7-methyl-3-methyleneocta-1,6-diene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 2001 to May 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP study equivalent or similar to OECD Guideline 408 with deviations: adrenals, spleen and brain weights not recorded, food consumption not followed, dosing 5 days/week instead of 7 days/week

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

adrenals, spleen and brain weights not recorded, food consumption not followed, dosing 5 days/week instead of 7 days/week

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:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methylideneocta-1,6-diene

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, USA)
- Age at study initiation: 5 weeks
- Housing: 5 animals/cage; polycarbonate cages; changed at least twice weekly
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardners, USA), ad libitum; changed at least weekly
- Water (e.g. ad libitum): Tap water (City of Columbus municipal supply) via automatic watering system, ad libitum
- Acclimation period: 11 (males) or 12 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3 °F
- Humidity (%): 50 ± 15 %
- Air changes (per h): 10/h
- Photoperiod (h dark / h light): 12 h dark / 12 h fluorescent light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared with corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Dose formulations were analyzed at the beginning, midpoint and end of the studies by GC-FID

Duration of treatment / exposure:

- Core study: 14 weeks
- Special study: 22 days

Frequency of treatment:

5 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500, 1000, 2000 and 4000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

20: 10 for core study + 10 for blood collection at Day 23

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Animals were distributed randomly into groups of approximately equal initial mean body weights
- Dose rationale: Based on published acute toxicity data

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Initially, weekly and at the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, weekly and at the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 23 and at the end of the study
- Anaesthetic used for blood collection: Yes (CO2/O2 mixture)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined: Hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 23 and at the end of the study
- Anaesthetic used for blood collection: Yes (CO2/O2 mixture)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined: Urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbital dehydrogenase, and total bile acids

REPRODUCTIVE PARAMETERS: Yes
- Time schedule for examinations: Sperm samples were collected at the end of the study; vaginal samples were collected for up to 12 consecutive days prior to the end of the studies
- How many animals: Sperm samples were collected from core study male animals in the 0, 250, 500, and 1000 mg/kg bw/day groups for sperm motility evaluations; vaginal samples were collected from core study females dosed with 0, 250, 500, and 1000 mg/kg bw/day for vaginal cytology evaluations
- Parameters examined: Sperm samples: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration; Vaginal samples: Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage; % of time spent in various estrous cycle stages and estrous cycle length were evaluated

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsies were performed on all core study animals and organs weighed were heart, right kidney, liver, lung, right testis, thymus; left cauda, left epididymis and left testis

HISTOPATHOLOGY: Yes
- Complete histopathologic examinations were performed on all core study rats in 0, 2000 and 4000 mg/kg bw/day groups and all animals that died early; tissues were fixed and preserved in 10% neutral buffered formalin and stained in hematoxylin/eosin
- In addition to gross lesions and tissue masses, the following tissues were examined to the no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), right testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus and right kidney

Other examinations:

None

Statistics:

- Survival: Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Organ and body weight data: Analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, spermatid and epididymal spermatozoal data: Analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964) or Jonckheere’s test (Jonckheere, 1954)
- Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across doses.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979) or chi-square statistics.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

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:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS
- No dose-related clinical findings were noted in animals surviving to the end of study.
- Dose-related clinical findings that occurred in animals that died early (some by moribund sacrifice) included thinness, lethargy, abnormal breathing, and ruffled fur. The cause of death of animals that died early was not determined. No clinical findings were seen in either sex receiving 1000 mg/kg bw/day or less.


MORTALITY
- Mortality in core study rats was 0/10 male and 0/10 female at 0 and 250 mg/kg bw/day; 1/10 males and 0/10 females at 500 mg/kg bw/day; 1/10 males and 1/10 females at 1000 mg/kg bw/day; 2/10 males and 4/10 females at 2000 mg/kg bw/day and 10/10 males and 10/10 females at 4000 mg/kg bw/day.
- Special study rats in the 4000 mg/kg bw/day groups died by the end of the first week. Survival of special study rats in the 250, 500 and 1000 mg/kg bw/day groups of both sexes was similar to that of the vehicle controls.


