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Toxicological information

Carcinogenicity

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Administrative data

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
The reliability is based on read across for a structural analogue compound, myrcene (CAS 123-35-3). The myrcene study report was conclusive and done to valid OECD and EU guidelines and the study was conducted under GLP conditions. The study for myrcene was conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
Myrcene and Farnesene have similar chemical structures and therefore are expected to have similar physical/chemical characteristics. Thus, read across of myrcene data is a reasonable approach.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
carcinogenicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
The reliability is based on read across for a structural analogue compound, myrcene (CAS 123-35-3). The myrcene study report was conclusive and done to valid OECD and EU guidelines and the study was conducted under GLP conditions. The study for myrcene was conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
Myrcene and Farnesene have similar chemical structures and therefore are expected to have similar physical/chemical characteristics. Thus, read across of myrcene data is a reasonable approach.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: NTP Specifications for Toxicology/Carcinogenicity Studies
Deviations:
no
Principles of method if other than guideline:
- Principle of test:
Long-term NTP studies usually involve exposing both sexes (rats and mice) to a substance for a period of 2 years. These two-year toxicology and carcinogenicity studies in rodents are the primary method NTP uses to identify substances that are hazardous to human health. Rodent studies along with epidemiology studies are the best way to identify potential human health hazards. Data from NTP rodent studies is used in risk assessments and contributes to the information used by regulatory agencies, such as the EPA, to establish regulations that protect human health.
- Short description of test conditions:
This study is used to determine the chronic toxicity and carcinogenic potential of a test article in animals. The high dose will be estimated by the NTP from the subchronic toxicity study and the lower doses will be a fraction of it.
Control animals of both sexes are required for all studies. The control animals may be untreated (as for most dosed feed and dosed water studies) or vehicle controls (as for most gavage or dermal studies).
The animals to be used shall be five to six weeks of age at the time of release from quarantine and start of this study. Fifty animals per dose group of both sexes and both species shall be started in each study group routinely. Three dose groups of test article plus controls shall be used routinely
- Parameters analysed / observed:
During the study, individual animal weights for treated and control groups shall be recorded on day one on study prior to initial treatment; weekly for the first 13 weeks; and at 4 week intervals thereafter, until the appearance of life-threatening tumors or a significant number of deaths occur in the groups, at which point weights shall be taken and recorded every two weeks.It is estimated that animals will be weighed every two weeks for the
final 3 months of the chronic study. The animals shall also be weighed at necropsy. Each animal shall be formally examined for clinical signs of toxicity at four-week intervals and these observations will be recorded on TDMS. Clinical observations made on an animal shall not be open-
ended or have gaps during the course of study. Animals shall be observed two times daily (once in the early morning and once in the late afternoon at least six hours apart, before 10:00 AM and after 2:00 PM, including holidays and weekends) for moribundity/mortality.

GLP compliance:
yes
Remarks:
according to Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, USA)
- Age at study initiation: 6-7 weeks
- Housing: Polycarbonate cages (Lab Products, Inc., Seaford, DE), changed at least twice weekly (female mice) or at least weekly (male mice)
- 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 exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- USP-grade corn oil was obtained in multiple lots from Spectrum Chemicals and Laboratory Products (Gardena, CA) and was used as the vehicle. Periodic analyses of the corn oil vehicle performed by the study laboratory using potentiometric titration demonstrated peroxide concentrations below the acceptable limit of 3 mEq/kg.

The appropriate volumes of beta-myrcene and corn oil were combined in a calibrated glass mixing container and stirred with a vigorous vortex for 10 minutes using an overhead stirrer. Water droplets, if noticed to have separated out in the container of bulk test article, were avoided. The dose formulations were prepared approximately monthly.

