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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1987

Materials and methods

Principles of method if other than guideline:
- Principle of test: Subacute toxicity study (1 or 4 weeks). Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-p-mentha-1,8-diene
EC Number:
227-813-5
EC Name:
(R)-p-mentha-1,8-diene
Cas Number:
5989-27-5
Molecular formula:
C10H16
IUPAC Name:
(4R)-isopropenyl-1-methylcyclohexene

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
5 or 26 days (5 days/week)
Frequency of treatment:
5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five males
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
- All animals were weighed daily and feed consumption was noted weekly.
- On Days 6 and 27, approximately 24 hours after the 5th and 20th doses, respectively, animals from the appropriate groups were weighed, anaesthetized by ethyl ether inhalation, and killed by exsanguinations from the posterior vena cava. The liver and kidneys were removed and weighed.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.

HISTOPATHOLOGY: Yes

-Transverse sections were fixed and preserved in 10% neutral buffered formalin and stained with haematoxylin/eosin. Additional kidney sections were treated with Mallory’s Heidenhain stain.
-Tissues from right kidney of three control and three d-limonene-treated (150 mg/kg bw/day) animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
daily in-life observation did not reveal any grossly evident indication of dose-related toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight gains values for treatment groups were, at both time points, similar to those of the vehicle control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption values for treatment groups were, at both time points, similar to those of the vehicle control groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Examination of animals at necropsy did not reveal any grossly evident indication of dose-related toxicity.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-related hyaline droplet formation associated with renal accumulation of α2μ-globulin was observed in all rats killed on Day 6.
Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male
Basis for effect level:
other: nephrotoxicity and accumulation of hyaline droplets (male rat specific)
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Executive summary:

In a subacute toxicity study, d-limonene was administered through gavage to groups of 5 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 75, 150 and 300 mg/kg bw/day for 1 or 4 weeks (5 days/week). Animals were weighed daily and feed consumption was recorded weekly. On Days 6 and 27, approximately 24 hours after the 5th and 20th doses, respectively, animals from the appropriate groups were subjected to gross necropsy during which weights of liver and kidneys were recorded and transverse sections were processed for histological examination. Tissues from right kidney of animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation. Neither daily in-life observation nor examination of animals at necropsy revealed any grossly evident indication of dose-related toxicity. Weight gains and feed consumption values for treatment groups were similar to those of the vehicle control groups. Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27. Dose-related hyaline droplet formation associated with renal accumulation of α2µ-globulin was observed in all rats killed on Day 6. Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27. As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.