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

Endpoint summary

Description of key information

In a three-month study conducted in rats, a LOAEL was set at 250 mg/kg bw/day based on 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.

In a three-month study conducted in mice, a NOAEL was set at 250 mg/kg/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day.

A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day.

In a three-month study conducted in mice, a NOAEL was set at 250 mg/kg bw/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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

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

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

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.

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.

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

Critical effects observed:

not specified

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

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.

Reference 2

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

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: 5-6 weeks
- Housing: 1 (males) or 5 (females) 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: 13 (females) or 14 (males) 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

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
- All dose formulations were within 10 % of the target concentrations

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

5 days/week

Remarks:

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

No. of animals per sex per dose:

10

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

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: At termination 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: At termination 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 mice in 0, 1000, 2000 and 4000 mg/kg bw/day groups; 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, gallbladder, 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. The bone marrow, lymph nodes (mandibular and mesenteric), nose, pancreas, spleen, and thymus were examined in all dosed groups of mice remaining.

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.

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.
- Clinical findings in animals that did not survive to the end of the study included thinness, lethargy, and abnormal breathing.


MORTALITY
- All 4000 mg/kg bw/day male and female mice died during week 1; 9/10 males and 8/10 females in 2000 mg/kg bw/day group died by week 4


BODY WEIGHT AND WEIGHT GAIN
- Mean body weights of 1000 mg/kg bw/day males and 500 and 2000 mg/kg bw/day females were significantly less than those of the vehicle controls


HAEMATOLOGY & CLINICAL CHEMISTRY:
- At week 14, an approximately 15-20 % decrease in hematocrit, hemoglobin, and erythrocyte count values occurred in the 1000 mg/kg bw/day male and female mice. However, the erythron effect may be secondary to the decreased body weights.


ORGAN WEIGHTS
- Liver: Relative liver weights of 500 and 1000 mg/kg bw/day males and the absolute and relative liver weights of 250 mg/kg bw/day males and 500 and 1000 mg/kg bw/day females were significantly greater than those of the vehicle controls
- Kidney: Absolute and relative right kidney weights of 1000 mg/kg bw/day females, and the relative kidney weights or 250 and 500 mg/kg bw/day females were significantly greater than those of the vehicle controls.


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 mice at any dose level


HISTOPATHOLOGY: NON-NEOPLASTIC
- No histopathology changes were observed in mice administered 1000 mg/kg bw/day or less.
- Mice in 2000 and 4000 mg/kg bw/day groups were not evaluated due to early deaths.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights at 250 and 500 mg/kg bw/day not related with any other toxic effect (bodyweight, hematology, histopathology)

Critical effects observed:

not specified

Table 1: Survival and body weights of mice in the 3‑month gavage study of β-myrcene

Dose (g/kg)	Survival^a	Mean Body Weight^b(g)			Final Weight Relative to Controls (%)
		Initial	Final	Change
Male
0	10/10	23.8 ± 0.3	34.6 ± 1.0	10.7 ± 0.8
0.25	10/10	23.1 ± 0.3	35.8 ± 0.6	12.7 ± 0.6	103
0.5	10/10	23.7 ± 0.3	34.6 ± 0.9	10.9 ± 0.9	100
1	10/10	23.4 ± 0.3	31.6 ± 0.8*	8.2 ± 0.7*	91
2	1/10^c	23.3 ± 0.2	26.4 ± 3.5	76
4	0/10^d	23.5 ± 0.3	-	-
Female
0	10/10	19.4 ± 0.3	28.0 ± 0.7	8.5 ± 0.5
0.25	10/10	19.0 ± 0.3	26.3 ± 0.6	7.3 ± 0.5	94
0.5	10/10	18.9 ± 0.2	25.7 ± 0.4*	6.8 ± 0.3*	92
1	10/10	18.6 ± 0.4	27.1 ± 0.4	8.5 ± 0.3	97
2	2/10^e	19.4 ± 0.3	22.0 ± 0.2**	2.7 ± 1.2**	78
4	0/10^d	18.7 ± 0.2	-	-

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

** P≤0.01

^aNumber of animals surviving at 3 months/number initially in group.