The appropriate volumes of beta-myrcene and corn oil were combined in a calibrated glass mixing container and mixed on a paint shaker for 5 minutes. The dose formulations were analyzed approximately every 3 months; animal room samples were also analyzed. All 27 dose formulations were within 10% of the target concentrations.
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:
104 (female mice) or 105 weeks (male mice)
Frequency of treatment:
5 days per week
Dose / conc.:
0 other: g/kg/bw
Remarks:
control vehicle
Dose / conc.:
0.25 other: g/kg/bw
Remarks:
low dose
Dose / conc.:
0.5 other: g/kg/bw
Remarks:
medium dose
Dose / conc.:
1 other: g/kg/bw
Remarks:
high dose
No. of animals per sex per dose:
Groups of 50 male and 50 female mice
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on decreased survival and body weight gains of the 2 and 4 g/kg groups observed in 3 month oral gavage study, the highest dose selected for the 2-year gavage study in mice was 1 g/kg. There were no significant histopathologic lesions observed at this dose in 3-month study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Animals were observed twice daily; animals were weighed initially, weekly for 13 weeks, monthly thereafter, and at the end of the studies.

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical findings were recorded monthly beginning at week 5 and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on study day 1, weekly for the first 13 weeks, at 4-week intervals thereafter, and at study termination.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Complete histopathologic examinations were performed on all mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice only), Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, 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), testis
with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Clinical pathology was not performed.
Statistics:
Analysis of Neoplasm and Nonneoplastic Lesion Incidences:

The Poly-k test was used to assess neoplasm and nonneoplastic lesion prevalence.
This test is a survival-adjusted quantal-response procedure that modifies the Cochran-Armitage linear trend test to take survival differences into account.
Variation introduced by the use of risk weights, which reflect differential mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended by Bieler and Williams (1993).
Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend.
Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented as 1– P with the letter N added (e.g., P = 0.99 is presented as P = 0.01N).

Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958)

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).

Clinical signs:
effects observed, non-treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival of 0.25 and 0.5 g/kg mice was similar to that of the vehicle controls.
Survival of 1 g/kg mice was significantly less than that of the vehicle controls; the cause of deaths was uncertain
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of 0.25 g/kg males and females and 0.5 g/kg males were generally similar to vehicle controls throughout the study.
Mean body weights of 1 g/kg males, 0.5 g/kg females, and 1 g/kg females were less than those of the vehicle controls after weeks 8, 17, and 11, respectively.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The incidences of fatty change were significantly decreased in the 0.5 g/kg groups of males and females,
and the incidence of chronic active inflammation was significantly decreased in 0.25 g/kg females.

A significantly decreased incidence of mixed cell focus occurred in the 0.25 g/kg males, and a significantly increased incidence of mixed cell focus occurred in the 0.5 g/kg females (Tables 17, C4, and D4).

In both males and females, the incidences of hepatocellular hypertrophy were significantly increased in the 0.5 g/kg groups. Also, there was a dose-related increase in the average severity of hepatocellular hypertrophy in males.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver:
In male mice, the incidences of hepatocellular adenoma in the 0.25 and 0.5 g/kg groups and hepatocellular carcinoma and hepatoblastoma in the 0.5 g/kg group were significantly greater than the vehicle control incidences.

The combined incidences of hepatocellular adenoma or hepatocellular carcinoma and of hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma in the 0.25 and 0.5 g/kg males were significantly greater than those in the vehicle controls.

