Peppermint, ext.

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

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

Author:

Paumgartten, F.J., De-Carvalho, R.R., Souza, C.A., Madi, K., Chahoudm, I.

Year:

1998

Bibliographic source:

Braz J Med Biol Res. 1998 Jul;31(7):955-65

Materials and methods

Test guideline

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

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-methyleneocta-1,6-diene

Details on test material:

- Name of test material (as cited in study report): β-myrcene
- Source: Sigma Chemical Co., St. Louis, MO
- Analytical purity: 95% (by methanol extraction and HPLC purification)

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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).

 

Applicant's summary and conclusion

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 to investigate the effects of β-myrcene on fertility and general reproductive performance in Wistar rats similarly to OECD Guideline 415.

 

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 were 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.

 

In conclusion, 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.