Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 290-058-5 | CAS number: 90063-97-1 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Mentha arvensis, Labiatae.
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
A Weight of Evidence approach with constituent data was followed which is described below.
No information is available for cornmint or peppermint oil, however, no effects on development and/or reproduction were observed for 71.1% of the constituents.
Based on the available information on constituents, cornmint oil does not have to be classified as toxic to the development or reproduction in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2008/EC.
Link to relevant study records
- Endpoint:
- one-generation reproductive toxicity
- Type of information:
- experimental study
- 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.
- 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
- 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
- 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
- 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.
- Endpoint:
- one-generation reproductive toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Sex:
- male/female
- Vehicle:
- peanut oil
- Positive control:
- No
- 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
- 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
- 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
- Conclusions:
- The reproductive toxicity of cornmint oil was assessed using read across from the source substance β-myrcene. 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:
The reproductive toxicity of cornmint oil was assessed using read across from the source substance β-myrcene. 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. 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.
- Endpoint:
- screening for reproductive / developmental toxicity
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
- Reproductive effects observed:
- not specified
- Endpoint:
- extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
- Data waiving:
- other justification
- Justification for data waiving:
- the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
- Reproductive effects observed:
- not specified
Referenceopen allclose all
- 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
- 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.
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).
- 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
- 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.
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).
Effect on fertility: via oral route
- Endpoint conclusion:
- no study available
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Justification for selection of Effect on fertility via oral route:
No selection is made as a Weight of Evidence approach with constituent data was followed which is described below.
Effects on developmental toxicity
Description of key information
No information is available for cornmint or peppermint oil, however, no effects on development and/or reproduction were observed for 71.1% of the constituents.
Based on the available information on constituents, cornmint oil does not have to be classified as toxic to the development or reproduction in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2008/EC.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1973
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study well documented Restriction: no full macroscopic examination; no data on statistical evaluation
- Qualifier:
- according to guideline
- Guideline:
- other: unspecified
- GLP compliance:
- no
- Species:
- rat
- Strain:
- Wistar
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg bw - Details on mating procedure:
- Virgin adult were mated with young adult males (observation of the vaginal sperm plug was considered Day 0 of gestation)
- Duration of treatment / exposure:
- gestation days 6-15
- Frequency of treatment:
- daily
- Duration of test:
- 10 consecutive days
- Remarks:
- Doses / Concentrations:
2.18 mg/kg bw/d
Basis:
no data - Remarks:
- Doses / Concentrations:
10.15 mg/kg bw/d
Basis:
no data - Remarks:
- Doses / Concentrations:
47.05 mg/kg bw/d
Basis:
no data - Remarks:
- Doses / Concentrations:
218.0 mg/kg bw/d
Basis:
no data - No. of animals per sex per dose:
- 25 females per dose
No of pregnant animals (2.18, 10.15, 47.05, 218 mg/kg bw): 22, 23, 23, 22 - Control animals:
- yes, sham-exposed
- other: NEGATIVE CONTROL: 25 pregnant... (see attached file)
- Maternal examinations:
- CAGE SIDE OBSERVATIONS:No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 11, 15, 20
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: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: urogenital tract - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes - Fetal examinations:
- - External examinations: Yes: 1/3 per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: No data
Other :
- Fetus weight : Yes: all per litter
- Abnormalities/malfunctions : Yes: all per litter - Details on maternal toxic effects:
- Maternal toxic effects:no effects
Details on maternal toxic effects:
No clinical signs of maternal toxicity - Dose descriptor:
- NOEL
- Effect level:
- 218 mg/kg bw/day
- Based on:
- no data
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
No clinical signs of fetotoxicity - Dose descriptor:
- NOEL
- Effect level:
- 218 mg/kg bw/day
- Based on:
- no data
- Basis for effect level:
- other: teratogenicity
- Dose descriptor:
- NOEL
- Effect level:
- 218 mg/kg bw/day
- Based on:
- no data
- Basis for effect level:
- other: fetotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- L-Menthol was not embryo- or fetotoxic and had no teratogenic properties in well performed gavage studies in rat at non-maternally toxic doses (218 mg/kg bw).