A significantly increased incidence of hepatocellular carcinoma or hepatoblastoma (combined) also occurred in 0.5 g/kg males. In female mice, significantly increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) occurred in the 0.25 g/kg group compared to those in the vehicle controls.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In female mice, a significantly increased incidence of bone marrow atrophy, manifested as a decrease in the density of hematopoietic cells,
occurred in the 0.5 g/kg group.
A significantly increased incidence of lymphoid follicle atrophy occurred in the spleen of 0.5 g/kg females, and dose related increases in severity were found in males and females. Lymphoid follicle atrophy was characterized by the decrease in the size and number of lymphoid follicles with total or near total loss of the white pulp in severe cases. A significantly increased incidence of atrophy in the mandibular lymph node occurred in 0.5 g/kg females. The features of lymph node atrophy were loss of lymphocytes from follicles and paracortical areas and a decrease in the size of the cross-section of the lymph
node in some cases. In females, significantly increased incidences of inflammation and epithelial hyperplasia of the forestomach occurred in the 0.5 g/kg group.
The incidence of pancreatic islet hyperplasia was significantly decreased in 0.5 g/kg male mice (Table C4). The incidences of uterine endometrial hyperplasia were decreased in 0.25 and 0.5 g/kg females when compared to the vehicle controls; the average severity in these groups was also decreased.
Relevance of carcinogenic effects / potential:
Beta-Myrcene and is not a genotoxic carcinogen as it does not exert any mutagenic or clastogenic properties. Further studies are needed to understand the mechanism of action of beta-myrcene-induced toxicity and carcinogenesis in mice.
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 0.25 other: g/kg/bw
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.25 other: g/kg/bw
System:
other: liver
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the conditions of the 2-year gavage study, there was clear evidence of carcinogenic activity of beta-myrcene in male in male B6C3F1 mice based
on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma. There was equivocal evidence of carcinogenic activity
of beta-myrcene in female B6C3F1 mice based on marginally increased incidences of hepatocellular adenoma and carcinoma.Beta-Myrcene and is not a genotoxic carcinogen as it does not exert any mutagenic or clastogenic properties.Therefore, the LOAEL in this chronic mouse study was established to be 250 mg/kg/d, based on the increase incidence in hepatocellular carcinoma with increasing beta-myrcene dose significantly higher than both the concurrent and the NTP historical controlIn addition, the time to observation of first hepatocellular carcinoma was also dose-related.  
Beta-Myrcene is calssified as carcinogen group 2B (Possibly carcinogenic to humans) by International Cancer Research on research on cancer (IARC).
Executive summary:

Groups of 50 male and 50 female mice were administered 0, 0.25, 0.5, or 1 g beta-myrcene/kg body weight in corn oil by gavage, 5 days per week for 104 or 105 weeks.

Survival:

Survival of 1 g/kg mice was significantly lessthan that of the vehicle controls; the cause of the deaths was uncertain. Due to the early mortality in 1 g/kg mice, data from this group was not presented.

 

Body weights:

Mean body weights of 1 g/kg males were at least 8% less than those of the vehicle controls between week 8 and week 56. Mean body weights of 0.5 g/kg females were at least 7% less than those of the vehicle controls after week 17, and those of 1 g/kg females were at least 8% less from week 11 to week 96.

Liver:

The liver was a primary target of beta-myrcene toxicity. and hepatoblastoma in males.

There was clear evidence of carcinogenicity in male mice based on increased incidences of liver neoplasms. The incidences of hepatocellular adenoma were significantly increased in 0.25 and 0.5 g/kg males, and the incidences of hepatocellular carcinoma and of hepatoblastoma were significantly increased in the 0.5 g/kg group. When these neoplasm types were combined, the increases were statistically significant in the 0.25 and 0.5 g/kg groups.

In female mice, the incidence of hepatocellular adenoma or carcinoma (combined) was increased in the 0.25 g/kg group compared to the vehicle controls, while those in the 0.5 g/kg group were similar to vehicle controls. The incidences of these neoplasms were within or slightly higher than the historical control range for 2-year corn oil gavage studies. The lack of a dose-response in the 1 g/kg group is likely due to the reduction in body weight gain combined with somewhat shorter survival in this

dose group. The marginally increased incidence in the 0.5 g/kg group was considered to be equivocal evidence of carcinogenicity in female mice.

Other results:

In addition, the incidences of bone marrow atrophy and lymph node follicle atrophy in the spleen were significantly increased in 0.5 g/kg females. In the forestomach, there were significantly increased incidences of inflammation and epithelial hyperplasia in 0.5 g/kg females.

Conclusions:

Under the conditions of the 2-year gavage study, there  was  clear  evidence  of  carcinogenic  activity  of beta-myrcene in male in male B6C3F1 mice based on  increased  incidences  of  hepatocellular  adenoma, hepatocellular carcinoma, and hepatoblastoma.  There was equivocal evidence  of carcinogenic  activity of beta-myrcene in female B6C3F1 mice based on marginally increased incidences of hepatocellular adenoma and carcinoma.

Beta-Myrcene and is not a genotoxic carcinogen as it does not exert any mutagenic or clastogenic properties. Therefore, the LOAEL in this chronic mouse study was established to be 250 mg/kg/d, based on the increase incidence in hepatocellular carcinoma with increasing beta-myrcene dose significantly higher than both the concurrent and the NTP historical control. In addition, the time to observation of first hepatocellular carcinoma was also dose-related.  

