3,7-dimethylocta-2,6-dienyl acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

Thirteen-week repeated dose study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

This information is used for read-across to Neryl acetate multi

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1982

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological, and Physical Agents in Laboratory Animals for the National Toxicology Program (NTP)

Principles of method if other than guideline:

As a pre-study for a carcinogenicity study, rats (n= 10/sex/dose) were exposed 5 days/week by gavage to geranyl acetate (71% geranyl acetate (CAS 105-87-3) and 29% citronellyl acetate (CAS 150-84-5)) in corn oil at doses of 250, 500, 1,000, 2,000 or 4,000 mg/kg bw/day for 13 weeks. Vehicle controls received corn oil alone. All animals were observed twice daily for signs of clinical signs, morbidity or mortality. Body weights were recorded every week. The mean body weight of each group was calculated by dividing the total weight of all animals in the group by the number of surviving animals in the group. Necropsies were performed on all animals. The following tissues were examined microscopically (controls and high dose group only): tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, sciatic nerve, bone marrow, thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles/ prostate/ testes or ovaries/ uterus, brain, pituitary, and spinal cord. The health of the animals was monitored during the course of the studies according to the protocols of the NTP Sentinel Animal Program.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Geranyl acetate

EC Number:

203-341-5

EC Name:

Geranyl acetate

Cas Number:

105-87-3

Molecular formula:

C12H20O2

IUPAC Name:

(2E)-3,7-dimethylocta-2,6-dien-1-yl acetate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Lot No. 36948 (Givaudan Corp.)
- Purity: ≥ 70.63% (impurities: 29% citronellyl acetate, remaining impurities (7 impurities, not further identified at appr. 0.37%).
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the dark at 5°C
- Stability under test conditions: stable (as shown by reanalysis)
- Solubility and stability of the test substance in the solvent/vehicle: Samples of the mixtures selected at random during the carcinogenicity study were analyzed periodically at Southern Research Institute, and the results indicated that all analyzed mixtures were properly formulated (within 10% of the target concentration).

Test animals

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Industries, Indianapolis, USA
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 101.3 - 109.8 g (mean weight males per dose group), 86.7 - 95.4 g (mean weight females per dose group)
- Fasting period before study: no
- Housing: group-housed (5 animals/ cage), polycarbonate cages with bedding of Beta Chips@ heat treated hardwood chips, Northeastern Products Corp., Warrensburg, NY, USA
- Diet: ad libitum, Wayne@ Lab Blox pellets, Allied Mills, Inc. Chicago, IL, USA
- Water: ad libitum, tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-27
- Humidity (%): 15-96
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Appropriate amounts of geranyl acetate were mixed with enough corn oil to give the desired concentration for the high dose groups. Gavage solutions for lower doses were prepared by diluting this stock solution with corn oil. Geranyl acetate/corn oil mixtures at the 2% (v/v) level were analyzed at Midwest Research Institute and found to be stable at room temperature for 7 days. Samples of the mixtures selected at random were analyzed periodically at Southern Research Institute, and the results indicated that all analyzed mixtures were properly formulated (within 10% of the target concentration). The results from the three analyses conducted at Midwest Research Institute also confirmed this finding.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/ week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

4 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A single dose test and a fourteen day exposure study were performed. Together with this 13-week exposure study these studies were used as pre-tests for a carcinogenicity study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily for clinical signs, morbidity, and mortality;

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all animals, major tissues or organs were examined)

HISTOPATHOLOGY: Yes (control and high dose animals). The following tissues or organs were examined microscopically for control and high dose groups: gross lesions, tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, scatic nerve, bone marrow, thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, seinal vesicles/prostate/ testes or ovaries/uterus, brain, pituitary, and spinal cord.

Statistics:

Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958). Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not sta- tistically censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) extensions of Cox's methods for testing for a dose-related trend. All reported P-values for the survival analyses are two-sided.
The incidence of neoplastic or nonneoplastic lesions has been given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. In most instances, the denominators included only those animals forwhich that site was examined histologically. However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumors) prior to histologic sampling, or when lesions could have appeared at multiple sites (e.g., lymphomas), the denominators consist of the number of animals on which necropsies were performed.
For the statistical analysis of tumor incidence data, two different methods of adjusting for intercurrent mortality were employed. Each used the classical method for combining contingency tables developed by Mantel and Haenszel (1959). Tests of significance included pairwise comparisons of high and low dose groups with controls and tests for overall dose-response trends.
In addition to these tests, one other set of statistical analyses was carried out and reported in the tables analyzing primary tumors: the Fisher’s exact test for pairwise comparisons and Cochran-Armitage linear trend test for dose- response trends (Armitage, 1971; Gart et al., 1979). These tests were based on the overall proportion of tumor-bearing animals. All reported P values for the tumor incidence analyses are one-sided.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No compound-related clinical signs were observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two of 10 male rats and 1/10 female rats died at 4000 mg/kg bw/day. One male rat in the 500 mg/kg bw/day group dies due to gavage error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 4000 mg/kg bw/day mean body weight compared to controls was depressed in males and in females (-19% and -8%, respectively, compared to controls). No significant effect on body weight was found for the other groups (males: -1%, -1%, 0% and -5% for groups dosed at 250, 500, 1000 and 2000 mg/kg bw/day, respectively; females: -1%, -4%, 0% and +1% for groups dosed at 250, 500, 1000 and 2000 mg/kg bw/day, respectively).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Reddened mucosa of the stomach was observed in 3/10 males that received 4000 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related histopathologic effects were observed.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

mortality

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Results of the pre-studies (rat):

Single dose study: All rats receiving 8000 mg/kg bw died on day 2. No further mortality occurred. All rats were inactive immediately after dosing.

