Essential Oil of Piper nigrum L. (Family: Piperaceae). Obtained by the steam distillation of dried unripe berries.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Study of the effects of β-myrcene on rat fertility and general reproductive performance

Author:

F.J.R. Paumgartten , R.R. De-Carvalho, C.A.M. Souza, K. Madi and I.Chahoud

Year:

1998

Bibliographic source:

Braz J Med Biol Res, July 1998, Volume 31(7) 955-965

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

Reproductive toxicity study of β-myrcene was performed on wistar rats by oral gavage for 91 days.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

204-622-5

EC Name:

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

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-Methyl-3-methylene-1,6-octadiene

Details on test material:

- Name of test material (as cited in study report):7-Methyl-3-methylene-1,6-octadiene
- Molecular formula : C10H16
- Molecular weight : 136.236 g/mol
- Substance type: organic
- purity:95%

Specific details on test material used for the study:

- Name of test material (as cited in study report):7-Methyl-3-methylene-1,6-octadiene
- Molecular formula : C10H16
- Molecular weight : 136.236 g/mol
- Substance type: organic
- purity:95%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animals and env. conditions
TEST ANIMALS
- Source: Bor: spf, TNO; Fa. Winkelmann, Borchen, Germany
- Age at study initiation: No data available
- Weight at study initiation:
- Fasting period before study: No data available
- Housing: Males were housed individually in a Macrolon type 3 cage with wood shavings as bedding
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): standard pelleted diet (Altromin 1324, Lage, Germany)
- Water (e.g. ad libitum): tap water ad libitum
Acclimation period: 3 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±1°C
- Humidity (%):50±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12h light –dark cycle (light on from 9:00 to 21:00h)
IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

Details on exposure
PREPARATION OF DOSING SOLUTIONS:
The test material diluted with peanut oil
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food ):
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): test material dissolved into peanut oil
- Concentration in vehicle: 0,100,300,500mg/kg/day
- Amount of vehicle (if gavage): 2.5ml/kg

- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

- M/F ratio per cage:1:3
- Length of cohabitation:2 h each day (7:00 to 9:00 h)
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:The first 24-h period following the mating procedure was called day 0 of pregnancy if sperm were detected in the smear
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:The mating procedure was repeated every working day until all three females became"sperm-positive" or, alternatively, for fifteen mating sessions extending over three weeks.
- Further matings after two unsuccessful attempts: [no / yes (explain)]:No data available

- After successful mating each pregnant female was caged (how)::No data available

- Any other deviations from standard protocol:No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Commercially available ß-myrcene (Sigma Chemical Co., St. Louis, MO) was purified up to 95% (methanol extraction and HPLC purification)

Duration of treatment / exposure:

a) Male rats for 91 days prior to mating and during the mating period; b) female rats for 21 days prior to mating, during the mating period, and during pregnancy and lactation until day 21 after parturition.

Frequency of treatment:

once a day

Details on study schedule:

No data available

Doses / concentrations

Remarks:

0, 100, 300 and 500 mg/kg bw

No. of animals per sex per dose:

Total:240
0 mg/kg bw:15 male and 45 female
100 mg/kg bw:15 male and 45 female
300mg/kg bw:15 male and 45 female
500 mg/kg bw:15 male and 45 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: Yes

Time schedule:


BODY WEIGHT: Yes
Time schedule for examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations:

OTHER: Pregnant females were also observed for weight gain, signs of abortion, dystocia and prolonged duration of pregnancy.

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

The litters’ were examined for numbers of live and stillborn pups and gross abnormalities. The weight gain of the pups was recorded on postnatal days 6, 11, 16 and 21. Each pup was examined for signs of physical development and the days on which developmental landmarks appeared were recorded as follows: a) incisor eruption: the first sign of eruption through the gums of both lower incisors; b) fur development: the first detection of downy hair; c) eye opening: total separation of the upper and lower eyelids and complete opening of both eyes.

Postmortem examinations (parental animals):

SACRIFICE :
Male animals: All males were sacrificed by decapitation and autopsied at the end of the mating period.
Maternal animals: yes
On day 21 of pregnancy one-third of the females in each group were anesthetized by ethyl ether inhalation and killed by decapitation.
At weaning (postnatal day 21) all mothers were anesthetized with ethyl ether, killed by decapitation and subjected to post-mortem examination

GROSS NECROPSY: in male all major organs were inspected macroscopically and weighed. Livers and one of the two testes were fixed in a 10% neutral buffered formalin solution for routine histological processing and light microscopic evaluation of sections stained with hematoxylin-eosin. The number of spermatozoa in the remaining testis and cauda epididymis was counted, while in female The gravid uterus was weighed with its contents. Resorption as well as living and dead fetuses were counted. The number of implantation sites was determined by the method of Salewski (16).

