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

Carcinogenicity

Description of key information

Two 2-year gavage studies have been conducted on β‑myrcene:

- there was evidence of carcinogenic activity of β-myrcene in male F344/N rats based on increased incidences of renal tubule neoplasms. There was equivocal evidence of carcinogenic activity of β‑myrcene in female F344/N rats based on increased incidences of renal tubule adenoma.

- there was evidence of carcinogenic activity of β‑myrcene in male B6C3F1 mice based on increased incidences of liver neoplasms. There was no evidence of carcinogenic activity of β‑myrcene in female B6C3F1 mice.

The effects found in male rat kidneys are sex and species specific. Therefore they are not relevant to humans. The background of naturally occurring liver tumours is high in male mice, therefore the observed effects are not relevant to humans.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Study conducted equivalent or similar to OECD Guideline 451 with minor deviations: high mortality in the high dose level males; only 2 doses available for analysis; blood count not performed and food consumption not followed.

Remarks:

Details of this NTP study are not all reported in the published article

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

high mortality in the high dose males; only 2 doses available for analysis

GLP compliance:

yes

Specific details on test material used for the study:

The purity of each lot was determined by elemental analyses, gas chromatography with flame ionization detection (GC-FID), and high performance liquid chromatography. The overall purity of the lot used for the 2-year study was greater than 93%.

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, NY)
- Age at reception: 5-6 weeks
- Housing: 5 animals/cage
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardners, PA), ad libitum; changed at least weekly
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 11 (males) or 12 (females) days

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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:

Not specified in the published article

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

5 days per week

Post exposure period:

No

Remarks:

Doses / Concentrations:
0, 250, 500 or 1000 mg/kg bw/day

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on adverse effects on survival and body weight gains at 2000 and 4000 mg/kg bw/day observed in a 3-month range-finding study, the highest dose selected for the 2-year gavage study in rats was 1000 mg/kg bw/day

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not specified in the published article
BODY WEIGHT: Yes
- Time schedule for examinations: not specified in the published article

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Organs examined: not specified in the published article

Other examinations:

For extended evaluation of proliferative renal lesions, additional sections of both kidneys from the residual formalin-fixed wet tissues were embedded in separate paraffin blocks and step sectioned at 1-mm intervals to produce up to 8 additional sections of each male and female kidney.

Statistics:

Done. No details of methods are presented in the published article

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival of male rats in the 250 and 500 mg/kg dose groups, and all dosed groups of females, was similar to the respective vehicle controls; but all males at the 1000 mg/kg dose had died by week 89 (20.5 months), with an average survival of 467 days (15.4 months) for the group.
Nephrosis was considered to be the cause of death in all cases.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Linear Papillary Mineralization: There was a statistically significant increase in the incidence of linear papillary mineralization in the 250- and 500-mg/kg male rats compared to controls with a concurrent increase in mean severity. There was papillary mineralization in some of the female rats (though with no treatment-related increase in incidence or severity) and in 1 control male rat.

- Nephrosis: Most of the male rats from the 250- and 500-mg/kg dose groups had some degree of nephrosis (42/50 and 46/50, respectively). In the 250-mg/kg group, the severity was minimal to mild. Only 2 females at the 250-mg/kg dose level had minimal nephrosis. In the 500-mg/kg group, the nephrosis was more severe with a mean severity grade of 2.7. There was minimal nephrosis in 27 of the 50 female rats. At 1,000 mg/kg, nephrosis in the outermost outer stripe of outer medulla was much more pronounced. The severity of nephrosis was, on average, marked (grade 4) in male rats, and mild (grade 2) in female rats. The kidneys of male rats with marked nephrosis were markedly enlarged.

- Chronic Progressive Nephropathy (CPN): In the control groups, the incidence of CPN in the males was 45/50 with a mean severity grade of 1.2. In the females, the incidence of CPN was 26/50 with a mean severity grade of 1.0. In male rats at 250 mg/kg, there was a modest enhancement of CPN to a mean severity of grade 2 (mild) relative to control males (incidence was 48/50). In the females, there was a slight exacerbation of CPN with an incidence of 43/50, but the mean severity remained at grade 1 (minimal). At 500 mg/kg, the grades of CPN severitywere elevated slightly, to grade 2.6 in males (incidence was 48/50), and 1.3 in females (incidence was 41/50). At 1000 mg/kg, CPN in male rats was mostly at grade 4 (marked), but it was difficult to distinguish from the kidney tissue involved in severe nephrosis. In female rats, the mean of CPN severity grade was elevated slightly to 1.7.

- Collecting duct hyperplasia: In male rats there was an increase in hyperplasia of the papilla lining reported as hyperplasia of the transitional epithelium lining of the pelvis and renal papilla was of the morphological type consistent with advanced stages of CPN. This lesion was therefore considered part of the CPN disease spectrum. This alteration was marked at 1000 mg/kg.

- Renal tubule hyperplasia: solitary foci of renal tubule hyperplasia were observed in low incidence in all groups except the high-dose (1,000 mg/kg) males, and particularly in the low-dose (250 mg/kg) males and females. In the lower dose groups, hyperplasia was often located within the cortex, but at higher doses, and in female rats, the lesions also appeared to involve the outer stripe of outer medulla.

- Chronic active inflammation in the nose: dose-related effect in male rats.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Renal tubule proliferative lesions: Adenomas and/or carcinomas were present at low incidence in all treated groups except the control males and females, but particularly evident in the low-dose and mid-dose (250 and 500 mg/kg) males, where there was up to a 30% incidence. The renal tubule tumors were basophilic lesions with a tendency to tubular differentiation. In the lower dose groups, small tumors were often located within the cortex, but at higher doses, and in female rats, the lesions also appeared to involve the outer stripe of outer medulla.