Fourteen-day study: All animals survived to the end of the dosing period. Weight gains by dosed and control groups were comparable. The activity of all rats that received 1000 mg/ kg decreased after dosing between days 2 and 4 of the studies. No compound-related effects were observed during necropsy.

 

Results in mice

Single dose study: Four of the five male mice receiving 8000 mg/kg bw/day died (two on day 2 and two on day 3), and 5/5 female mice that received this dose died (four on day 2 and one on day 3). All mice administered 1000-8000 mg/kg bw were inactive immediately after dosing.

Fourteen-day study: Three female mice that received 2000 mg/kg bw/day died. All other animals survived to the end of the dosing period. Mean body weight gains by dosed groups were not adversely affected by administration of geranyl acetate. All mice that received 1000 mg/kg bw/day or more were inactive after the dose was administered but they returned to normal within 24 hours. One of five male mice that received 2000 mg/kg bw/day had a thickened duodenal wall, and 3/ 5 female mice receiving 2000 mg/kg bw/day had a thickened wall of the cardiac stomach.

Thirteen-week study: Seven of ten males and 9/10 females receiving 2000 mg/kg bw/day died. Three female mice at lower doses died as a result of gavage error. All other animals survived to the end of the studies. Overall, mean body weights of dosed groups were comparable with those of the controls. Cytoplasmic vacuolization of the liver, kidney, and myocardium was observed in male and female mice at the 2000 mg/kg bw/day dose level (liver: 7/10 males and 8/9 females; kidney: 2/10 males and 4/9 females; myocardium: 2/10 males and 1/9 females). The vacuoles were found to contain lipids. In the liver, the lipid droplets varied in size from barely visible to larger than the nuclei of the hepatocytes. The nuclei of the hepatocytes remained in the center of the cells. The lipidosis was present throughout the lobules, particularly in the periportal area. The lipid droplets in the kidney were present in the cytoplasm of the proximal tubules in a subnuclear location. The myocardium contained fine lipid droplets within the fibers and the myofibriles. Stomach lesions, consisting of focal suppurative inflammation, focal ulcerative inflamma tion, or submucosal edema, were found in 2/10 males and 6/10 females that received 2000 mg/kg bw/day.

Since the results in mice were not considered to affect the conclusion based on the rat studies, the results were not further summarized in detail.

Applicant's summary and conclusion

Conclusions:

A 13-week repeated dose study was conducted with food grade Geranyl Acetate in rats. Mortality was seen (2/10 males; 1/10 females) and the body weight gain was decreased in the high dose group (dosed at 4000 mg/kg bw/day) in males and females, The NOAEL is 2000 mg/kg bw/d.

Executive summary:

In a 13-week study with 10 rats/sex/dose, and test doses of 0, 250, 500, 1000, 2000 and 4000 mg/kg bw/day, rats were observed for clinical signs, morbidity, and mortality twice daily; rats were weighed weekly; necropsies were performed on all animals, histological examinations were performed on control and high-dose groups. In this study no test substance-related histopathological effects or increase in tumour-incidence were noted. Since the survival and the body weight gain was decreased in the high dose group (dosed at 4000 mg/kg bw/day) in males and females, the NOAEL of the 13-week study was established to be 2000 mg/kg bw/day.

In parallel the same set of tests (including the two-year carcinogenicity study) were performed with mice. The results in the mice studies were not considered to affect the conclusion based on the rat studies.

Other available information

This 13 wk study was a pre-study for a carcinogenicity study which is shortly presented below as well as an other preliminary study

A carcinogenicity study was conducted in which rats (n= 50/sex/dose) were exposed daily by gavage to Geranyl Acetate in corn oil at doses of 1,000 and 2,000 mg/kg bw/day for two years. Several pre-tests were performed in order to determine the final test concentrations in the carcinogenicity test. These pre-studies were reported to be performed with Geranyl Acetate containing 6-17% Citronellyl Acetate (‘mono’).

In a 14-day study with 5 rats/sex/dose, with test doses of 0, 62, 125, 250, 500 and 1000 mg/kg bw/day, rats were observed for clinical signs, morbidity, and mortality twice daily; rats were weighed weekly; necropsy was performed on all animals. No mortality was seen, weight gains by dosed and control groups were comparable. The activity of all rats that received 1,000 mg/ kg bw/day decreased after dosing between days 2 and 4 of the study. No compound-related effects were observed during necropsy.