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: As soon as possible after birth the numbers of viable and dead new borns were recorded, and the pups were sexed and weighed. Any newborn death on postnatal day 1 was considered to be a stillbirth. The weight gain of the pups was recorded on postnatal days 6, 11, 16 and 21. Each pup was examined for signs of physical development and the days on which developmental landmarks appeared were recorded as follows: a) incisor eruption: the first sign of eruption through the gums of both lower incisors; b) fur development: the first detection of downy hair; c) eye opening: total separation of the upper and lower eyelids and complete opening of both eyes.


GROSS NECROPSY
All living fetuses were numbered with a marker pen, examined for externally visible malformations and fixed in a 5% formalin solution. All fetuses were examined for skeletal anomalies after clearing with potassium hydroxide and staining with Alizarin Red S

Statistics:

Data were analysed by one-way analysis of variance or, alternatively, by the Kruskal-Wallis test whenever the data did not fit a normal distribution. Differences between groups were tested by the two-tailed Student t-test or Mann-Whitney U-test. Proportions were analysed by the chi-square test or, alternatively, by the Fischer exact test. Statistical evaluation was performed using a MINITAB program (MTB, University of Pennsylvania, 1984), and a difference was considered statistically significant at P<0.05.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicity were apparent in male rats treated orally in dose concentration 100, 300 and 500 mg/kg body weight for 91 days prior to mating and during the mating period.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No deaths were induced at dose level 100, 300 and 500 mg/kg body weight.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in body weight gain between the control and the treated male and female rats during the premating (21 days) and mating periods.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

no effect of treatment was found either on the number of spermatids in the testis or on the number of spermatozoa in the cauda epididymis

Reproductive performance:

no effects observed

Description (incidence and severity):

The test material did not present any adverse effect on fertility indices at the dose 100, 300 and 500 mg/kg body weight .The proportion of females impregnated by male rats (mating index), and the ratio of pregnant to sperm positive females (pregnancy index) did not differ between control and treated groups. Thus, no indication was found that test material administered orally at doses as high as 500 mg/kg could impair male or female fertility.
Duration of pregnancy was not affected by treatment with dose level100, 300 and 500 mg/kg body weight. Also no adverse effect on labor was noted.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: No efects on reproductive performance

2

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: slight increase in both absolute and relative weights of liver and kidneys of males exposed to the dose 500 mg/kg body weight.

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

pup mortality in the treated groups was not above that observed in the vehicle-control group on the first day of life (stillbirths) or throughout lactation, i.e. from postnatal day 2 through day 21

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight of treated fetuses did not differ from that of the control group at dose level 100, 300 and 500 mg/kg body weight .

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

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

At the highest dose tested 500 mg/kg, it produced a slight increase in the resorption rate and a parallel decrease in the ratio of live fetuses per implantation site.The effects of prenatal exposure to test material on the occurrence of fetal skeleton abnormalities No differences between control and treated groups were observed at doses up to 300 mg per kg body weight, but the frequency of skeletal malformations was increased at 500 mg/kg. Nonetheless, the higher incidence of skeletal abnormalities observed at this dose level seems to have been due, to a large extent, to an increase in the occurrence of anomalies such as fused os zygomatic, dislocated sternum (non-aligned sternebrae)and lumbar extra ribs, the spontaneous frequencies of which are high in our rat strain. Anyhow, the higher ncidence of skeletal abnormalities as well as the embryolethal effect clearly indicated that a dose as high as 500 mg /kg is embryotoxic to rats.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetal skeleton abnormalities such as fused os zygomatic, dislocated sternum (non-aligned sternebrae) and lumbar extra ribs, the spontaneous frequencies of which are high in rat strain.

Remarks on result:

other: incidence of skeletal abnormalities as well as the embryolethal effect clearly indicated that a dose as high as 500 mg /kg is embryotoxic to rats.

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

musculoskeletal system

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In reproductive toxicity study, the NOAEL was considered to be 300 mg/kg bw as no dose related effects on reproductive and developmental parameters were observed and LOAEL was considered to be 500 mg/kg bw as incidence of skeletal abnormalities as well as the embryolethal effect was observed as well as slight increase in both absolute and relative weights of liver and kidneys of males, When male and female wistar rats treated with β-myrcene (123-35-3) by oral gavage for 91 days.

Executive summary:

The reproductive and developmental toxicity study ofβ-myrcene (123-35-3) was performed on male and female wistar rats obtain from Bor: spf, TNO; Fa. Winkelmann, Borchen, Germany. The animals received a standard pelleted diet (Altromin 1324, Lage, Germany) and tap waterad libitumduring the experiment. All rats were adapted to the conditions of our animal quarters for three weeks before starting the experiment. The test material dissolved in peanut oil and administered in dose concentration 100, 300 and 500 mg/kg body weight via oral gavage route once a day to 3 experimental group each contain 15 male and 45 female animals while the control group received a similar treatment but with vehicle only (peanut oil, 2.5 ml/kg body weight).The exposure period for male rats for 91 days prior to mating and during the mating period while for female rats for 21 days prior to mating, during the mating period, and during pregnancy and lactation until day 21 after parturition.