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From April 28 to August 5, 1992
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP study conducted according to OECD Guideline 414 with a minor deviation as only 2 doses were tested but up to the limit dose of 1000 mg/kg/day
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- yes
- Remarks:
- (only 2 doses tested but up to the limit dose of 1000 mg/kg/day)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Lippische Versuchstierzucht, Hagemann GmbH, Germany
- Stock: Tif: RAI f (SPF)
- Age at study initiation: 54 days
- Weight at study initiation: 180-190 g
- Fasting period before study:
- Housing: Individually housed in MAKROLON cages type III
- Diet (e.g. ad libitum): ALTROMIN 1314 (ALTROMIN GmbH, Lage/Lippe, Germany), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 15%
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- other: sesame oil, DAB 10
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: Test solutions were freshly prepared in sesame oil each day immediately before dosing
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material was soluble in sesame oil
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 2009
- Source: Henry Lamotte, Bremen, Germany - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Concentrations: 50 and 200 mg/mL suspension
- Sampling interval: Sampled at start and termination of treatment
- Sample storage conditions before analysis: Samples were deep-frozen at -20 °C or cooler until dispatch
- Analytical method: Gas chromatography with FID detector - Details on mating procedure:
- - Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Overnight
- Proof of pregnancy: Sperm in vaginal smear referred to as Day 0 of pregnancy. If the smear was negative, mating was repeated.
- Fertile (proved) male rats of the same breed were repeatedly employed for mating, at the earliest 3 days after successful copulation - Duration of treatment / exposure:
- Day 6-15 of pregnancy
- Frequency of treatment:
- Once daily
- Duration of test:
- 20 days
- Remarks:
- Doses / Concentrations:
250 mg/kg bw/day
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 20 pregnant females per dose (plus 5 females per dose as reserve)
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected on the basis of a pilot study. In this limit test study, camphene (1000 mg/kg bw/day) was administered to 3 pregnant rats from Day 6 to 15 of pregnancy.
Except for a slight transient reaction after the first dosing, 1000 mg/kg bw/day was well-tolerated by the dams and did not influence the prenatal development.
- Rationale for animal assignment (if not random): Rats were assigned to their respective group according to their mating day i.e. in a cyclic way following the listing of positive findings in the vaginal smear. - Maternal examinations:
- - Time schedule: Twice daily
- Cage side observations were included: Behaviour, external appearance, mortality and faeces
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Daily
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
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20 under ether
- Organs examined: Ovaries and uteri - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations and location of fetuses in the uterus: Yes
- Number and size of resorptions: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses and placentae count, sex and viability of fetuses, weight of ovaries, weight and length of fetuses and weight of placentae - Fetal examinations:
- - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter] - Statistics:
- - Comparison of malformation and variation rates was carried out using R.A. Fisher’s exact test (p ≤ 0.05).
- Homogeneity of variances was tested by the Bartlett chi-square test followed by a one-way analysis of variance (ANOVA).
- When the results indicated a significant difference among groups Dunnett test (p ≤ 0.01) was used to compare the experimental groups with the control group. - Indices:
- - Resorption rate in % = (Resorptions / implantations) X 100
- Malformation rate in % = (malformed fetuses / fetuses) X 100
- Variation rate in % = (fetuses with variations / fetuses) X 100
- Pre-implantation loss in % = (corpora lutea-implantations / corpora lutea) X 100
- Post-implantation loss in % = (implantations-living fetuses / implantations) X 100
- Conception rate in % = (number of pregnant animals / number of animals mated) X 100 - Historical control data:
- Yes; summarized results of teratology studies in Sprague-Dawley rats (1988-1991) were attached as Appendix-4 to the study
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
- Mortality: No treatment-related mortality was observed.
- Clinical symptoms: At 1000 mg/kg bw/day, 6/20 dams showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5-20 minutes
after administration and lasted for 20-60 minutes, 1-2 hours or 2-6 hours. No clinical signs were observed in the remaining high-dosed and the low-dosed dams.
- Body weight, body weight change: Body weights remained within the normal range, body weight gain showed no influence of the test material.
- Food consumption: Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively.
Food consumption had normalised from the 10th gestation day onwards.
- Drinking-water consumption: Treatment did not influence drinking-water consumption.
