, or 250 ppm (0, 0.25 or 1.23 mg/L) test chemical 6 hr/day, 5 days/week for 4 weeks. The exposure was performed with the rats in their normal active state in the dark phase of the light: dark cyclus. During exposure, the animals were placed in stainless steel wire cages without food and water, and exposed to vapours of test chemical in chambers with walls of stainless steel and glass. The air exchange in the chambers during exposure was 13 times/hr. Synaptosomes were isolated from whole brain minus cerebellum and used as an ex situ model for in situ conditions at the level of the presynaptic nerve terminal. There were no clinical signs of overt toxicity during the exposure. There was no difference in body weight between the three groups of animals at any time during the investigation. There was no test chemical-induced effects on the terminal weight of whole brain minus cerebellum, cerebellum, or whole brain. Exposure to p-cymene did not affect the terminal weight of whole brain minus cerebellum, cerebellum, or the whole brain. There were no treatment-induced effects on the enzyme activities in whole brain minus cerebellum, cerebellum, or the whole brain when expressed per g tissue or per mg protein. The concentration of NA, DA, and 5-HT in whole brain minus cerebellum, cerebellum, and the whole brain was unaffected by the treatment. Regional protein concentrations were unaffected, whereas the relative yield and the total amount of synaptosomal protein were statistically significantly reduced in an exposure concentration-related manner. The relative yield of synaptosomal protein may reflect the synapse density in the whole brain minus cerebellum in situ. The total amount of synaptosomal protein may reflect the total number of synapses in the whole brain minus cerebellum in situ. Yield of synaptosomal protein was statistically significantly reduced in an exposure concentration-related manner. Synaptosomal NA and DA concentrations and acetylcholinesterase, butyrylcholinesterase, and lactate dehydrogenase activities were statistically significantly increased when expressed relative to synaptosomal protein. It is hypothesized that a reduced density and number of synapses in situ are functionally compensated for by increased NA and DA release from noradrenergic and dopaminergic presynaptic nerve terminals. The changes to the neurochemical parameters are not considered to be adverse treatment-related effects. Based on the details of the study and lack of overt toxicity, the no observed adverse effect level (NOAEL) for the test chemical is considered to be 1.23 mg/L.