 

 

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: NTP Specifications for Toxicology/Carcinogenicity Studies
Deviations:
no
Principles of method if other than guideline:
- Principle of test:
Long-term NTP studies usually involve exposing both sexes (rats and mice) to a substance for a period of 2 years. These two-year toxicology and carcinogenicity studies in rodents are the primary method NTP uses to identify substances that are hazardous to human health. Rodent studies along with epidemiology studies are the best way to identify potential human health hazards. Data from NTP rodent studies is used in risk assessments and contributes to the information used by regulatory agencies, such as the EPA, to establish regulations that protect human health.
- Short description of test conditions:
This study is used to determine the chronic toxicity and carcinogenic potential of a test article in animals. The high dose will be estimated by the NTP from the subchronic toxicity study and the lower doses will be a fraction of it.
Control animals of both sexes are required for all studies. The control animals may be untreated (as for most dosed feed and dosed water studies) or vehicle controls (as for most gavage or dermal studies).
The animals to be used shall be five to six weeks of age at the time of release from quarantine and start of this study. Fifty animals per dose group of both sexes and both species shall be started in each study group routinely. Three dose groups of test article plus controls shall be used routinely
- Parameters analysed / observed:
During the study, individual animal weights for treated and control groups shall be recorded on day one on study prior to initial treatment; weekly for the first 13 weeks; and at 4 week intervals thereafter, until the appearance of life-threatening tumors or a significant number of deaths occur in the groups, at which point weights shall be taken and recorded every two weeks.It is estimated that animals will be weighed every two weeks for the
final 3 months of the chronic study. The animals shall also be weighed at necropsy. Each animal shall be formally examined for clinical signs of toxicity at four-week intervals and these observations will be recorded on TDMS. Clinical observations made on an animal shall not be open-
ended or have gaps during the course of study. Animals shall be observed two times daily (once in the early morning and once in the late afternoon at least six hours apart, before 10:00 AM and after 2:00 PM, including holidays and weekends) for moribundity/mortality.

GLP compliance:
yes
Remarks:
according to Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): beta-myrcene
- Molecular formula (if other than submission substance): C10H16
- Molecular weight (if other than submission substance): 136.24
- Smiles notation (if other than submission substance): See attached
- Structural formula attached as image file (if other than submission substance): see attached
Name of test material (as cited in study report): β-Myrcene
- Source: Millennium Specialty Chemicals (Jacksonville, USA)
- Analytical purity: > 90 %
- Impurities (identity and concentrations): Total impurities (9.55 %): psi-limonene (4.4 %), dl-limonene (1.4 %), water (< 0.016 %)
- Lot/batch No.: 1WB503
- Physical state: Clear to slightly yellow liquid
- Stability under test conditions: No degradation was detected during storage period
- Storage condition of test material: Stored in amber glass bottles sealed with Teflon®-lined lids ≤ –20 °C

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, USA)
- Age at study initiation: 6-7 weeks
- Housing: Polycarbonate cages (Lab Products, Inc., Seaford, DE), changed at least twice weekly (female mice) or at least weekly (male mice)
- 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- USP-grade corn oil was obtained in multiple lots from Spectrum Chemicals and Laboratory Products (Gardena, CA) and was used as the vehicle. Periodic analyses of the corn oil vehicle performed by the study laboratory using potentiometric titration demonstrated peroxide concentrations below the acceptable limit of 3 mEq/kg.

The appropriate volumes of beta-myrcene and corn oil were combined in a calibrated glass mixing container and stirred with a vigorous vortex for 10 minutes using an overhead stirrer. Water droplets, if noticed to have separated out in the container of bulk test article, were avoided. The dose formulations were prepared approximately monthly.