Other effects:

not specified

Relevance of carcinogenic effects / potential:

Under the conditions of the 2-year study conducted for NTP, there was clear evidence of carcinogenic activity of β-myrcene in male F344/N rats based on increased incidences of renal tubule neoplasms and there was equivocal evidence of carcinogenic activity of β-myrcene in female F344/N rats based on increased incidences of renal tubule adenoma (only one female above historical control data at the highest dose).
Renal tubule tumours were observed in males due to the well-known sex- and species-specific mechanism of α2-globulin accumulation. This type of nephropathy is likely in male rats in this study up to 1000 mg/kg and can explain the increased incidence of neoplastic lesions in this sex for all dose levels.
Increased incidences of Chronic Progressive Nephropathy (CPN) and nephrosis reported in both male and female rats are mediated via an unknown mechanism. Nephrosis seems to be complicated by an apparent excerbation of CPN with increasing dose, especially in the male rats. At the highest dose level (1000 mg/kg), the incidence of nephrosis was approximately the same in both sexes (48/50 in males and 45/50 in females) but was more severe in males than in females (mean grade of 3.9 in males and 1.2 in females). However in males, the carcinogenic activity is already explained by a mechanism which is sex- and species-speciific. The increased incidence of renal tubule adenomas in females is not dose-related and no carcinomas were observed.
As no biologically relevant increase in adenoma/carcinoma compared to historical data was observed in females, it is concluded that this mechanism is not sufficient alone to lead to the development of renal tumours.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Clear evidence of carcinogenic activity in male rats based on increased incidences of renal tubule neoplasms

Remarks on result:

other:

Remarks:

Effect type: other: carcinogenicity (not relevant for humans) (migrated information)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No evidence of carcinogenic activity in female rats based on not biologically relevant increased incidence of renal tubule adenoma

Remarks on result:

other:

Remarks:

Effect type: other: carcinogenicity (not relevant for humans) (migrated information)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Table 1: Summary of the 2 -year carcinogenesis study of β-myrcene in rats

	Male F344/N Rats	Female F344/N Rats
Doses in corn oil by gavage	0, 250, 500 or 1000 mg/kg bw/day	0, 250, 500 or 1000 mg/kg bw/day
Nonneoplastic effects	Kidney: renal tubule, nephrosis (0/50, 42/50, 46/50, 48/50); papilla, mineralization (1/50, 48/50, 40/50, 4/50); chronic progressive nephropathy (45/50, 48/50, 48/50, 49/50);severity of nephropathy (1.2, 2.0, 2.6, 3.9); collecting duct, hyperplasia (0/50, 0/50, 7/50, 48/50); renal tubule, hyperplasia, standard sigle section (0/50, 0/50, 2/50, 0/50)	Kidney: renal tubule, nephrosis (0/50, 2/50, 27/50, 45/50); chronic progressive nephropathy (26/50, 43/50, 41/50, 44/50); severity of nephropathy (1.0, 1.0, 1.3, 1.7)
Neoplastic effects	Kidney: renal tubule adenoma (standard single section - 0/50, 4/50, 8/50, 2/50; standard single section and step sections combined - 0/50, 12/50, 13/50, 4/50); renal tubule adenoma or carcinoma (standard   single section - 0/50, 7/50, 9/50, 2/50; standard single section and step sections combined - 0/50, 14/50, 13/50, 4/50)	None
Equivocal findings	None	Kidney: renal tubule adenoma (standard evaluation - 0/50, 1/50, 0/50, 2/50; extended evaluation - 0/50, 1/50, 1/50, 2/50; standard and extended evaluation - 0/50, 2/50, 1/50, 3/50)
Level of evidence of carcinogenic activity	Clear evidence	Equivocal evidence

Table 2: Incidences of neoplasms and nonneoplastic lesions of the kidney in rats in the 2-year gavage study of β-myrcene

	Vehicle Control	0.25 g/kg	0.5 g/kg	1.0 g/kg
Male
Single Sections (Standard Evaluation)
Number Examined Microscopically	50	50	50	50
Renal Tubule, Hyperplasia	0	0	2	0
Renal Tubule, Nephrosis	0	42 (1.8)	46 (2.7)	48 (3.9)
Papilla, Mineralization	1 (1.0)	48 (2.1)	40 (1.9)	4 (1.0)
Chronic progressive nephropathy	45 (1.2)	48 (2.0)	48 (2.6)	49 (3.9)
Renal Tubule, Adenoma	0	4	8	2
Renal Tubule, Carcinoma	0	3	1	0
Renal tubule, Ad/Ca combined	0	7*	9**	2
Single Sections and Step Sections (Combined)
Number Examined Microscopically	50	50	50	50
Renal Tubule, Hyperplasia	1	5	3	0
Renal Tubule, Adenoma	0	12***	13***	4
Renal Tubule, Carcinoma	0	3	1	0
Renal Tubule, Ad/Ca combined	0	14***	13***	4

	Vehicle Control	0.25 g/kg	0.5 g/kg	1 g/kg
Female
Single Sections (Standard Evaluation)
Number Examined Microscopically	50	50	50	50
Renal Tubule, Hyperplasia	0	0	0	1
Renal Tubule, Nephrosis	0	2 (1.0)	27 (1.0)	45 (1.2)
Papilla, Mineralization	5 (1.0)	3 (1.0)	1 (1.0)	0
Chronic progressive nephropathy	26 (1.0)	43 (1.0)	41 (1.3)	44 (1.7)
Collecting duct, Hyperplasia	0	0	0	0
Renal Tubule, Adenoma	0	1	0	2
Renal Tubule, Ad/Ca combined	0	1	0	2
Single Sections and Step Sections (Combined)
Number Examined Microscopically	50	50	50	50
Renal Tubule, Hyperplasia	1	4	1	2
Renal Tubule, Adenoma	0	2	1	3
Renal Tubule, Ad/Ca combined	0	2	1	3

* p = 0.01

** p = 0.002

*** P ≤ 0.001

Conclusions:

It is concluded that the only carcinogenic effect found in kidneys in rats is sex and species specific and is therefore not relevant to humans.

Executive summary:

A chronic study was conducted to evaluate the carcinogenic potential of β‑myrcene in F344/N rats by the National Toxicology Program. This paper focuses on the effects of kidneys.

Groups of 50 male and 50 female rats were administered β‑myrcene at concentrations of 0, 250, 500 or 1000 mg/kg bw/day in corn oil by gavage, 5 days per week for 105 weeks.

All 1000 mg/kg bw/day male rats died before the end of the study due to renal toxicity.

In the standard evaluation of the kidney, the incidence of renal tubule adenoma was increased with a dose related trend in male rats, and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in 250 and 500 mg/kg bw/day males. In the combined standard single and step sections evaluation, the incidences of renal tubule adenoma and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in the 250 and 500 mg/kg bw/day groups of males.

The incidences of renal tubule nephrosis were markedly increased in all dosed groups of both sexes except in 250 mg/kg bw/day females. The incidences of papillary mineralization in 250 and 500 mg/kg bw/day males were increased. Increased incidences of chronic progressive nephropathy occurred in dosed females, and the severity was increased in the 500 and 1000 mg/kg bw/day males and females. The incidence of collecting duct hyperplasia increased in males from the 500 mg/kg bw dose level.

