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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The no-observed-adverse-effect level (NOAEL) for toxic effects on fertility and general reproductive performance of β-myrcene via oral route were considered to be 300 mg/kg bw/day in Wistar rats.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study well documented and conducted similar to OECD Guideline 415 with deviations: mating conditions (1 male for 3 females); food consumption not followed; bodyweight frequency not adequate

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 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

mating conditions (1 male for 3 females); food consumption not followed; bodyweight frequency not adequate

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fa. Winkelmann, Borchen, Germany
- Housing: Housed in macrolon type 3 cage with wood shavings as bedding
- Diet (e.g. ad libitum): Standard pelleted diet (Altromin 1324, Lage, Germany), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: Three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1 °C
- Humidity (%): 50 ± 5%
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test material was dissolved in peanut oil

Details on mating procedure:

- M/F ratio per cage: 1:3
- Length of cohabitation: 2 hours/day
- Proof of mating: Sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after two unsuccessful attempts: Yes, mating procedure was repeated every working day until all three females became sperm-positive or, alternatively, for 15 mating sessions extending over 3 weeks

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

- Male rats: 91 days prior to mating and during the mating period
- Female rats: 21 days prior to mating, during the mating period and during pregnancy and lactation until Day 21 after parturition

Frequency of treatment:

Once daily

Details on study schedule:

None

Remarks:

Doses / Concentrations:
0, 100, 300 and 500 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

15 males and 45 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

None

Positive control:

No

Parental animals: Observations and examinations:

- All Fo-males and -females were evaluated for weight development, mortality and signs of toxicity

- Pregnant females were observed for weight gain, signs of abortion, dystocia and prolonged duration of pregnancy

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in all male parental generations: Testis weight, sperm count in testes and cauda epididymis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, signs of physical development and the days on which developmental landmarks (incisor eruption, fur development or eye opening) appeared

GROSS EXAMINATION OF DEAD PUPS:
- No; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed by decapitation and autopsied at the end of the mating period
- Maternal animals: One-third of surviving females were sacrificed on Day 21 of pregnancy for cesarean examinations. All mothers were killed for postmortem examination on postnatal Day 21

GROSS NECROPSY
- All major organs were inspected macroscopically

HISTOPATHOLOGY / ORGAN WEIGHTS
Male animals:
- Organ weights: Liver, kidney, spleen, heart, thymus, brain and testes were weighed
- Histological examinations: Livers and one of the two testes were histologically examined

OTHERS
Cesarean examination:
- Gravid uterus was weighed with its contents
- Resorption as well as living and dead fetuses were counted
- Number of implantation sites were determined
- All living fetuses were immediately weighed and examined for externally visible malformations
- All fetuses were examined for skeletal anomalies

Postmortem examinations (offspring):

Not applicable

Statistics:

Statistical evaluation was performed using a MINITAB program (MTB, University of Pennsylvania, 1984), and a difference was considered statistically significant at P < 0.05.
- Data were analyzed by one-way analysis of variance (ANOVA) followed by Kruskal-Wallis test
- Differences between groups were tested by two-tailed Student t-test or Mann-Whitney U-test
- Proportions were analyzed by the chi-square test or, alternatively, by the Fischer exact test

Reproductive indices:

- Mating index = [No. of sperm-positive females ÷ No. of mated females] x 100
- Pregnancy index = [No. of pregnant females ÷ No. of sperm-positive females] x 100

Offspring viability indices:

- % of stillbirths = [No. of stillbirths/total of pups born] x 100
- % of pups dead = [No. of pups dead/No. of viable pups on Day 1] x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

food consumption not examined

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

food consumption not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Mortality: No deaths were observed
- Clinical signs: No signs of toxicity were apparent

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- No statistically significant differences in body weight gain between the control and the treated rats were observed

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- No treatment-related effect was found either on the number of spermatids in the testis or on the number of spermatozoa in the cauda epididymis

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No treatment-related effects were observed on mating and pregnancy index
- No treatment-related adverse effects were observed on duration of pregnancy or labor

ORGAN WEIGHTS (PARENTAL ANIMALS)
- At 500 mg/kg bw/day: Slight increase in both absolute and relative weights of liver and kidneys were observed

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Microscopic evaluation revealed no morphological alterations in the liver or testicular tissues of male rats

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increase in liver and kidney weights in parental animals at 500 mg/kg bw/day

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- Slight increase in the resorption rate (3.0% vs. 10.5% in control vs. 500 mg/kg bw group, respectively) and a parallel decrease in the ratio of live fetuses per implantation site (97.0% vs. 89.5% in control vs. 500 mg/kg bw group, respectively) were observed at 500 mg/kg bw/day.
- A slight retardation in the appearance of incisor eruption, primary coat and eye opening were observed but this effect was not considered to be dose-related and the delay was more evident with incisor eruption (300 mg/kg bw/day) and eye opening (100 and 300 mg/kg bw/day).
- See table 1 for detailed data

MORTALITY (OFFSPRING)
- No differences were observed between control and treatment groups on the first day of life (stillbirths) or throughout lactation (postnatal Day 2-21).