The appropriate volumes of beta-myrcene and corn oil were combined in a calibrated glass mixing container and mixed on a paint shaker for 5 minutes. The dose formulations were analyzed approximately every 3 months; animal room samples were also analyzed. All 27 dose formulations were within 10% of the target concentrations.
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:
104 (female mice) or 105 weeks (male mice)
Frequency of treatment:
5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: g/kg/bw
Remarks:
control vehicle
Dose / conc.:
0.25 other: g/kg/bw
Remarks:
low dose
Dose / conc.:
0.5 other: g/kg/bw
Remarks:
medium dose
Dose / conc.:
1 other: g/kg/bw
Remarks:
high dose
No. of animals per sex per dose:
Groups of 50 male and 50 female mice
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on decreased survival and body weight gains of the 2 and 4 g/kg groups observed in 3 month oral gavage study, the highest dose selected for the 2-year gavage study in mice was 1 g/kg. There were no significant histopathologic lesions observed at this dose in 3-month study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Animals were observed twice daily; animals were weighed initially, weekly for 13 weeks, monthly thereafter, and at the end of the studies.

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical findings were recorded monthly beginning at week 5 and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on study day 1, weekly for the first 13 weeks, at 4-week intervals thereafter, and at study termination.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Complete histopathologic examinations were performed on all mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice only), Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, 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), testis
with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.

Clinical pathology was not performed.
Statistics:
Analysis of Neoplasm and Nonneoplastic Lesion Incidences:

The Poly-k test was used to assess neoplasm and nonneoplastic lesion prevalence.
This test is a survival-adjusted quantal-response procedure that modifies the Cochran-Armitage linear trend test to take survival differences into account.
Variation introduced by the use of risk weights, which reflect differential mortality, was accommodated by adjusting the variance of the Poly-3 statistic as recommended by Bieler and Williams (1993).
Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend.
Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented as 1– P with the letter N added (e.g., P = 0.99 is presented as P = 0.01N).

Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958)

Analysis of Continuous Variables:
Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival of 0.25 and 0.5 g/kg mice was similar to that of the vehicle controls.
Survival of 1 g/kg mice was significantly less than that of the vehicle controls; the cause of deaths was uncertain
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of 0.25 g/kg males and females and 0.5 g/kg males were generally similar to vehicle controls throughout the study.
Mean body weights of 1 g/kg males, 0.5 g/kg females, and 1 g/kg females were less than those of the vehicle controls after weeks 8, 17, and 11, respectively.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The incidences of fatty change were significantly decreased in the 0.5 g/kg groups of males and females,
and the incidence of chronic active inflammation was significantly decreased in 0.25 g/kg females.

A significantly decreased incidence of mixed cell focus occurred in the 0.25 g/kg males, and a significantly increased incidence of mixed cell focus occurred in the 0.5 g/kg females (Tables 17, C4, and D4).

In both males and females, the incidences of hepatocellular hypertrophy were significantly increased in the 0.5 g/kg groups. Also, there was a dose-related increase in the average severity of hepatocellular hypertrophy in males.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver:
In male mice, the incidences of hepatocellular adenoma in the 0.25 and 0.5 g/kg groups and hepatocellular carcinoma and hepatoblastoma in the 0.5 g/kg group were significantly greater than the vehicle control incidences.

The combined incidences of hepatocellular adenoma or hepatocellular carcinoma and of hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma in the 0.25 and 0.5 g/kg males were significantly greater than those in the vehicle controls.

A significantly increased incidence of hepatocellular carcinoma or hepatoblastoma (combined) also occurred in 0.5 g/kg males. In female mice, significantly increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) occurred in the 0.25 g/kg group compared to those in the vehicle controls.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In female mice, a significantly increased incidence of bone marrow atrophy, manifested as a decrease in the density of hematopoietic cells,
occurred in the 0.5 g/kg group.
A significantly increased incidence of lymphoid follicle atrophy occurred in the spleen of 0.5 g/kg females, and dose related increases in severity were found in males and females. Lymphoid follicle atrophy was characterized by the decrease in the size and number of lymphoid follicles with total or near total loss of the white pulp in severe cases. A significantly increased incidence of atrophy in the mandibular lymph node occurred in 0.5 g/kg females. The features of lymph node atrophy were loss of lymphocytes from follicles and paracortical areas and a decrease in the size of the cross-section of the lymph
node in some cases. In females, significantly increased incidences of inflammation and epithelial hyperplasia of the forestomach occurred in the 0.5 g/kg group.
The incidence of pancreatic islet hyperplasia was significantly decreased in 0.5 g/kg male mice (Table C4). The incidences of uterine endometrial hyperplasia were decreased in 0.25 and 0.5 g/kg females when compared to the vehicle controls; the average severity in these groups was also decreased.
Relevance of carcinogenic effects / potential:
Beta-Myrcene and is not a genotoxic carcinogen as it does not exert any mutagenic or clastogenic properties. Further studies are needed to understand the mechanism of action of beta-myrcene-induced toxicity and carcinogenesis in mice.