In conclusion, under the conditions of this test, there was clear evidence of carcinogenic activity of β-myrcene in male F344/N rats based on increased incidences of renal tubule neoplasms due to α2u-globulin accumulation. There was no evidence of carcinogenic activity of β-myrcene in female F344/N rats based on not biologically relevant increased incidences of renal tubule adenoma.

As the carcinogenic effect found in kidneys in rats is sex and species specific, it is concluded therefore that it is not relevant to humans.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 2002 to March 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP Study conducted equivalent or similar to OECD Guideline 451 with minor deviations: high mortality in the high dose level; only 2 doses available for analysis; blood count not performed and food consumption not followed.

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 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

high mortality in the high doses; only 2 doses available for analysis; blood count not performed and food consumption not followed

GLP compliance:

yes

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: 5-6 weeks
- Housing: 3 (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: 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 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 approximately every 3 months
- All dose formulations were within 10 % of the target concentrations

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

5 days per week

Post exposure period:

No

Remarks:

Doses / Concentrations:
0, 250, 500 or 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on adverse effects on survival and body weight gains at 2000 and 4000 mg/kg bw/day observed in 3-months range-finding study, the highest dose selected for the 2‑year gavage study in rats was 1000 mg/kg bw/day
- Rationale for animal assignment: Animals were distributed randomly into groups of approximately equal initial mean body weights
- Rationale for selecting sentinel groups: Five male and five female animals were randomly selected for parasite evaluation and gross observation of disease

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Monthly beginning at week 5 and at the end of the study


BODY WEIGHT: Yes
- Time schedule for examinations: Initially on Day 1, weekly for 13 weeks, monthly thereafter and at the end of the study

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Organs examined: In addition to gross lesions and tissue masses, the following tissues were examined: Adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, 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

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.
- Neoplasm and nonneoplastic lesion incidences: Poly‑k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence.
- Body weight data: Analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Three males administered 1 g/kg died before week 60, and the remainder died by week 89. Death in male rats was attributed to renal toxicity. Survival of 0.25 and 0.5 g/kg males and all dosed groups of females was similar to that of the vehicle controls.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three males administered 1 g/kg died before week 60, and the remainder died by week 89. Death in male rats was attributed to renal toxicity. Survival of 0.25 and 0.5 g/kg males and all dosed groups of females was similar to that of the vehicle controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of 1 g/kg males and females were less than those of the vehicle controls after weeks 7 and 13, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Details on results:

MORTALITY
- All 1000 mg/kg bw/day male rats died before the end of the study due to renal toxicity. Survival of 250 and 500 mg/kg bw/day males and all dosed groups of females was similar to that of the vehicle controls.
- See table 1 for more details


CLINICAL SIGNS
- Thinness, lethargy, ruffled fur, and ocular and nasal discharge


BODY WEIGHT
- Compared to vehicle controls, the mean body weights of 250 and 500 mg/kg bw/day males were slightly greater, and mean body weights of 1000 mg/kg bw/day males were clearly less at the end of the study.


HISTOPATHOLOGY: NON-NEOPLASTIC/NEOPLASTIC
- Kidney: In the standard evaluation of the kidney, the incidence of renal tubule adenoma was significantly increased in 500 mg/kg bw/day male rats, and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in 250 and 500 mg/kg bw/day males. In both the extended evaluation and the combined standard and extended evaluations, the incidences of renal tubule adenoma and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in the 250 and 500 mg/kg bw/day groups of males. The incidences of renal tubule nephrosis were markedly increased in all dosed groups of both sexes except in 250 mg/kg bw/day females. The incidences of papillary mineralization in 250 and 500 mg/kg bw/day males were significantly increased. Significantly increased incidences of nephropathy occurred in dosed females, and the severity was increased in the 500 and 1000 mg/kg bw/day males and females. The incidences of hyperplasia of the transitional epithelium lining the pelvis and overlying the renal papilla were significantly increased in all dosed groups of males and females. In male rats, the incidences of focal suppurative inflammation were significantly increased in the 250 and 500 mg/kg bw/day groups.
- Nose: A significantly increased incidence of chronic active inflammation of the nose occurred in 500 mg/kg bw/day males, likely due to regurgitation of β-myrcene during gavage.
- Stomach: In 500 mg/kg bw/day males, the incidence of chronic active inflammation of the forestomach was increased.
- Thyroid: Significant increase in thyroid gland C-cell adenoma was observed in females of the 250 mg/kg bw/day group.
- Liver: Decreased incidence of hepatic foci except the incidence of eosinophilic focus in females of the 500 and 1000 mg/kg bw/day groups; negative trends in incidences of bile duct hyperplasia in males and incidences of chronic inflammation in both sexes
- Uterus: The incidence of cystic endometrial hyperplasia of the uterus was significantly increased in the 1000 mg/kg bw/day group.


HISTORICAL CONTROL DATA (if applicable):
- Yes, NTP historical database contains all studies that use the NTP 2000 diet with histopathology findings completed until November 19, 2008. Additionally, historical database includes studies using the same route of administration, and the overall incidences of neoplasms for all routes of administration

Relevance of carcinogenic effects / potential:

Under the conditions of this test, there was clear evidence of carcinogenic activity of β-myrcene in male F344/N rats based on increased incidences of renal tubule neoplasms. However, there was equivocal evidence of carcinogenic activity of β-myrcene in female F344/N rats based on increased incidences of renal tubule adenoma (only one female above historical control data at the highest dose).
Renal tubular tumours were observed in males due to the well-known sex- and species-specific mechanism of α2uglobulin accumulation. Increased incidences of Chronic Progressive Nephropathy (CPN) and nephrosis reported in both male and female rats are mediated via an unknown mechanism. However, as no biologically relevant increase in adenoma/carcinoma compared to historical data was observed in females, it is concluded that this mechanism is not sufficient alone to lead to the development of renal tumours.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Clear evidence of carcinogenic activity in male rats based on increased incidences of renal tubule neoplasms

Remarks on result:

other:

Remarks:

Effect type: other: carcinogenicity (not relevant for humans) (migrated information)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No evidence of carcinogenic activity in female rats based on not biologically relevant increased incidence of renal tubule adenoma

Remarks on result:

other:

Remarks:

Effect type: other: carcinogenicity (not relevant for humans) (migrated information)