- Autopsy findings: No treatment-related pathological changes were detected at autopsy. - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
- Mortality: No dead fetuses observed in control or treatment groups.
- Uterus and placentae weights, conception rate, mean number of corpora lutea and implantation sites, pre-implantation loss and number of viable fetuses, sex distribution of fetuses and weight of fetuses:
No treatment-related effects
- Number of resorptions and consequently the post-implantation loss: Slightly but not significantly (at p ≤ 0.01) increased at 1000 mg/kg bw/day
- External macroscopic examination: No treatment-related effects; one malformed fetus at 1000 mg/kg bw/day (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail,
omphalocele) belongs to the spontaneous range as to type and number of affected fetuses (control: one fetus with stump tail)
- Soft tissue examination: No treatment-related effects; very common variations (uni- or bilateral dilated renal pelvis, haemorrhages of the liver, 4th cerebral ventricle enlarged) were observed in all groups
without any dose-response relationships
- Skeletal examination: No treatment-related effects; variations (accessory 14th ribs, wavy ribs and bipartite/misaligned sternum) were observed in all groups and/or retardations (incomplete or missing
ossification of hyoid, skull, vertebral bodies and/or sternebrae) were found biologically non-significant - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: fetotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- In conclusion, the no-observed-effect level (NOEL) for maternal and fetotoxicity of camphene administered orally (gavage) was considered to be 250 mg/kg bw/day in Sprague-Dawley rats. Further,
camphene is not considered to be teratogenic. - Executive summary:
In a prenatal developmental toxicity study performed in accordance with OECD guideline 414 and in compliance with GLP, camphene in sesame oil was administered through gavage to groups of Sprague- Dawley pregnant rats (20/dose) at dose levels of 0 (vehicle control), 250 and 1000 mg/kg bw/day from Day 6 to 15 of pregnancy. Dams were observed twice daily for behaviour, external appearance, mortality and faeces. Body weights, food and water consumption were noted daily. Caesarean sections were performed on Day 20 of pregnancy and the ovaries and uterine contents were recorded. Foetuses were weighed and examined for external, visceral and skeletal malformations. No treatment-related mortality was observed at any dose level. At 1000 mg/kg bw/day, 6/20 dams showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5-20 minutes after administration and lasted for 20-60 minutes, 1-2 hours or 2-6 hours. Body weights remained within the normal range and body weight gain showed no influence of the test material. Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively. Treatment did not influence drinking-water consumption. No treatment-related pathological changes were detected at autopsy. No dead fetuses observed in control or treatment groups. No treatment-related effects were observed on uterus and placentae weights, conception rate, mean number of corpora lutea and implantation sites, pre-implantation loss and number of viable fetuses, sex distribution of fetuses and weight of fetuses. External macroscopic examination, examination of soft tissue and skeletal examination revealed no treatment-related biologically and/or statistically significant variations/retardations. Number of resorptions and consequently the post-implantation loss were increased slightly (statistically non- significant) at 1000 mg/kg bw/day. In conclusion, the no-observed-effect level (NOEL) for maternal and fetotoxicity of camphene administered orally (gavage) was considered to be 250 mg/kg bw/day in Sprague-Dawley rats. Therefore, camphene is not considered to be teratogenic.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1977
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Original reference in Japanese language
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- Principles of method if other than guideline:
- Prenatal developmental toxicity study: Groups of pregnant Japanese white rabbits were administered orally with d-limonene at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation and evaluated for teratogenicity.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rabbit
- Strain:
- other: Japanese white
- Details on test animals or test system and environmental conditions:
- no data
- Route of administration:
- oral: unspecified
- Vehicle:
- not specified
- Details on exposure:
- no data
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- no data
- Details on mating procedure:
- No data
- Duration of treatment / exposure:
- 13 days (gestation Day 6-18)
- Frequency of treatment:
- Once daily
- Duration of test:
- Gestatation Day 0 to postnatal Day 49
- Remarks:
- Doses / Concentrations:
0, 250, 500 or 1000 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 10 (in 0-500 mg/kg bw/day groups) or 18 (in 1000 mg/kg bw/day group) pregnant females
- Control animals:
- yes
- Details on study design:
- no data
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: No data
General behaviour observed, but no data regarding the frequency of observation
BODY WEIGHT: Yes
- Time schedule for examinations: daily
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
mean daily food consumption by treatment group is reported
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
POST-MORTEM EXAMINATIONS: No data
OTHER: - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight - Fetal examinations:
- - External examinations: Yes: about 90% per litter
- Visceral examinations: Yes: about 90% per litter
- Skeletal examinations: Yes: about 90% per litter - Statistics:
- statistical significance difference of effects from controls were calculated at 5% level.