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
ca. 0.25 other: g/kg/bw
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.25 other: g/kg/bw
System:
other: liver
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
Under the conditions of the 2-year gavage study, there was clear evidence of carcinogenic activity of beta-myrcene in male in male B6C3F1 mice based
on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma. There was equivocal evidence of carcinogenic activity
of beta-myrcene in female B6C3F1 mice based on marginally increased incidences of hepatocellular adenoma and carcinoma.Beta-Myrcene and is not a genotoxic carcinogen as it does not exert any mutagenic or clastogenic properties.Therefore, the LOAEL in this chronic mouse study was established to be 250 mg/kg/d, based on the increase incidence in hepatocellular carcinoma with increasing beta-myrcene dose significantly higher than both the concurrent and the NTP historical controlIn addition, the time to observation of first hepatocellular carcinoma was also dose-related.  
Beta-Myrcene is calssified as carcinogen group 2B (Possibly carcinogenic to humans) by International Cancer Research on research on cancer (IARC).
Executive summary:

Groups of 50 male and 50 female mice were administered 0, 0.25, 0.5, or 1 g beta-myrcene/kg body weight in corn oil by gavage, 5 days per week for 104 or 105 weeks.

Survival:

Survival of 1 g/kg mice was significantly lessthan that of the vehicle controls; the cause of the deaths was uncertain. Due to the early mortality in 1 g/kg mice, data from this group was not presented.

 

Body weights:

Mean body weights of 1 g/kg males were at least 8% less than those of the vehicle controls between week 8 and week 56. Mean body weights of 0.5 g/kg females were at least 7% less than those of the vehicle controls after week 17, and those of 1 g/kg females were at least 8% less from week 11 to week 96.

Liver:

The liver was a primary target of beta-myrcene toxicity. and hepatoblastoma in males.

There was clear evidence of carcinogenicity in male mice based on increased incidences of liver neoplasms. The incidences of hepatocellular adenoma were significantly increased in 0.25 and 0.5 g/kg males, and the incidences of hepatocellular carcinoma and of hepatoblastoma were significantly increased in the 0.5 g/kg group. When these neoplasm types were combined, the increases were statistically significant in the 0.25 and 0.5 g/kg groups.

In female mice, the incidence of hepatocellular adenoma or carcinoma (combined) was increased in the 0.25 g/kg group compared to the vehicle controls, while those in the 0.5 g/kg group were similar to vehicle controls. The incidences of these neoplasms were within or slightly higher than the historical control range for 2-year corn oil gavage studies. The lack of a dose-response in the 1 g/kg group is likely due to the reduction in body weight gain combined with somewhat shorter survival in this

dose group. The marginally increased incidence in the 0.5 g/kg group was considered to be equivocal evidence of carcinogenicity in female mice.

Other results:

In addition, the incidences of bone marrow atrophy and lymph node follicle atrophy in the spleen were significantly increased in 0.5 g/kg females. In the forestomach, there were significantly increased incidences of inflammation and epithelial hyperplasia in 0.5 g/kg females.

Conclusions:

Under the conditions of the 2-year gavage study, there  was  clear  evidence  of  carcinogenic  activity  of beta-myrcene in male in male B6C3F1 mice based on  increased  incidences  of  hepatocellular  adenoma, hepatocellular carcinoma, and hepatoblastoma.  There was equivocal evidence  of carcinogenic  activity of beta-myrcene in female B6C3F1 mice based on marginally increased incidences of hepatocellular adenoma and carcinoma.

Beta-Myrcene and is not a genotoxic carcinogen as it does not exert any mutagenic or clastogenic properties. Therefore, the LOAEL in this chronic mouse study was established to be 250 mg/kg/d, based on the increase incidence in hepatocellular carcinoma with increasing beta-myrcene dose significantly higher than both the concurrent and the NTP historical control. In addition, the time to observation of first hepatocellular carcinoma was also dose-related.