Table 1: Summary of the 2 -year carcinogenesis study of β-myrcene in rats*

	Male F344/N Rats	Female F344/N Rats
Doses in corn oil by gavage	0, 250, 500 or 1000 mg/kg bw/day	0, 250, 500 or 1000 mg/kg bw/day
Body weights	250 and 500 mg/kg bw/day groups greater than vehicle control group after week 81	1000 mg/kg bw/day group less than vehicle control group after week 11; 500 mg/kg bw/day group less than vehicle control group after week 17
Survival rates	29/50, 36/50, 28/50, 0/50	31/50, 33/50, 28/50, 33/50
Nonneoplastic effects	Kidney: renal tubule, nephrosis (0/50, 42/50, 46/50); papilla, mineralization (1/50, 48/50, 40/50); severity of nephropathy (1.2, 2.0, 2.6); transitional epithelium, hyperplasia (0/50, 21/50, 19/50); inflammation, suppurative, focal (1/50, 22/50, 22/50)   Nose: inflammation, chronic active (14/50, 19/50, 27/50)	Kidney: renal tubule, nephrosis (0/50, 2/50, 27/50, 45/50); nephropathy (26/50, 43/50, 41/50, 44/50); severity of nephropathy (1.0, 1.0, 1.3, 1.7); transitional epithelium, hyperplasia (1/50, 12/50, 15/50, 19/50)
Neoplastic effects	Kidney: renal tubule adenoma (standard evaluation - 0/50, 4/50, 8/50; extended evaluation - 0/50, 8/50, 7/50; standard and extended evaluations combined - 0/50, 12/50, 13/50); renal tubule adenoma or carcinoma (standard evaluation - 0/50, 7/50, 9/50; extended evaluation - 0/50, 10/50, 7/50; standard and extended evaluations combined - 0/50, 14/50, 13/50)	None
Equivocal findings	None	Kidney: renal tubule adenoma (standard evaluation - 0/50, 1/50, 0/50, 2/50; extended evaluation - 0/50, 1/50, 1/50, 2/50; standard and extended evaluation - 0/50, 2/50, 1/50, 3/50)
Level of evidence of carcinogenic activity	Clear evidence	Equivocal evidence

* Neoplasm and nonneoplastic lesion incidences are not presented for 1000 mg/kg bw/day male rats due to high mortality

Table 2: Incidences of neoplasms and nonneoplastic lesions of the kidney in rats in the 2-year gavage study of β-myrcene^a

	Vehicle Control	0.25 g/kg	0.5 g/kg
Male
Single Sections (Standard Evaluation)
Number Examined Microscopically	50	50	50
Renal Tubule, Hyperplasia^b	0	0	2 (1.0)^c
Renal Tubule, Nephrosis	0	42** (1.8)	46** (2.7)
Papilla, Mineralization	1 (1.0)	48** (2.1)	40** (1.9)
Nephropathy	45 (1.2)	48 (2.0)	48 (2.6)
Transitional Epithelium, Hyperplasia	0	21** (1.4)	19** (1.4)
Inflammation, Suppurative, Focal	1 (1.0)	22** (1.0)	22** (1.0)
Vein, Thrombosis	0	0	3 (2.3)
Renal Tubule, Adenoma, Multiple	0	2	1
Renal Tubule, Adenoma (includes multiple)^d
Overall rate^e	0/50 (0%)	4/50 (8%)	8/50 (16%)
Adjusted rate^f	0.00%	8.80%	18.70%
Terminal rate^g	0/29 (0%)	3/36 (8%)	5/28 (18%)
First incidence (days)	-^i	717	551
Poly-3 test^h	P=0.002	P=0.068	P=0.003
Renal Tubule, Carcinoma^j	0	3	1
Renal Tubule, Adenoma or Carcinoma^k
Overall rate	0/50 (0%)	7/50 (14%)	9/50 (18%)
Adjusted rate	0.00%	15.40%	21.00%
Terminal rate	0/29 (0%)	5/36 (14%)	6/28 (21%)
First incidence (days)	-	652	551
Poly-3 test	P=0.002	P=0.010	P=0.002
Single Sections and Step Sections (Combined)
Number Examined Microscopically	50	50	50
Renal Tubule, Hyperplasia	1 (1.0)	5 (1.6)	3 (1.0)
Renal Tubule, Adenoma
Overall rate	0/50 (0%)	12/50 (24%)	13/50 (26%)
Adjusted rate	0.00%	26.50%	30.20%
Terminal rate	0/29 (0%)	11/36 (31%)	9/28 (32%)
First incidence (days)	-	717	551
Poly-3 test	P<0.001	P<0.001	P<0.001
Renal Tubule, Carcinoma	0	3	1
Renal Tubule, Adenoma or Carcinoma
Overall rate	0/50 (0%)	14/50 (28%)	13/50 (26%)
Adjusted rate	0.00%	30.80%	30.20%
Terminal rate	0/29 (0%)	12/36 (33%)	9/28 (32%)
First incidence (days)	-	652	551
Poly-3 test	P<0.001	P<0.001	P<0.001

	Vehicle Control	0.25 g/kg	0.5 g/kg	1 g/kg
Female
Single Sections (Standard Evaluation)
Number Examined Microscopically	50	50	50	50
Renal Tubule, Hyperplasia	0	0	0	1 (1.0)
Renal Tubule, Nephrosis	0	2 (1.0)	27** (1.0)	45** (1.2)
Papilla, Mineralization	5 (1.0)	3 (1.0)	1 (1.0)	0*
Nephropathy	26 (1.0)	43** (1.0)	41** (1.3)	44** (1.7)
Transitional Epithelium, Hyperplasia	1 (1.0)	12** (1.3)	15** (1.3)	19** (1.2)
Inflammation, Suppurative, Focal	0	1 (1.0)	0	1 (1.0)
Renal Tubule, Adenoma^l	0	1	0	2
Single Sections and Step Sections (Combined)
Number Examined Microscopically	50	50	50	50
Renal Tubule, Hyperplasia	1 (1.0)	5 (1.4)	1 (1.0)	2 (1.0)
Renal Tubule, Adenoma
Overall rate	0/50 (0%)	2/50 (4%)	1/50 (2%)	3/50 (6%)
Adjusted rate	0.00%	4.80%	2.50%	7.20%
Terminal rate	0/31 (0%)	1/33 (3%)	0/28 (0%)	3/33 (9%)
First incidence (days)	-	689	701	730 (T)
Poly-3 test	P=0.105	P=0.239	P=0.491	P=0.121

* Significantly different (P≤0.05) from the vehicle control group by the Poly-3 test

** P≤0.01

(T) Terminal sacrifice

^a Data for the 1 g/kg male group are not presented due to early mortality.