^bWeights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study. No statistical analysis performed for 2 g/kg males because less than two measurements available.

^cWeeks of death: 1, 1, 3, 3, 3, 3, 4, 4, 4

^dWeek of deaths: 1

^eWeeks of death: 1, 1, 1, 1, 4, 4, 4, 4 Table 2: Selected hematology data for mice in the 3-month gavage study of β-myrcene^a

	Vehicle control	0.25 g/kg	0.5 g/kg	1 g/kg
Male
n	10	10	9	10
Hematocrit (auto) (%)	48.7 ± 0.3	48.7 ± 0.5	48.4 ± 0.4	47.0 ± 0.4**
Hemoglobin (g/dL)	16.4 ± 0.1	16.4 ± 0.1	16.3 ± 0.1	15.8 ± 0.2**
Erythrocytes (10^6/μL)	10.78 ± 0.10	10.65 ± 0.11	10.50 ± 0.07	10.11 ± 0.10**
Female
n	10	10	10	10
Hematocrit (auto) (%)	47.5 ± 0.5	48.0 ± 0.6	47.4 ± 0.4	46.8 ± 0.2
Hemoglobin (g/dL)	16.1 ± 0.2	16.3 ± 0.2	16.1 ± 0.1	15.7 ± 0.1
Erythrocytes (10^6/μL)	10.37 ± 0.11	10.50 ± 0.14	10.29 ± 0.10	9.92 ± 0.07*

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

** P ≤ 0.01

^a Mean ± standard error. Statistical tests were performed on unrounded data. No data presented for 2 or 4 g/kg males or females due to early mortality.

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

	Vehicle Control	0.25 g/kg	0.5 g/kg	1 g/kg
Male
n	10	10	9	10
Necropsy body wt	35.8 ± 1.0	36.6 ± 0.7	35.8 ± 0.9	32.7 ± 0.8*
Liver
Absolute	1.453 ± 0.043	1.593 ± 0.036*	1.537 ± 0.042	1.557 ± 0.038
Relative	40.689 ± 0.735	43.580 ± 0.850*	43.104 ± 1.201*	47.687 ± 0.516**
Female
n	10	10	10	10
Necropsy body wt	27.9 ± 0.7	26.1 ± 0.5	26.1 ± 0.5	26.9 ± 0.3
R. Kidney
Absolute	0.164 ± 0.004	0.175 ± 0.004	0.173 ± 0.003	0.193 ± 0.004**
Relative	5.901 ± 0.150	6.719 ± 0.178**	6.621 ± 0.084**	7.197 ± 0.116**
Liver
Absolute	1.127 ± 0.024	1.143 ± 0.036	1.236 ± 0.025*	1.369 ± 0.019**
Relative	40.604 ± 1.024	43.862 ± 1.481	47.458 ± 1.076**	50.985 ± 0.531**

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

** P≤0.01

^a Organ 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 presented for 2 or 4 g/kg males or females due to early mortality.

Conclusions:

A NOAEL was set at 250 mg/kg/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights were observed at 250 and 500 mg/kg bw/day but they were not associated with any other toxic effect (bodyweight, hematology, histopathology).

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 B6C3F1 mice (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. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.

All 4000 mg/kg bw/day (male and female mice) died during week 1; 9/10 males and 8/10 females in 2000 mg/kg bw/day group died by week 4. The mean body weights of 1000 mg/kg bw/day males and 500 and 2000 mg/kg bw/day females were significantly lower than those of the vehicle controls. Clinical findings in animals that did not survive to the end of the study included thinness, lethargy, and abnormal breathing. Absolute and relative right kidney weights of 1000 mg/kg bw/day females, and the relative kidney weights of 250 and 500 mg/kg bw/day females were significantly greater than those of the vehicle controls. Relative liver weights of 500 and 1000 mg/kg bw/day males and the absolute and relative liver weights of 250 mg/kg bw/day males and 500 and 1000 mg/kg bw/day females were significantly greater than those of the vehicle controls. No histopathology changes were observed in mice administered 1000 mg/kg bw/day or less. Mice in 2000 and 4000 mg/kg bw/day groups were not evaluated due to early deaths.

