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Toxicological information

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:
key study
Study period:
From September 18 to October 04, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 407 but with deviations: study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 109-117 g; females: 97-103 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 12 or 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-76 °F
- Humidity (%): 80-90%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
12 doses over 16 days
Frequency of treatment:
Once per day; 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Random
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on seven rats from survivors of highest dose groups
Other examinations:
No
Statistics:
No data

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats in 6600 mg/kg bw/day group and 5/5 males and 3/5 females in 3300 mg/kg bw/day group died within the first 2 days.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls.
- Final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls.
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:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
825 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
1 650 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Survival and mean body weights of rats in the 16-day gavage studies of d-limonene

 

Dose (mg/kg bw/day)

Survival (a)

Mean body weights (grams)

Final weight relative
to vehicle controls (%)

Initial (b)

Final

Change (c)

Male

0

  5/5

115 ± 2

173 ± 3

58 ± 3

-

413

  5/5

113 ± 2

171 ± 4

58 ± 3

99

825

  5/5

113 ± 3

173 ± 4

60 ± 5

100

1650

  5/5

113 ± 3

156 ± 4

43 ± 3

90

3300

 (d) 0/5

114 ± 2

(e)

(e)

(e)

6600

 (f) 0/5

111 ± 2

(e)

(e)

(e)

Female

0

  5/5

98 ± 1

123 ± 1

25 ± 1

-

413

  5/5

101 ± 2

(g) 139 ± 2

38 ± 3

113

825

  5/5

100 ± 2

131 ± 3

31 ± 4

107

1650

  5/5

101 ± 2

127 ± 1

27 ± 2

103

3300

 (f) 2/5

102 ± 2

113 ± 8

10 ± 5

92

6600

 (f) 0/5

103 ± 4

(e)

(e)

(e)

(a) Number surviving/number initially in the group

(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.

(c) Mean body weight change of the survivors ± standard error of the mean

(d)Day of death: 1, 1, 1, 1, 2

(e) No data are reported due to the 100% mortality in this group.

(f) Day of death all 1

(g) One final body weight not recorded; weight change based on remaining four animals.

Applicant's summary and conclusion

Conclusions:
The NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.
Executive summary:

In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene was administered through gavage to groups of 5 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on seven rats from survivors of highest dose groups.

 

All rats that received 6600 mg/kg bw/day and 5/5 males and 3/5 females that received 3300 mg/kg bw/day of d-limonene died within the first 2 days. The final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls. The final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls. No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower. No treatment-related lesions were observed.

 

Therefore, the NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.