^b Number of animals with lesion

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

^d Historical incidence for 2-year gavage studies with corn oil vehicle control groups (mean ± standard deviation): 1/150 (0.7% ± 1.2%), range 0%‑2%; all routes: 8/1394 (0.6% ± 0.9%), range 0%-2%

^e Number of animals with neoplasm per number of animals with kidney examined microscopically

^f Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality

^g Observed incidence at terminal kill

^h Beneath the vehicle control incidence is the P value associated with the trend test. Beneath the dosed group incidence are the P values corresponding to pairwise comparisons between the vehicle controls and that dosed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice.

ⁱ Not applicable; no neoplasms in animal group

^j Historical incidence for corn oil gavage studies: 0/150; all routes: 2/1394 (0.1% ± 0.5%), range 0%‑2%

^k Historical incidence for corn oil gavage studies: 1/150 (0.7% ± 1.2%), range 0%‑2%; all routes: 10/1394 (0.7% ± 1.2%), range 0%-4%

^l Historical incidence for corn oil gavage studies: 0/150; all routes, 1/1,340 (0.1% ± 0.4%), range 0%‑2%

Conclusions:

It is concluded that the only carcinogenic effect found in kidneys in rats is sex and species specific and is therefore not relevant to humans.

Executive summary:

A chronic study was conducted to evaluate the carcinogenic potential of β‑myrcene in F344/N rats according to method equivalent or similar to OECD Guideline 451 in compliance with Good Laboratory Practices.

Groups of 50 male and 50 female rats were administered β‑myrcene at concentrations of 0, 250, 500 or 1000 mg/kg bw/day in corn oil by gavage, 5 days per week for 105 weeks. Parameters evaluated included survival, body weights, clinical observations, gross necropsy and histopathological (non-neoplastic and neoplastic) findings in all animals.

All 1000 mg/kg bw/day male rats died before the end of the study due to renal toxicity. Compared to vehicle controls, the mean body weights of 250 and 500 mg/kg bw/day males were slightly greater and mean body weights of 1000 mg/kg bw/day males were clearly less at the end of the study. In the standard evaluation of the kidney, the incidence of renal tubule adenoma was significantly increased in 500 mg/kg bw/day male rats, and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in 250 and 500 mg/kg bw/day males. In both the extended evaluation and the combined standard and extended evaluations, the incidences of renal tubule adenoma and the combined incidences of renal tubule adenoma or carcinoma were significantly increased in the 250 and 500 mg/kg bw/day groups of males. The incidences of renal tubule nephrosis were markedly increased in all dosed groups of both sexes except in 250 mg/kg bw/day females. The incidences of papillary mineralization in 250 and 500 mg/kg bw/day males were significantly increased. Significantly increased incidences of nephropathy occurred in dosed females, and the severity was increased in the 500 and 1000 mg/kg bw/day males and females. The incidences of hyperplasia of the transitional epithelium lining the pelvis and overlying the renal papilla were significantly increased in all dosed groups of males and females. In male rats, the incidences of focal suppurative inflammation were significantly increased in the 250 and 500 mg/kg bw/day groups. A significantly increased incidence of chronic active inflammation of the nose occurred in 500 mg/kg bw/day males. Also in 500 mg/kg bw/day males, the incidence of chronic active inflammation of the forestomach was increased.

In conclusion, under the conditions of this test, there was clear evidence of carcinogenic activity of β-myrcene in male F344/N rats based on increased incidences of renal tubule neoplasms due to α2u-globulin accumulation. There was no evidence of carcinogenic activity of β-myrcene in female F344/N rats based on not biologically relevant increased incidences of renal tubule adenoma.

As the carcinogenic effect found in kidneys in rats is sex and species specific, it is concluded therefore that it is not relevant to humans.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 2002 to April 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP study conducted equivalent or similar to OECD Guideline 451 with minor deviations: blood count not performed and food consumption not followed

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 451 (Carcinogenicity Studies)

Deviations:

yes

Remarks:

blood count not performed and food consumption not followed

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

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: 6-7 weeks
- Housing: 1 (males) or 5 (females) animals/cage; polycarbonate cages; changed once or 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 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 approximately every 3 months
- All dose formulations were within 10 % of the target concentrations

Duration of treatment / exposure:

104 weeks for females, 105 weeks for males

Frequency of treatment:

5 days/week

Post exposure period:

No

Remarks:

Doses / Concentrations:
0, 250, 500 or 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on adverse effects on survival and body weight gains at 2000 and 4000 mg/kg bw/day observed in 3-months range-finding study, the highest dose selected for the 2‑year gavage study in mice was 1000 mg/kg bw/day
- Rationale for animal assignment: Animals were distributed randomly into groups of approximately equal initial mean body weights
- Rationale for selecting sentinel groups: Five male and five female animals were randomly selected for parasite evaluation and gross observation of disease

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Monthly beginning at week 5 and at the end of the study


BODY WEIGHT: Yes
- Time schedule for examinations: Initially on Day 1, weekly for 13 weeks, monthly thereafter and at the end of the study

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY (NEOPLASTIC and NON-NEOPLASTIC LESIONS): Yes
- Organs examined: In addition to gross lesions and tissue masses, the following tissues were examined: Adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, gallbladder, 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

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.
- Neoplasm and nonneoplastic lesion incidences: Poly‑k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence.
- Body weight data: Analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See detailed table below.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See detailed table below.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See detailed table below.

Details on results:

MORTALITY
- Survival of 1000 mg/kg bw/day mice was significantly less than that of the vehicle controls; the cause of the deaths was uncertain
- See table 1 for more details


CLINICAL SIGNS
- No treatment-related clinical effects were observed


BODY WEIGHT
- Mean body weights of 1000 mg/kg bw/day males and females and 500 mg/kg bw/day females were less than those of the vehicle controls after weeks 8, 11, and 17, respectively


HISTOPATHOLOGY: NON-NEOPLASTIC/NEOPLASTIC
- Liver: Incidences of liver neoplasms were significantly increased in 250 and/or 500 mg/kg bw/day males and 250 mg/kg bw/day females. Liver neoplasms included hepatocellular adenoma and hepatocellular carcinoma in males and females and hepatoblastoma in males. The incidences of hepatocellular hypertrophy were significantly increased in 500 mg/kg bw/day males and females, as was the incidence of mixed cell focus in 500 mg/kg bw/day females.
- Spleen: The incidences of bone marrow atrophy and lymph node follicle atrophy in the spleen were significantly increased in 500 mg/kg bw/day females.
- Stomach: In the forestomach, there were significantly increased incidences of inflammation and epithelial hyperplasia in 500 mg/kg bw/day females.