BODY WEIGHT (OFFSPRING)
- No differences between control and treated groups were found with regard to maternal or offspring weight changes during the lactation period.

GROSS PATHOLOGY (OFFSPRING)
- Frequency of skeletal malformations: No differences between control and treated groups were observed at doses up to 300 mg/kg bw/day, but the frequency of skeletal malformations was increased at 500 mg/kg bw/day (35.4% vs. 64.7% in control vs. 500 mg/kg bw group, respectively). However, this effect was attributed to spontaneous strain-specific increase in the occurrence of anomalies such as fused os zygomatic, dislocated sternum (non-aligned sternebrae) and lumbar extra ribs.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased resorption rate and a higher frequency of fetal skeleton anomalies observed in the 500 mg/kg bw/day group

Reproductive effects observed:

not specified

Table 1: Physical signs of postnatal development of offspring of rats treated orally with β-myrcene (0, 100, 300 and 500 mg/kg bw/day)

Postnatal day	Primary coat (%)				Incisor eruption (%)				Eye opening (%)
	0	100	300	500	0	100	300	500	0	100	300	500
7	78	67*	48*	59*	1.4	-	-	-	-	-	-	-
8	99	94	95	90	2.8	38	4.2	1.5	-	-	-	-
9	100	100	100	97	41	79	27*	33*	-	-	-	-
10	-	-	-	100	78	99	57*	81	-	-	-	-
11	-	-	-	-	98	100	87*	100	-	-	-	-
12	-	-	-	-	100	-	98	-	-	-	-	-
13	-	-	-	-	-	-	100	-	3.5	4.1	0.6	3
14	-	-	-	-	-	-	-	-	26	12	5.4	40
15	-	-	-	-	-	-	-	-	68	43*	37*	50*
16	-	-	-	-	-	-	-	-	93	73*	73*	87
17	-	-	-	-	-	-	-	-	100	83*	100	99
18	-	-	-	-	-	-	-	-	-	98	-	100
19	-	-	-	-	-	-	-	-	-	100	-	-

* P < 0.05 compared to controls (chi-square test).

Conclusions:

The no-observed-adverse-effect level (NOAEL) for toxic effects on fertility and general reproductive performance of β-myrcene via oral route were considered to be 300 mg/kg bw/day in Wistar rats.

Executive summary:

A study was conducted similarlly to OECD Guideline 415 to investigate the effects of β-myrcene on fertility and general reproductive performance in Wistar rats.

 

β-Myrcene (0, 100, 300 and 500 mg/kg bw/day) in peanut oil was administered daily by gavage to male Wistar rats (15/group) for 91 days prior to mating and during the mating period, as well as to females (45/group) continuously for 21 days before mating, during mating and pregnancy, and throughout the period of lactation up to postnatal Day 21. All Fo-males and -females were evaluated for weight development, mortality and signs of toxicity. Pregnant females were observed for weight gain, signs of abortion, dystocia and prolonged duration of pregnancy. Parameters examined in all male parental generations include testis weight, sperm count in testes and cauda epididymis. On Day 21 of pregnancy one-third of the females of each group were submitted to cesarean section. Resorption, implantation, as well as dead and live fetuses were counted. All fetuses were examined for external malformations, weighed and examined for skeleton evaluation. The remaining dams were allowed to give birth to their offspring. The progeny was examined at birth and subsequently up to postnatal Day 21. Mortality, weight gain and physical signs of postnatal development were evaluated.

 

An increase in liver and kidney weights was observed in male and female rats at 500 mg/kg bw/day. No other sign of toxicity was noted in male and female rats exposed to β-myrcene. The test material did not affect the mating index or the pregnancy index. No sign of maternal toxicity and no increase in externally visible malformations were observed at any dose level. Only at the highest dose tested (500 mg/kg bw/day) increased resorption rate and a higher frequency of fetal skeleton anomalies were observed. No adverse effect on postnatal weight gain was noted but days of appearance of primary coat, incisor eruption and eye opening were slightly delayed in the exposed offspring.