All the animals were observed for mortality, and signs of toxicity and weight development, Pregnant females were also observed for weight gain, signs of abortion, dystocia and prolonged duration of pregnancy. All males were sacrificed by decapitation and autopsied at the end of the mating period. All major organs were inspected macroscopically and weighed. Livers and one of the two testes were fixed in a 10% neutral buffered formalin solution for routine histological processing and light microscopic evaluation of sections stained with hematoxylin-eosin. The number of spermatozoa in the remaining testis and cauda epididymis was counted as described elsewhere (15). The following indices were used: 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 On day 21 of pregnancy one-third of the females in each group were anesthetized by ethyl ether inhalation and killed by decapitation. The gravid uterus was weighed with its contents. Resorption as well as living and dead fetuses were counted. The number of implantation sites was determined by the method of Salewski (16). All living fetuses were immediately weighed, numbered with a marker pen, examined for externally visible malformations and fixed in a 5% formalin solution. All fetuses were examined for skeletal anomalies after clearing with potassium hydroxide and staining with Alizarin Red S (17). All the remaining pregnant females were allowed to give birth to their offspring. From pregnancy day 20 on the dam's cages were inspected for births and the day of birth was designated as postnatal day 1. As soon as possible after birth the numbers of viable and dead new born were recorded, and the pups were sexed and weighed. Any new born death on postnatal day 1 was considered to be a stillbirth. The weight gain of the pups was recorded on postnatal days 6, 11, 16 and 21. Each pup was examined for signs of physical development and the days on which developmental landmarks appeared were recorded as follows: a) incisor eruption: the first sign of eruption through the gums of both lower incisors; b) fur development: the first detection of downy hair; c) eye opening: total separation of the upper and lower eyelids and complete opening of both eyes. At weaning (postnatal day 21) all mothers were anesthetized with ethyl ether, killed by decapitation and subjected to post mortem examination.

 

No signs of toxicity were apparent in male rats treated orally in dose concentration 100, 300 and 500 mg/kg body weight for 91 days prior to mating and during the mating period. There were no statistically significant differences in body weight gain between the control and the treated male and female rats during the premating (21 days) and mating periods. No deaths were induced atdoselevel 100, 300 and 500 mg/kg body weight.No other treatment-related abnormality was noted in treated rats at autopsy. Light microscopy evaluation of sections stained with hematoxylin and eosin revealed no morphological alterations in the liver or testicular tissue of male rats exposed to test material in doselevel 100, 300 and 500 mg/kg body weight.The test material did not present any adverse effect on fertility indices at the dose100, 300 and 500 mg/kg body weight .The proportion of females impregnated by male rats (mating index), and the ratio of pregnant to sperm positivefemales (pregnancy index) did not differ between control and treated groups. Thus, no indicationwas found that test material administered orally at doses as high as 500 mg/kg could impair male or female fertility.Duration of pregnancy was not affected by treatment with dose level100, 300 and 500 mg/kg body weight. Also no adverse effect on labor was noted.Except for a slight increase in both absolute and relative weights of liver and kidneys of males exposed to the dose500 mg/kg body weight.

 

The pup mortality in the treated groups was not above that observed in the vehicle-control group on the first day of life (stillbirths) or throughout lactation, i.e.from postnatal day 2 through day 21. The body weight of treated fetuses did not differ from that of the control group at dose level 100, 300 and 500 mg/kg body weight.The effects of prenatal exposure to test material on the occurrence of fetal skeleton abnormalities. No differences between control and treated groups were observed at doses up to 300 mg per kg body weight, but the frequency of skeletal malformations was increased at 500 mg/kg. Nonetheless, the higher incidence of skeletal abnormalities observed at this dose level seems to have been due, to a large extent, to an increase in the occurrence of anomalies such as fused os zygomatic, dislocated sternum (non-aligned sternebrae) and lumbar extra ribs, the spontaneous frequencies of which are high in our rat strain. Anyhow, the higher incidence of skeletal abnormalities as well as the embryolethal effect clearly indicated that a dose as high as 500 mg /kg is embryotoxic to rats. Hencethe NOAEL was considered to be 300 mg/kg bw as no dose related effects on reproductive and developmental parameters were observed and LOAEL was considered to be 500 mg/kg bw asincidence of skeletal abnormalities as well as the embryolethal effect was observed as well as slight increase in both absolute and relative weights of liver and kidneys of males,When male and femalewistar rats treated withβ-myrcene (123-35-3)by oral gavage for 91 days.