HISTORICAL CONTROL DATA (if applicable):
- Yes, NTP historical database contains all studies that use the NTP 2000 diet with histopathology findings completed until November 19, 2008. Additionally, historical database includes studies using the same route of administration, and the overall incidences of neoplasms for all routes of administration

Relevance of carcinogenic effects / potential:

The incidence of hepatocellular adenoma and carcinoma was increased in males and females. However, this increase of neoplasms was within historical data and was not dose-related in females. A high background of liver tumours naturally occurring is described in B6C3F1 male mice and no increase in the incidence of liver tumours was reported in F344 rats, therefore this carcinogenic effect in the liver of male mice seems specific to this species and has no relevance for humans.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increased incidences of liver neoplasms

Remarks on result:

other:

Remarks:

Effect type: other: carcinogenicity (not relevant for humans) (migrated information)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Increased incidences of hepatocellular neoplasms not dose-related and/or within the historical control data; mortality at 1000 mg/kg bw/day

Remarks on result:

other:

Remarks:

Effect type: other: carcinogenicity (not relevant for humans) (migrated information)

Table 1: Summary of the 2 -year carcinogenesis study of β-myrcene in mice*

	Male B6C3F1 Mice	Female B6C3F1 Mice
Doses in corn oil by gavage	0, 250, 500 or 1000 mg/kg bw/day	0, 250, 500 or 1000 mg/kg bw/day
Body weights	1000 mg/kg bw/day group less than vehicle control group after week 8	1000 mg/kg bw/day group less than vehicle control group after week 11; 500 mg/kg bw/day group less than vehicle control group after week 17
Survival rates	35/50, 35/50, 31/50, 21/50	39/50, 34/50, 35/50, 17/50
Nonneoplastic effects	Liver: hepatocyte, hypertrophy (1/50, 2/50, 16/50)	Liver: hepatocyte, hypertrophy (0/50, 0/50, 6/50); mixed cell focus (1/50, 4/50, 6/50)
Neoplastic effects	Liver: hepatocellular adenoma (26/50, 41/50, 43/50); hepatocellular carcinoma (14/50, 20/50, 28/50); hepatocellular adenoma or carcinoma (33/50, 44/50, 48/50); hepatoblastoma (4/50, 6/50, 11/50); hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma (34/50, 45/50, 48/50)	None
Equivocal findings	None	Liver: hepatocellular adenoma (6/50, 13/50, 6/50); hepatocellular carcinoma (1/50, 7/50, 2/50); hepatocellular adenoma or carcinoma (7/50, 18/50, 8/50)
Level of evidence of carcinogenic activity	Clear evidence	Equivocal evidence

* Neoplasm and nonneoplastic lesion incidences are not presented for 1000 mg/kg bw/day male or female mice due to high mortality

Table 2: Incidences of neoplasms and nonneoplastic lesions of the kidney in mice in the 2-year gavage study of β-myrcene^a

	Vehicle Control	0.25 g/kg	0.5 g/kg
Male
Number Examined Microscopically	50	50	50
Clear Cell Focus^b	15	21	21
Eosinophilic Focus	16	23	21
Mixed Cell Focus	13	3**	6
Hepatocyte, Hypertrophy	1 (1.0)^c	2 (1.5)	16** (1.7)
Fatty Change	25 (1.6)	18 (1.6)	16* (1.6)
Inflammation, Chronic Active	26 (1.0)	24 (1.1)	23 (1.0)
Hepatocellular Adenoma, Multiple	15	31**	30**
Hepatocellular Adenoma (includes multiple)^d
Overall rate^e	26/50 (52%)	41/50 (82%)	43/50 (86%)
Adjusted rate^f	57.80%	88.20%	89.30%
Terminal rate^g	23/34 (68%)	33/35 (94%)	29/31 (94%)
First incidence (days)	468	533	525
Poly-3 test^h	P<0.001	P<0.001	P<0.001
Hepatocellular Carcinoma, Multiple	1	4	9**
Hepatocellular Carcinoma, (includes multiple)^i
Overall rate	14/50 (28%)	20/50 (40%)	28/50 (56%)
Adjusted rate	30.60%	42.80%	58.80%
Terminal rate	8/34 (24%)	13/35 (37%)	16/31 (52%)
First incidence (days)	611	533	450
Poly-3 test	P=0.003	P=0.158	P=0.004
Hepatocellular Adenoma or Carcinoma^j
Overall rate	33/50 (66%)	44/50 (88%)	48/50 (96%)
Adjusted rate	71.00%	92.60%	96.60%
Terminal rate	25/34 (74%)	33/35 (94%)	30/31 (97%)
First incidence (days)	468	533	450
Poly-3 test	P<0.001	P=0.003	P<0.001
Hepatoblastoma^k
Overall rate	4/50 (8%)	6/50 (12%)	11/50 (22%)
Adjusted rate	9.00%	13.30%	25.00%
Terminal rate	3/34 (9%)	3/35 (9%)	7/31 (23%)
First incidence (days)	599	582	660
Poly-3 test	P=0.027	P=0.382	P=0.041
Hepatocellular Carcinoma or Hepatoblastoma^l
Overall rate	16/50 (32%)	22/50 (44%)	31/50 (62%)
Adjusted rate	34.70%	46.70%	65.10%
Terminal rate	9/34 (27%)	14/35 (40%)	19/31 (61%)
First incidence (days)	599	533	450
Poly-3 test	P=0.002	P=0.163	P=0.002
Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastoma^m
Overall rate	34/50 (68%)	45/50 (90%)	48/50 (96%)
Adjusted rate	72.50%	94.00%	96.60%
Terminal rate	25/34 (74%)	33/35 (94%)	30/31 (97%)
First incidence (days)	468	533	450
Poly-3 test	P<0.001	P=0.003	P<0.001
Female
Number Examined Microscopically	50	50	50
Clear Cell Focus	0	1	1
Eosinophilic Focus	4	5	6
Mixed Cell Focus	1	4	6*
Hepatocyte, Hypertrophy	0	0	6* (1.5)
Fatty Change	29 (1.6)	35 (1.4)	16* (1.3)
Inflammation, Chronic Active	43 (1.0)	35* (1.1)	34 (1.0)
Hepatocellular Adenoma, Multiple	0	2	0
Hepatocellular Adenoma (includes multiple)^n
Overall rate	6/50 (12%)	13/50 (26%)	6/50 (12%)
Adjusted rate	13.00%	29.80%	14.60%
Terminal rate	6/39 (15%)	12/34 (35%)	6/35 (17%)
First incidence (days)	727 (T)	709	727 (T)
Poly-3 test	P=0.418	P=0.042	P=0.534
Hepatocellular Carcinoma^o
Overall rate	1/50 (2%)	7/50 (14%)	2/50 (4%)
Adjusted rate	2.20%	15.90%	4.80%
Terminal rate	1/39 (3%)	5/34 (15%)	1/35 (3%)
First incidence (days)	727 (T)	608	640
Poly-3 test	P=0.334	P=0.025	P=0.461
Hepatocellular Adenoma or Carcinoma^p
Overall rate	7/50 (14%)	18/50 (36%)	8/50 (16%)
Adjusted rate	15.10%	40.90%	19.30%
Terminal rate	7/39 (18%)	15/34 (44%)	7/35 (20%)
First incidence (days)	727 (T)	608	640
Poly-3 test	P=0.297	P=0.005	P=0.406