A NOAEL was set at 250 mg/kg/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights were observed at 250 and 500 mg/kg bw/day but they were not associated with any other toxic effect (bodyweight, hematology, histopathology).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Rats treated only 14 days, very limited observations performed, only 3-4 rats used.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Rats were given test substance mixed in feed for 14 days and the following parameters were compared to a control group: food intake, body weight gain, liver weight, cholesterol, triacylglycerol and Apo A-I.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
1 %
Basis:
nominal in diet

No. of animals per sex per dose:

3 to 4

Control animals:

yes, concurrent no treatment

Critical effects observed:

not specified

Table 1: Results on the parameters observed

	Food intake (g/day)	Body weight gain (g/14days)	Liver weight (g/100 g bw)	Cholesterol (mg/dL)	Triacylglycerol (mg/dL)	Apo A-I (mg/dL)
Control	20.7 ± 0.7	68 ± 8	3.3 ± 0.1	91 ± 1	98 ± 7	66 ± 4
Myrcene	21.3 ± 0.9	70 ± 4	3.9 ± 0.1 *	102 ± 67	156 ± 31	63 ± 4

* significantly different from control group at p<0.05 by Student’s t test

Conclusions:

Myrcene given at 1% in feed for 14 days significantly increased liver weight in rats.

Executive summary:

Myrcene was given at 1% in feed for 14 days to 3 to 4 Wistar male rats. Food intake, body weight gain, liver weight, cholesterol, triacylglycerol and Apo A-I were evaluated at the end of the treatment.

The only statistically significant difference with control group was an increase in liver weight in rats.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

Purity: 93.3 %

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

None

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of test substance in the diet was evaluated by analysing dietary levels of myrcene on days 0, 4, 7, and 10 following preparation, as well as daily, for seven days, in an independent stability assessment. At the beginning, middle, and end of the study, diet preparation samples were analysed to verify the concentration of myrcene in the diet over the course of the study.
The myrcene content at the 700 mg/kg feed level was found to decrease from 72 to 18 % from day 0 to day 7, with an average intake of 45 % of the target concentration over the course of the study. Similarly, the myrcene content at the 2100 mg/kg feed level decreased from 65 to 30 %, with an average intake of 44 % of the target concentration over the course of the study. The myrcene content at the 4200 mg/kg feed level decreased from 69 to 17 % from day 0 to day 7, with an average intake of 43 % of the target concentration over the course of the study.
The concentration on the last day of the week for quantification of the exposure was considered for test item intake rather than the (logarithmic) mean over the whole week. Based on the stability data, weekly diet refreshment, and measured dietary intake, an adjusted calculated mean daily intake of 8.0, 40 , and 44 mg/kg bw per day for males, and 9.6, 48 and 53 mg/kg bw per day for females for 90 days was calculated.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous

Dose / conc.:

700 mg/kg diet

Dose / conc.:

2 100 mg/kg diet

Dose / conc.:

4 200 mg/kg diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control:

None

Observations and examinations performed and frequency:

Prior to study initiation and again on day 88, the eyes of all rats were examined by focal illumination and indirect ophthalmoscopy. The animals were observed for viability, signs of gross toxicity and behavioural changes at least once daily during the study and weekly for a battery of detailed clinical observations. Body weights were recorded twice during acclimation, including prior to test initiation (day 0) and together with food consumption, weekly thereafter and prior to terminal sacrifice. Blood and urine samples were collected on day 89 from all surviving study animals for urinalysis, hematology, and clinical chemistry determinations. Coagulation assessments were performed on day 92 or 93, preceding necropsy.

Sacrifice and pathology:

Gross necropsies were performed on all study animals and histological evaluation of selected organs and tissues from control animals (0 mg/kg feed), animals administered the highest dietary level, and tissues with gross macroscopic observations.