BODY WEIGHT AND WEIGHT GAIN
- Mean body weights were significantly decreased in the 500, 1000 and 2000 mg/kg bw/day groups.


HAEMATOLOGY & CLINICAL CHEMISTRY:
- In rats on Day 23, a decrease (approximately 25 %) in leukocyte counts, characterized by a decrease in lymphocytes, occurred in 2000 mg/kg bw/day males and females and was consistent with a transient, physiological (corticosteroid-induced-type) response. Such a response is consistent with the decreases in final body weights in the 2000 mg/kg bw/day males and females.
- In core study rats at week 14, dose-related decreases (up to 30 %) in creatinine concentration occurred in males and females and would be consistent with the decreased body weights. The leukon and creatinine effects were likely secondary biological effects.
- Other changes in hematology and clinical chemistry parameters seem inconsistent between sexes and in comparison to similar or complimentary markers.


ORGAN WEIGHTS
- Absolute and relative right kidney and liver weights of both sexes in all dosed groups were significantly greater than those of the vehicle controls with the exception of the absolute liver weight of the 2000 mg/kg bw/day males.
- Decreased absolute and relative thymus weights occurred in the 2000 mg/kg bw/day males.


REPRODUCTIVE PARAMETERS:
- No significant changes seen in the weights neither of the reproductive organs nor in the sperm parameters or estrous cyclicity of the male or female rats at any dose level


HISTOPATHOLOGY: NON-NEOPLASTIC
- Kidney: In rats evaluated on Day 23, the incidences and severities of chronic progressive nephropathy (CPN) and renal tubule necrosis were increased in 2000 mg/kg bw/day males. At the end of the 3 month study, the incidences of renal tubule necrosis were significantly increased in all dosed groups of males and females. Treatment-related increases in the incidences and severities of hyaline droplet accumulation were found in 250, 500 and 1000 mg/kg bw/day males; hyaline droplet accumulation was not observed in the 2000 mg/kg bw/day males and in any of the females
- Nose: At 3 months, the incidences and severities of olfactory epithelium degeneration in 2000 mg/kg bw/day males and females were significantly increased, and the severities were increased. The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased.
- Spleen: All 2000 mg/kg bw/day males and females had splenic atrophy.
- Mesenteric lymph node: Significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females.
- Stomach: Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females.
- Eye: Incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Selected clinical pathology data for rats in the 3‑month gavage study of β-myrcene^a

	Vehicle Control	0.25 g/kg	0.5 g/kg	1 g/kg	2 g/kg
Male
Hematology
n
Day 23	10	10	10	9	5
Week 14	9	10	9	9	8
Leukocytes (10^3/μL)
Day 23	10.54 ± 0.55	10.24 ± 0.40	9.65 ± 0.37	10.09 ± 0.64	7.70 ± 0.46**
Week 14	7.32 ± 0.28	8.49 ± 0.50	7.86 ± 0.55	7.52 ± 0.61	7.33 ± 0.39
Lymphocytes (10^3/μL)
Day 23	9.22 ± 0.53	8.78 ± 0.42	8.43 ± 0.33	8.65 ± 0.54	6.00 ± 0.41**
Week 14	6.15 ± 0.29	7.31 ± 0.50	6.70 ± 0.56	6.45 ± 0.59	5.66 ± 0.19
Clinical Chemistry
n
Day 23	10	10	10	9	6
Week 14	10	10	9	9	8
Creatinine (mg/dL)
Day 23	0.44 ± 0.02	0.49 ± 0.01	0.47 ± 0.02	0.49 ± 0.01*	0.52 ± 0.02**
Week 14	0.56 ± 0.02	0.58 ± 0.01	0.50 ± 0.00*	0.47 ± 0.02**	0.40 ± 0.00**
Female
Hematology
n
Day 23	9	7	8	8	5
Week 14	10	9	10	9	6
Leukocytes (10^3/μL)
Day 23	11.08 ± 0.37	12.14 ± 0.67	10.45 ± 0.36	9.60 ± 0.58	8.36 ± 0.48**
Week 14	7.83 ± 0.66	7.53 ± 0.29	8.45 ± 0.62	8.92 ± 0.66	7.72 ± 0.52
Lymphocytes (10^3/μL)
Day 23	9.74 ± 0.34	10.76 ± 0.63	9.26 ± 0.34	8.46 ± 0.51	7.27 ± 0.63**
Week 14	6.76 ± 0.52	6.23 ± 0.24	7.14 ± 0.47	7.83 ± 0.58	6.42 ± 0.43
Clinical Chemistry
n	10	10	10	9	6
Creatinine (mg/dL)
Day 23	0.49 ± 0.01	0.48 ± 0.01	0.48 ± 0.01	0.48 ± 0.02	0.48 ± 0.02
Week 14	0.57 ± 0.02	0.50 ± 0.02**	0.48 ± 0.01**	0.49 ± 0.01**	0.43 ± 0.02**