* Significantly different (P≤0.05) from the vehicle control group by the Poly-3 test

** P≤0.01

(T) Terminal sacrifice

^a Data for the 1 g/kg mice are not presented due to high mortality.

^b Number of animals with lesion

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

^d Historical incidence for 2-year gavage studies with corn oil vehicle control groups (mean ± standard deviation): 94/200 (47.0% ± 3.8%), range 44%-52%; all routes: 733/1,447 (50.7% ± 13.9%), range 22%-72%

^e Number of animals with neoplasm per number of animals with liver examined microscopically

^f Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality

^g Observed incidence at terminal kill

^h Beneath the vehicle control incidence is the P value associated with the trend test. Beneath the dosed group incidence are the P values corresponding to pairwise comparisons between the vehicle controls and that dosed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice.

ⁱ Historical incidence for corn oil gavage studies: 52/200 (26.0% ± 7.5%), range 16%-34%; all routes: 415/1,447 (28.7% ± 8.8%), range 16%-52%

^j Historical incidence for corn oil gavage studies: 124/200 (62.0% ± 5.9%), range 56%-68%; all routes: 961/1,447 (66.4% ± 12.3%), range 36%-84%

^k Historical incidence for corn oil gavage studies: 10/200 (5.0% ± 2.6%), range 2%-8%; all routes: 48/1,447 (3.3% ± 6.4%), range 0%-34%

^l Historical incidence for corn oil gavage studies: 59/200 (29.5% ± 7.0%), range 22%-38%; all routes: 446/1,447 (30.8% ± 9.7%), range 16%-54%

^m Historical incidence for corn oil gavage studies: 127/200 (63.5% ± 5.3%), range 58%-68%; all routes: 972/1,447 (67.2% ± 13.1%), range 36%-92%

ⁿ Historical incidence for corn oil gavage studies: 33/197 (16.8% ± 9.4%), range 6%-27%; all routes: 396/1,494 (26.5% ± 15.2%), range 2%-54%

^o Historical incidence for corn oil gavage studies: 11/197 (5.6% ± 3.5%), range 2%-10%; all routes: 137/1,494 (9.2% ± 6.7%), range 0%-28%

^p Historical incidence for corn oil gavage studies: 41/197 (20.9% ± 12.0%); range 8%-35%; all routes: 481/1,494 (32.2% ± 17.3%); range 6%-64%

Conclusions:

There was no evidence of carcinogenic activity of β-myrcene in female B6C3F1 mice based on marginally increased incidences of hepatocellular neoplasms that were not dose-related and/or within the historical control data. There was clear evidence of carcinogenic activity of β-myrcene in male B6C3F1 mice based on increased incidences of liver neoplasms. As the background of naturally occurring liver tumours is high in male mice, the carcinogenic effect found is considered as not relevant for humans.

Executive summary:

A chronic study was conducted to evaluate the carcinogenic potential of β‑myrcene in B6C3F1 mice according to method equivalent or similar to OECD Guideline 451 in compliance with Good Laboratory Practices.

Groups of 50 male and 50 female mice were administered β‑myrcene at concentrations of 0, 250, 500 or 1000 mg/kg bw/day in corn oil by gavage, 5 days per week for 104 weeks for females and 105 weeks for males. Parameters evaluated included survival, body weights, clinical observations, gross necropsy and histopathological (non-neoplastic and neoplastic) findings in all animals.

Survival of 1000 mg/kg bw/day mice was significantly less than that of the vehicle controls; the cause of the deaths was uncertain. Mean body weights of 1000 mg/kg bw/day males and females and 500 mg/kg bw/day females were less than those of the vehicle controls after weeks 8, 11, and 17, respectively. The incidences of liver neoplasms were significantly increased in 250 and/or 500 mg/kg bw/day males and 250 mg/kg bw/day females. Liver neoplasms included hepatocellular adenoma and hepatocellular carcinoma in males and females and hepatoblastoma in males. The incidences of hepatocellular hypertrophy were significantly increased in 500 mg/kg bw/day males and females, as was the incidence of mixed cell focus in 500 mg/kg bw/day females. The incidences of bone marrow atrophy and lymph node follicle atrophy in the spleen were significantly increased in 500 mg/kg bw/day females. In the forestomach, there were significantly increased incidences of inflammation and epithelial hyperplasia in 500 mg/kg bw/day females.

In conclusion, under the conditions of this test, there was no evidence of carcinogenic activity of β-myrcene in female B6C3F1 mice based on marginally increased incidences of hepatocellular neoplasms that were not dose-related and/or within the historical control data. There was clear evidence of carcinogenic activity of β-myrcene in male B6C3F1 mice based on increased incidences of liver neoplasms. However, a high background of liver tumours naturally occurring is described in B6C3F1 male mice and no increase in the incidence of liver tumours was reported in F344 rats, therefore this carcinogenic effect in the liver of male mice seems specific to this species and has no relevance for humans.

Endpoint conclusion

Dose descriptor:

LOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

other: rats and mice

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The β‑myrceneis a monoterpene hydrocarbon occurring naturally in many plant species. It did not show any evidence of genotoxicity in all the studies conducted.