- Indices:
- no data
- Historical control data:
- no data
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams with less than 40% mortality. The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg of d-limonene, but no anomalies were observed in the general behaviour of dams given 250 and 500 mg/kg of d-limonene during the gestation. - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
- External examination of fetuses showed no anormalies.
- Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.
- Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene. - Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: fetotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.
- Executive summary:
In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant Japanese white rabbits at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation. Food consumption and bodyweights of pregnant rabbits were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations.
Treatment with the highest dose level (1000 mg/kg bw/day) of d-limonene resulted in death of 6/18 dams (33% mortality). The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg bw/day of d-limonene, but no anormalies were observed in the general behavior of dams given 250 and 500 mg/kg bw/day of d-limonene during the gestation. External examination of fetuses showed no anormalies. Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.
Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be higher than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1977
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Original reference in Japanese language
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- Principles of method if other than guideline:
- Prenatal developmental toxicity study: Groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation and evaluated for developmental and postnatal development toxicity.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- mouse
- Strain:
- ICR
- Details on test animals or test system and environmental conditions:
- No data
- Route of administration:
- oral: unspecified
- Vehicle:
- not specified
- Details on exposure:
- No data
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- No data
- Details on mating procedure:
- no data
- Duration of treatment / exposure:
- 6 days (gestation Day 7-12)
- Frequency of treatment:
- Once daily
- Duration of test:
- Gestatation Day 0 to postnatal week 7
- Remarks:
- Doses / Concentrations:
0, 591 and 2363 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 15
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- No data
- Maternal examinations:
- See results tables
- Ovaries and uterine content:
- See results tables
- Fetal examinations:
- See results tables
- Statistics:
- statistical significance difference of effects from controls were calculated at 5% and 1% levels.
- Indices:
- No data
- Historical control data:
- No data
- Details on maternal toxic effects:
- Details on maternal toxic effects:
See results tables - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: Body weight gain
- Description (incidence and severity):
- decreased bodyweight gain
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
See results tables - Dose descriptor:
- LOAEL
- Effect level:
- 2 363 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- skeletal malformations
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- skeletal: rib
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 2 363 mg/kg bw/day
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.
- Executive summary:
In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation. Bodyweights of pregnant mice were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, sensory functions, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.
A significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day. However, no anomalies were observed in the general behavior of dams during the period of gestation. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control. In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded ossifications were restored to normal during postnatal development. A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.
Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal skeletal formation in fetuses at 2363 mg/kg bw/day.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1975
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Original reference in Japanese language
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- Principles of method if other than guideline:
- Prenatal developmental toxicity study: Groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation and evaluated for developmental toxicity.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- No data
- Route of administration:
- oral: unspecified
- Vehicle:
- other: 1% gum-arabic solution
- Details on exposure:
- Volume administered: 5 mL/kg bw for all doses
- Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- None
- Details on mating procedure:
- no data
- Duration of treatment / exposure:
- 7 days (gestation Day 9-15)
- Frequency of treatment:
- Once daily
- Duration of test:
- Gestation Day 0 to postnatal week 7
- Remarks:
- Doses / Concentrations:
0, 591 and 2869 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 20 pregnant rats
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- No data
- Maternal examinations:
- See result tables
- Ovaries and uterine content:
- See result tables
- Fetal examinations:
- See result tables
- Statistics:
- statistical significance difference of effects from controls were calculated at 5% level.
- Indices:
- No data
- Historical control data:
- No data
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Key result
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: decreased body weight gain, and death
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Prolongation of the ossification of metacarpals and proximal phalanges in fetuses, decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- skeletal malformations
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- fetal/pup body weight changes
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- other: decreased organ weights
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: Skeletal: metacarpals and proximal phalanges
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 2 869 mg/kg bw/day
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.