 

Therefore, the no-observed-adverse-effect level (NOAEL) for toxic effects on fertility and general reproductive performance of β-myrcene by the oral route were considered to be 300 mg/kg bw/day in Wistar rats.

Effect on fertility: via oral route

Dose descriptor:

NOAEL

300 mg/kg bw/day

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study similar to OECD Guideline 415 was conducted in Wistar rats. β-Myrcene was tested at 0, 100, 300 and 500 mg/kg bw/day. The toxicity signs observed in F0 animals were increases in liver and kidney weights in males and females at 500 mg/kg bw/day. The test material did not affect the mating index or the pregnancy index. Among reproductive parameters observed, only increased resorption rate and a slight decrease in live fetuses were observed at the highest dose tested (Paumgartten et al., 1998).

Moreover, no effect was found in either weight of testes, cauda epididymis and prostate, or on sperm count in males whose mothers were treated with myrcene from day 15 of gestation until post-natal day 21 (Delgado et al., 1993b). Female pups from these females had decreased fertility at very high doses (1000 and 1500 mg/kg bw/d; the decrease observed at 500 mg/kg bw/d was not attributable to test item). Due to lack of individual and historical control data, these effects are inconclusive.

Finally, in 3-month repeated dose toxicity studies in rats and mice (NTP, 2009; see section 7.5.1), no effects on the following reproductive parameters were observed in males and females up to 1000 mg/kg bw/day: weights of left cauda epididymis, left epididymis and left testis, spermatid measurements (spermatid heads), epididymal spermatozoal measurements (sperm motility and sperm heads) and estrous cycle (length of estrous cycle and stages: diestrus, proestrus, estrus and metestrus).

Effects on developmental toxicity

Description of key information

In a study similar to OECD Guideline 415, beta-myrcene (tested at 100, 300 and 500 mg/kg bw/d) increased liver and kidney weights

of male and female rats at 500 mg/kg bw/d without any effect on body weight. One third of the females were sacrificed for foetus examination. Higher frequency of fetal skeleton anomalies were observed at the highest dose tested, associated with maternal toxicity.
In a study conducted similarly to OECD Guideline 414 on rats treated with β-Myrcene at 250, 500 and 1200 mg/kg bw/day, a higher incidence of signs of retardation and of anomalies in the foetal skeleton was observed at 1200 mg/kg bw/day, a dose-level that was also toxic to the dams (decreased weight gain during the first days of treatment and death of one dam).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study well documented and conducted similarly to OECD Guideline 414 with deviations: mating conditions (1 male for 3 females); food consumption not followed; bodyweight only recorded at Day 0, 6, 16 and 20 of gestation

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

mating conditions (1 male for 3 females); food consumption not followed; bodyweight only recorded at Day 0, 6, 16 and 20 of gestation

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Oswaldo Cruz Foundation Central Animal House breeding stock
- Housing: Housed in plastic cages with wood shavings as bedding
- Diet (e.g. ad libitum): Nuvital diet for laboratory animals, Nuvilab Ltd, Curitiba, Brazil; ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 °C
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test material was dissolved in corn oil

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:3
- Length of cohabitation: 2 h
- Proof of mating: Sperm in vaginal smear referred to as Day 0 of pregnancy

Duration of treatment / exposure:

10 days (Day 6-15 of pregnancy)

Frequency of treatment:

Once daily

Duration of test:

20 days

No. of animals per sex per dose:

Treated females: 22-36; pregnant females: 16-29

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

Control animals: as no statistically significant difference was found between the untreated and the vehicle treated control animals, in any of the parameters analysed, the data of both groups were pooled and were thereafter designated the control group.

