(S)-p-mentha-1,8-diene

Repeated dose toxicity: oral

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:

other: 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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study 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

Test material information

Constituent 1

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:

4-isopropenyl-1-methylcyclohexene

Details on test material:

- Name of test material (as cited in study report): d-limonene
- Lot/batch No.: 1F57A
- Source: SCM Corporation (Jacksonville, USA)
- Physical state: Clear, colorless liquid
- Boiling point: 177 °C
- Density (at 21 °C): 0.84207 g/mL
- Analytical purity: > 99%; purity determined by elemental analysis, water analysis, the Food Chemicals Codex (1972) test for peroxide content and GC)
- Stability under test conditions: Stable as a bulk chemical for 2 weeks at temperatures up to 60 °C; no degradation was seen over the course of the study
- Storage condition of test material: Room temperature
- Maximum storage time: 8 days

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, 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

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

413 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

825 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 650 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

3 300 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

6 600 mg/kg bw/day (nominal)

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

825 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: mortality at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 1650 mg/kg bw/day

2

Key result

Dose descriptor:

LOAEL

Effect level:

1 650 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

mortality

3

Key result

Dose descriptor:

NOAEL

Effect level:

1 650 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: mortalities at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 3300 mg/kg bw/day

4

Key result

Dose descriptor:

LOAEL

Effect level:

3 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

mortality

Target system / organ toxicity

Key result

Critical effects observed:

not specified

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:

Under the test conditions, 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 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.

 

Under the test conditions, 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.