In the rat NTP study there is a clear development of a Male Rat Nephropathy Syndrom (MRNS) which is known to be due to accumulation of α2u-globulin in the cells of the proximal tubule, a mechanism not relevant to human. When important, this accumulation leads to cell tubular lesion with necrosis which, in turn, can lead to kidney tumors. These tumors are now well known to be also non relevant to human.

Although MRNS is clearly present with β-myrcene, it is probably not the only cause of nephropathy as some lesions are also observed in female rats (and also in male and female mice).

There is no increase in malignant kidney tumors in females and the slight increase in renal tubule adenoma (0/50, 1/50, 0/50, 2/50) is in the range of historical controls for NTP studies. On the other hand, there is a clear increased incidence of malignant (0/50,12/50, 13/50 ) and combined (0/50, 12/50, 13/50) kidney tumors in males. It can then be concluded that although nephropathy is not a pure MRNS, the tumors observed in male rats are due to this syndrome and are then no relevant to human and the small increase of benign tumors in females appears to be non biologically relevant.

In male and female mice, liver was a primary target of β-myrcene toxicity. In the 3-month study, absolute and relative liver weights were increased inmale and female mice. In the 2-year study, there is a clear increase in hepatocellular adenoma (26/50, 41/50, 43/50); hepatocellular carcinoma (14/50, 20/50, 28/50) hepatocellular adenoma or carcinoma (33/50, 44/50, 48/50) in males. However, due to the very high background incidence (66% for adenoma or carcinoma in the control group), the significance of this increase at levels where there is a clear hepatic hypertrophy is unclear. It must also be noted that these incidences are well within the historical control range (upper limit of the historical control range (all routes considered) reaching 72%, 52%, 84% and 34%, for adenoma, carcinoma, combined adenoma and carcinoma, and hepatoblastoma, respectively). In females where the background incidence is much lower (14% for adenoma or carcinoma in the control group), there is no dose related increase in either malignant or benign tumors. Based on the above considerations, the relevance of these increased liver tumor incidence in male mice should be considered as not relevant to humans.

 

In conclusion, β-myrcene administered by gavage at toxic dose in rats and mice induces increased incidence of benign and malignant tumors on the target organ for toxic effect in both species but none of them appear relevant for cancer classification in human. Thus it is not deemed necessary toclassify β-myrcene as carcinogen.

Justification for classification or non-classification

When considering each 2-year study:

- In mice: the “clear evidence of carcinogenic activity of β-myrcene in male B6C3F1 mice” is based on an increased incidence of liver neoplasms. High background incidence of liver tumors is typically observed in studies with B6C3F1 male mice and a very high background rate of liver tumors (66%) in controls was actually observed during this study.

The relevance of mouse liver tumors can be seriously questioned for a non-genotoxic substance like β-myrcene. Indeed, negative results were obtained with myrcene in the following four in vitro and two in vivo genotoxicity tests: reverse mutation in bacterial strains (TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA/pKM101), chromosome aberration test in human lymphocytes, gene mutation in CHO cells, sister chromatid exchange in human lymphocytes, chromosome aberration test on rat bone marrow cells and micronucleus test in mouse erythrocytes.

A genetic drift can be observed in the mice strain used in the most recent NTP bioassays on the basis of historical data: B6C3F1 mice are not a reliable indicator of carcinogenic hazard for β-myrcene. The European Food Safety Authority [1] and Commonwealth of Australia [2] have taken a consistent approach to considering such liver tumors not relevant to humans.

The 2-year mouse data are not a sufficient basis to support any classification for carcinogenicity.

- In rats: the “clear evidence of carcinogenic activity of β-myrcene in male F344/N rats” is based on an increased incidence of renal tubule adenomas (and not carcinomas). Actually, this results in an increased incidence of combined renal tubule adenomas and carcinomas in males, even if the incidence of carcinomas was slightly increased at the low dose level only. Renal tubule tumors were observed in males due to the well-known sex- and species-specific mechanism of α2u-globulin accumulation. This type of nephropathy is likely in male rats in this study up to the high dose level and can explain the increased incidence of neoplastic lesions in this sex.

The presence of renal neoplasms in female rats suggests a mechanism of carcinogenesis that may be related to nephrosis and is distinct from the α2u-globulin mechanism. However, even if α2u-globulin is not the sole mechanism of action, the other most likely mechanism of action involves chronic progressive nephropathy (CPN). But, CPN, like α2u-globulin, is a mechanism of action considered by many not to be relevant to humans [3]. Also, the increased incidence of renal tubule adenomas in females is not dose-related and no carcinomas were observed. As no biologically relevant increase in adenoma/carcinoma compared to historical data was observed in females, it is concluded that this “new” mechanism (if any) is not sufficient alone to lead to the development of renal tumors.

Therefore, the 2-year rat data are not a sufficient basis to support any classification for carcinogenicity.

In addition, as clearly reminded in the NTP Technical Report (TR) 557, “the interpretive conclusions presented in NTP Technical Reports are based only on the results of these NTP studies. Extrapolation of these results to other species, including characterization of hazards and risks to humans, requires analyses beyond the intent of these reports.” This means that these conclusions cannot be considered alone but together with other information. Within the NTP, NTP Technical Reports are used to prepare Reports on Carcinogens (RoCs) which take into consideration “the weight of the scientific evidence, including studies not conducted by NTP, and the human relevance of findings in animal studies”. The 14th Report on Carcinogens (RoC) was released by the U.S. Department of Health and Human Services on November 3, 2016. β-myrcene does not appear in this RoC and does not appear in the list of substances under evaluation or under consideration to this day. If there is any concern about the possible carcinogenic effect of β-myrcene, this is clearly not a priority to go ahead to clarify any mechanism of action and/or to classify it in the RoC.

[1] EFSA (2011). European Food Safety Authority; EFSA Statement on the scientific evaluation of two studies related to the safety of artificial sweeteners (question no EFSA-Q-2011-00064, approved on 25 February 2011 by European Food Safety Authority). EFSA J 9(2):2089 [16 pp;]. doi: 10.2903/j.efsa.2011.2089. Available at http://www.efsa.europa.eu/en/efsajournal/pub/2089.htm.

[2] Commonwealth of Australia, 2000. National Industrial Chemical Notification and Assessment Scheme (NICNAS), December 2000, Commonwealth of Australia, 134 pp.

[3] Hard GC et al. 2013 Consideration of rat chronic progressive nephropathy in regulatory evaluations for carcinogenicity. Toxicol Sci. Apr;132(2):268 -75.doi:10.1096/toxsci/kfs305.