- Executive summary:
In a developmental toxicity study, d-limonene was administered orally to groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation. Bodyweight of pregnant rats were recorded on Days 0, 9, 12, 16 and 20 during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.
At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed. Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth. A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group. Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg.
Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1993
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: 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
- Remarks:
- Doses / Concentrations:
0, 250, 500 and 1200 mg/kg bw/day
Basis:
actual ingested - 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
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
At 250 and 500 mg/kg bw /day: No treatment-related effects
At 1200 mg/kg bw/day:
- Mortality: 1/29 dams died on Day 11 of pregnancy
- Body weights: 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)
- Cesarean examination: 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:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: other:
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: decreased weight gain, death 1/29 animals
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
- Body weight: 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 examinations: Increased frequency of irregularly positioned hind paws was observed in 1200 mg/kg bw/day group
- Visceral examinations: 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)
- Skeletal examinations: Increased incidence of delayed ossification and minor gross structural anomalies in the foetal skeleton were observed in 1200 mg/kg bw/day group - Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: signs of retardation
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- visceral/soft tissue: urinary
- other: delayed ossification and minor gross structural anomalies
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 1 200 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- 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 was conducted to evaluate the embryo-foetotoxic potential of β-myrcene in the Wistar rats similarly to OECD Guideline 414.
β -Myrcene (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 .
In conclusion, 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.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Key result
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: decreased body weight gain, and death
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Prolongation of the ossification of metacarpals and proximal phalanges in fetuses, decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- skeletal malformations
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- fetal/pup body weight changes
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- not specified
- Basis for effect level:
- other: decreased organ weights
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: Skeletal: metacarpals and proximal phalanges
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 2 869 mg/kg bw/day
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- Conclusions:
- The developmental toxicity of cornmint oil was assessed using read across from the source substance d-limonene. Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.
- Executive summary:
The developmental toxicity of cornmint oil was assessed using read across from the source substance d-limonene. In a developmental toxicity study, d-limonene was administered orally to groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation. Bodyweight of pregnant rats were recorded on Days 0, 9, 12, 16 and 20 during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.
At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed. Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth. A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group. Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg.
Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- he read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
At 250 and 500 mg/kg bw /day: No treatment-related effects
At 1200 mg/kg bw/day:
- Mortality: 1/29 dams died on Day 11 of pregnancy
- Body weights: 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)
- Cesarean examination: 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:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: other:
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: decreased weight gain, death 1/29 animals
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
- Body weight: 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 examinations: Increased frequency of irregularly positioned hind paws was observed in 1200 mg/kg bw/day group
- Visceral examinations: 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)
- Skeletal examinations: Increased incidence of delayed ossification and minor gross structural anomalies in the foetal skeleton were observed in 1200 mg/kg bw/day group - Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: signs of retardation
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- visceral/soft tissue: urinary
- other: delayed ossification and minor gross structural anomalies
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 1 200 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- Conclusions:
- The developmental toxicity of cornmint oil was assessed using read across from the source substance β-myrcene. 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:
The developmental toxicity of cornmint oil was assessed using read across from the source substance β-myrcene. A study was conducted to evaluate the embryo-foetotoxic potential of β-myrcene in the Wistar rats similarly to OECD Guideline 414.
β -Myrcene (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 .
In conclusion, 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.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
- Mortality: No treatment-related mortality was observed.
- Clinical symptoms: At 1000 mg/kg bw/day, 6/20 dams showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5-20 minutes
after administration and lasted for 20-60 minutes, 1-2 hours or 2-6 hours. No clinical signs were observed in the remaining high-dosed and the low-dosed dams.
- Body weight, body weight change: Body weights remained within the normal range, body weight gain showed no influence of the test material.
- Food consumption: Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively.
Food consumption had normalised from the 10th gestation day onwards.
- Drinking-water consumption: Treatment did not influence drinking-water consumption.
- Autopsy findings: No treatment-related pathological changes were detected at autopsy. - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
- Mortality: No dead fetuses observed in control or treatment groups.