Other examinations:

None

Statistics:

Not specified

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some incidental changes in hematology parameters were within approximate historical control values, did not correlate with macroscopic or histopathological findings, were without biologic impact, and were considered not toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some incidental changes in clinical chemistry parameters were within approximate historical control values, did not correlate with macroscopic or histopathological findings, were without biologic impact, and were considered not toxicologically relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A few histopathological changes were considered incidental, spontaneous in nature as observed for the age and strain of rat used in this study, and had no established relationship to administration of the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects up to the highest dose tested

Remarks on result:

other: underestimated exposure based on minimum concentration before diet replacement

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects up to the highest dose tested

Remarks on result:

other: underestimated exposure based on minimum concentration before diet replacement

Key result

Critical effects observed:

no

Conclusions:

Based on the toxicological endpoints evaluated in this study, the no-adverse effect level (NOAEL) for administration of myrcene in the diet was determined to be the highest dose group, calculated to provide an estimated daily intake of 44 mg/kg bw per day for males and 53 mg/kg bw per day for females, respectively.

Executive summary:

In an OECD 408 study, four groups of adult Sprague-Dawley rats (10/sex/group) were maintained on diets prepared to contain 0, 700, 2100, or 4200 mg/kg feed of myrcene. Prior to study initiation and again on day 88, the eyes of all rats were examined by focal illumination and indirect ophthalmoscopy. The animals were observed for viability, signs of gross toxicity and behavioural changes at least once daily during the study and weekly for a battery of detailed clinical observations. Body weights were recorded twice during acclimation, including prior to test initiation (day 0) and together with food consumption, weekly thereafter and prior to terminal sacrifice. Blood and urine samples were collected on day 89 from all surviving study animals for urinalysis, hematology, and clinical chemistry determinations. Coagulation assessments were performed on day 92 or 93, preceding necropsy. Gross necropsies were performed on all study animals and histological evaluation of selected organs and tissues from control animals (0 mg/kg feed), animals administered the highest dietary level, and tissues with gross macroscopic observations.

There were no mortalities, clinical, or ophthalmological changes attributable to myrcene administration. There were no statistically significant, dietary concentration-dependent changes in body weight, body weight gain, food consumption, or food efficiency in males and females attributed to the administration of myrcene during the study.

There were no clinical pathological findings, changes in macroscopic or microscopic histopathology, or organ weight changes in the groups that received myrcene. Some incidental changes in clinical chemistry and hematology parameters were within approximate historical control values, did not correlate with macroscopic or histopathological findings, were without biologic impact, and were considered not toxicologically relevant. A few histopathological changes were considered incidental, spontaneous in nature as observed for the age and strain of rat used in this study, and had no established relationship to administration of the test substance.

Therefore, the no-adverse effect level (NOAEL) for administration of myrcene in the diet was determined to be the highest dose group, calculated to provide an estimated daily intake of 44 mg/kg bw per day for males and 53 mg/kg bw per day for females, respectively.

Endpoint conclusion

Dose descriptor:

LOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Organ:

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a three-month study conducted in rats, 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.

In a three-month study conducted in rats by diet, no toxicological effects related to the test item were observed up to the highest dose tested; a NOAEL was set at the underestimated daily intake of 44 mg/kg bw/day for males and 53 mg/kg bw/day for females, respectively, based on minimal concentrations analysed in the diet. This NOAEL would correspond to 111 and 134 mg/kg bw/day for males and females, respectively, when considering an average intake of 43% of target concentration.

In a three-month study conducted in mice, a NOAEL was set at 250 mg/kg bw/day in females based on statistically significant increase in relative and absolute liver weights and relative kidney weights associated with a statistically significant decrease in bodyweight at 500 mg/kg bw/day. A NOAEL was set at 500 mg/kg bw/day in males based on mortality and statistically significant decreased bodyweight at 1000 mg/kg bw/day. Slight increases in relative liver weights were observed at 250 and 500 mg/kg bw/day but they were not associated with any other toxic effect (bodyweight, hematology, histopathology).

Justification for classification or non-classification

In a three-month study conducted in rats by diet, no toxicological effects related to the test item were observed up to the highest dose tested, corresponding to an average intake of 111 and 134 mg/kg bw/day for males and females, respectively.

As no adverse effects on target organs were observed below the threshold of 100 mg/kg bw/day, myrcene is not deemed to be classified for repeated dose toxicity according to CLP Regulation (EC) No 1272/2008.