* Significantly different (P≤0.05) from the vehicle control group by Dunn’s or Shirley’s test

** P≤0.01

^aMean ± standard error. Statistical tests were performed on unrounded data. No data available for 4 g/kg males or females due to 100% mortality.

Table 2: Selected organ weights and organ-weight-to-body-weight ratios for rats in the 3‑month gavage study of β-myrcene^a

	Vehicle Control	0.25 g/kg	0.5 g/kg	1 g/kg	2 g/kg
Male
n	10	10	9	9	8
Necropsy body wt	341 ± 7	335 ± 7	318 ± 5*	300 ± 8**	255 ± 8**
R. Kidney
Absolute	0.964 ± 0.020	1.186 ± 0.021**	1.306 ± 0.028**	1.524 ± 0.033**	1.792 ± 0.064**
Relative	2.826 ± 0.049	3.545 ± 0.033**	4.109 ± 0.045**	5.099 ± 0.092**	7.014 ± 0.121**
Liver
Absolute	11.47 ± 0.21	12.76 ± 0.35*	12.78 ± 0.29*	13.44 ± 0.32**	12.55 ± 0.43
Relative	33.688 ± 0.742	38.084 ± 0.398**	40.205 ± 0.563**	44.930 ± 0.653**	49.121 ± 0.647**
Thymus
Absolute	0.350 ± 0.016	0.340 ± 0.018	0.285 ± 0.009**	0.272 ± 0.015**	0.205 ± 0.017**
Relative	1.024 ± 0.040	1.013 ± 0.048	0.899 ± 0.038	0.913 ± 0.052	0.795 ± 0.052**
Female
n	10	10	10	9	6
Necropsy body wt	196 ± 3	196 ± 3	187 ± 3	188 ± 3	185 ± 5
R. Kidney
Absolute	0.633 ± 0.012	0.799 ± 0.012**	0.828 ± 0.019**	0.953 ± 0.027**	1.197 ± 0.043**
Relative	3.229 ± 0.056	4.091 ± 0.062**	4.418 ± 0.056**	5.055 ± 0.085**	6.483 ± 0.143**
Liver
Absolute	5.990 ± 0.162	6.717 ± 0.109**	7.022 ± 0.164**	7.819 ± 0.219**	9.421 ± 0.326**
Relative	30.533 ± 0.641	34.407 ± 0.622**	37.463 ± 0.463**	41.499 ± 0.831**	51.003 ± 0.867**
Thymus
Absolute	0.265 ± 0.009	0.256 ± 0.009	0.248 ± 0.012	0.266 ± 0.009	0.224 ± 0.008*
Relative	1.353 ± 0.046	1.313 ± 0.050	1.321 ± 0.057	1.410 ± 0.036	1.213 ± 0.042

* Significantly different (P≤0.05) from the vehicle control group by Williams’ or Dunnett’s test

** P ≤ 0.01

^aOrgan weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error). No data available for 4 g/kg males or females due to 100% mortality.