Maternal examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 16 and 20 of pregnancy

Ovaries and uterine content:

The uterine content was examined after termination: Yes, animals were sacrificed on Day 20 for caesarean examinations
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of living and dead foetuses: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes [all living foetuses]; weighed and examined for externally visible malformations
- Soft tissue examinations: Yes [5-7 litters per group]: evaluated for visceral malformations; heart, lungs, thymus, liver, spleen and kidneys were weighed and microdissected
- Skeletal examinations: Yes [all the remaining litters]

Statistics:

- Statistical analysis was by one way analysis of variance or, alternatively, by the Kruskal-Wallis test whenever the data did not fit a normal distribution
- Statistically significant differences between groups were tested by using a two sided Student's t-test or the Mann-Whitney U-test
- Proportions were analysed by the chi-square test
- Difference was considered to be statistically significant at P < 0.05

Indices:

No data

Historical control data:

No

Mortality:

mortality observed, treatment-related

Description (incidence):

At 1200 mg/kg bw/d: 1/29 dams died on Day 11 of pregnancy

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in weight gain during days 6-11 of pregnancy (17.3 ± 7 g vs. 3.2 ± 13.2 g in control vs. 1200 mg/kg group, respectively)

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Number of visible implantation sites was significantly reduced (12.6 ± 2.2 vs. 10.2 ± 2.9 in control vs. 1200 mg/kg group, respectively)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

mortality

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment-related effects on foetal weight were only very slight, or even insignificant, if the litter is taken as the statistical unit of analysis

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Increased frequency of irregularly positioned hind paws was observed in 1200 mg/kg bw/day group

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Visceral malformations such as enlarged ureters associated with an enlarged renal pelvis (one control foetus and one in 1200 mg/kg group), shorter ureter (one control foetus and one in 250 mg/kg bw/day group) and accessory (seventh) lobe in the liver (three foetus in 250 mg/kg bw/day group)

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Increased incidence of delayed ossification and minor gross structural anomalies in the foetal skeleton were observed in 1200 mg/kg bw/day group

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: higher incidence of signs of retardation and of anomalies in the foetal skeleton observed at 1200 mg/kg bw/day

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1. Occurrence of delayed ossification in foetuses of rats treated orally with β-myrcene on Days 6-15 of pregnancy

	β-Myrcene (mg/kg bw)
Treatment	0	250	500	1200
Foetuses examined (no.)	114	106	116	209
Percentage of foetuses with signs of delayed ossification^αin:
Skull bones	4.4	0.9	3.4	9.6*
Caudal vertebrae	7	2.8	6	37.8*
Forelimbs/metacarpus	2.6	0	0.9	9.1*
Hind limbs/metatarsus	5.3	2.8	3.4	29.2*

^α Signs of delayed ossification: not ossified (whole bone is not stained), poorly ossified (whole bone is poorly stained), and irregular spongy bones.

* P < 0.05; chi-square test

Conclusions:

The no-observed-adverse-effect level (NOAEL) for maternal toxicity and for embryo-foetotoxicity of β-myrcene administered orally (gavage) was considered to be 500 mg/kg bw/day in Wistar rats.

Executive summary:

A study comparable to Guideline OECD 414 was conducted to evaluate the embryo-foetotoxic potential of β-myrcene in the Wistar rats.

β -Myrcene (at 250, 500 and 1200 mg/kg bw/day) in corn oil was given orally to Wistar rats from Day 6 to 15 of pregnancy. Two control groups, one received vehicle only and another without treatment, were also studied simultaneously. All rats were weighed on Day 0, 6, 16 and 20 of pregnancy. Caesarean sections were performed on Day 20 of pregnancy, and the number of resorptions and implantation sites were recorded. Foetuses were weighed and examined for external, visceral and skeletal malformations.

No adverse effects on the offspring were seen with the lowest dose tested, but at 500 mg/kg bw/day and higher doses, decreased birth weight, increased perinatal mortality and delayed day of appearance of landmarks of postnatal development were observed. Moreover, fertility was impaired in female offspring exposed to the two highest doses of β-myrcene. However it is difficult to know if this effect was related to reprotoxicity or due to the general toxicity observed at these dose-levels.

Therefore, the no-observed-adverse-effect level (NOAEL) for maternal toxicity and for embryo-foetotoxicity of β -myrcene administered orally (gavage) was considered to be 500 mg/kg bw/day in Wistar rats.

 

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

500 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted similarly to OECD Guideline 414 on rats treated with β-Myrcene at 250, 500 and 1200 mg/kg bw/day. Decreased weight gain during the first days of treatment and the death of one of 29 treated dams indicated that the highest dose tested (1200 mg/kg bw/day) induced maternal toxicity. A higher incidence of signs of retardation and of anomalies in the foetal skeleton indicated that 1200 mg/kg bw/day was also toxic to the rat embryo (Delgado et al., 1993a).

Justification for classification or non-classification

All potential effects observed on reproduction and development were either associated with maternal toxicity, or not dose-related or data were not enough detailed to be conclusive. In conclusion, myrcene does not need to be classified for toxicity to reproduction.