Table 3: Incidences of selected nonneoplastic lesions in rats in the 3‑month gavage study of β-myrcene^a

	Vehicle Control	0.25 g/kg	0.5 g/kg	1 g/kg	2 g/kg
Male
Kidney^b	7	10	9	10	10
Renal Tubule, Necrosis^c	0	10** (1.0)^d	9** (1.1)	10** (1.8)	10** (2.9)
Nephropathy	7 (1.0)	10 (1.0)	9 (1.3)	8 (1.0)	9 (1.9)
Nephrosis	0	0	1 (1.0)	10** (1.0)	9** (2.7)
Renal Tubule, Accumulation, Hyaline Droplet	0	10** (2.0)	9** (2.4)	10** (2.1)	0
Nose	10	10	10	10	10
Olfactory Epithelium, Degeneration	0	0	0	2 (1.0)	8** (2.6)
Inflammation, Suppurative	0	0	0	1 (1.0)	3 (1.0)
Inflammation, Chronic	0	0	1 (1.0)	6** (1.0)	8** (1.1)
Spleen	10	10	10	10	10
Atrophy	0	0	1 (2.0)	0	10** (1.8)
Mesenteric Lymph Node	10	0	1	1	10
Atrophy	0		0	1 (1.0)	6** (1.7)
Harderian Gland	10	10	10	10	9
Pigmentation, Porphyrin	1 (1.0)	3 (1.0)	7** (1.0)	8** (1.1)	9** (1.3)
Female
Kidney	10	10	10	10	10
Renal Tubule, Necrosis	0	10** (1.0)	10** (1.0)	9** (2.2)	9** (2.4)
Nephropathy	1 (1.0)	2 (1.0)	3 (1.0)	4 (1.0)	1 (1.0)
Nephrosis	0	0	0	10** (1.0)	7** (1.1)
Nose	10	0	10	10	10
Olfactory Epithelium, Degeneration	0		0	2 (1.5)	7** (2.9)
Inflammation, Suppurative	0		0	1 (1.0)	6** (1.7)
Inflammation, Chronic	0		0	9** (1.0)	4* (1.5)
Spleen	10		10	10	10
Atrophy	0		0	1 (1.0)	10** (1.8)
Mesenteric Lymph Node	10		0	3	10
Atrophy	0			2** (1.0)	4* (1.8)
Forestomach	10		0	10	10
Inflammation, Acute	0			0	4* (1.8)

* Significantly different (P≤0.05) from the vehicle control group by the Fisher exact test

** P ≤ 0.01

^a No data presented for 4 g/kg males or females due to early mortality.

^b Number of animals with tissue examined microscopically

^c Number of animals with lesion

^d Average severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked

Applicant's summary and conclusion

Conclusions:

A significant increase in liver and kidney weights in males and females, associated with minimal renal tubule necrosis and only in males, hyaline droplet accumulation, were observed at 250 mg/kg bw/day. Therefore, no NOAEL was identified and a LOAEL was set at 250 mg/kg bw/day.

Executive summary:

A GLP study was conducted to evaluate the cumulative toxic effects of repeated exposure to β‑myrcene according to a method equivalent or similar to OECD Guideline 408.

 

Groups of 20 F334/N rats (10/sex/dose) were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day of β-myrcene in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses for 22 days. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.

 

All rats exposed to 4000 mg/kg bw/day died and 2/10 males and 4/10 females died in the 2000 mg/kg bw/day group. Dose-related clinical findings in animals that died early included thinness, lethargy, abnormal breathing and ruffled fur. Mean body weights were significantly decreased in the 500, 1000 and 2000 mg/kg bw/day groups. A significant increase in liver and kidney weights was observed in all dosed animals as well as renal tubule necrosis. A significant decrease in creatinine was also detected in all dosed females and in males at 500 mg/kg bw/day and higher doses. The incidences and severities of olfactory epithelium degeneration were increased in males and females of the 2000 mg/kg bw/day group.The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased. All 2000 mg/kg bw/day males and females had splenic atrophy. In the mesenteric lymph node, significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females. Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females. The incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.

 

As a significant increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females, as well as hyaline droplet accumulation only in males, were observed at 250 mg/kg bw/day, no NOAEL could be identified in this study and a LOAEL was set at 250 mg/kg bw/day.