- Uterus and placentae weights, conception rate, mean number of corpora lutea and implantation sites, pre-implantation loss and number of viable fetuses, sex distribution of fetuses and weight of fetuses:
No treatment-related effects
- Number of resorptions and consequently the post-implantation loss: Slightly but not significantly (at p ≤ 0.01) increased at 1000 mg/kg bw/day
- External macroscopic examination: No treatment-related effects; one malformed fetus at 1000 mg/kg bw/day (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail,
omphalocele) belongs to the spontaneous range as to type and number of affected fetuses (control: one fetus with stump tail)
- Soft tissue examination: No treatment-related effects; very common variations (uni- or bilateral dilated renal pelvis, haemorrhages of the liver, 4th cerebral ventricle enlarged) were observed in all groups
without any dose-response relationships
- Skeletal examination: No treatment-related effects; variations (accessory 14th ribs, wavy ribs and bipartite/misaligned sternum) were observed in all groups and/or retardations (incomplete or missing
ossification of hyoid, skull, vertebral bodies and/or sternebrae) were found biologically non-significant - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: fetotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The developmental toxicity of cornmint oil was assessed using read across from the source substance camphene.
In conclusion, the no-observed-effect level (NOEL) for maternal and fetotoxicity of camphene administered orally (gavage) was considered to be 250 mg/kg bw/day in Sprague-Dawley rats. Further,
camphene is not considered to be teratogenic. - Executive summary:
The developmental toxicity of cornmint oil was assessed using read across from the source substance camphene. In a prenatal developmental toxicity study performed in accordance with OECD guideline 414 and in compliance with GLP, camphene in sesame oil was administered through gavage to groups of Sprague- Dawley pregnant rats (20/dose) at dose levels of 0 (vehicle control), 250 and 1000 mg/kg bw/day from Day 6 to 15 of pregnancy. Dams were observed twice daily for behaviour, external appearance, mortality and faeces. Body weights, food and water consumption were noted daily. Caesarean sections were performed on Day 20 of pregnancy and the ovaries and uterine contents were recorded. Foetuses were weighed and examined for external, visceral and skeletal malformations. No treatment-related mortality was observed at any dose level. At 1000 mg/kg bw/day, 6/20 dams showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5-20 minutes after administration and lasted for 20-60 minutes, 1-2 hours or 2-6 hours. Body weights remained within the normal range and body weight gain showed no influence of the test material. Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively. Treatment did not influence drinking-water consumption. No treatment-related pathological changes were detected at autopsy. No dead fetuses observed in control or treatment groups. No treatment-related effects were observed on uterus and placentae weights, conception rate, mean number of corpora lutea and implantation sites, pre-implantation loss and number of viable fetuses, sex distribution of fetuses and weight of fetuses. External macroscopic examination, examination of soft tissue and skeletal examination revealed no treatment-related biologically and/or statistically significant variations/retardations. Number of resorptions and consequently the post-implantation loss were increased slightly (statistically non- significant) at 1000 mg/kg bw/day. In conclusion, the no-observed-effect level (NOEL) for maternal and fetotoxicity of camphene administered orally (gavage) was considered to be 250 mg/kg bw/day in Sprague-Dawley rats. Therefore, camphene is not considered to be teratogenic.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams with less than 40% mortality. The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg of d-limonene, but no anomalies were observed in the general behaviour of dams given 250 and 500 mg/kg of d-limonene during the gestation. - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
- External examination of fetuses showed no anormalies.
- Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.
- Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene. - Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: fetotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- The developmental toxicity of cornmint oil was assessed using read across from the source substance d-limonene. Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.
- Executive summary:
The developmental toxicity of cornmint oil was assessed using read across from the source substance d-limonene. In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant Japanese white rabbits at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation. Food consumption and bodyweights of pregnant rabbits were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations.
Treatment with the highest dose level (1000 mg/kg bw/day) of d-limonene resulted in death of 6/18 dams (33% mortality). The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg bw/day of d-limonene, but no anormalies were observed in the general behavior of dams given 250 and 500 mg/kg bw/day of d-limonene during the gestation. External examination of fetuses showed no anormalies. Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.
Under the test conditions, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be higher than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- The read across justification is presented in the document attached to this record.
- Reason / purpose for cross-reference:
- read-across source
- Species:
- mouse
- Strain:
- ICR
- Details on test animals or test system and environmental conditions:
- No data
- Route of administration:
- oral: unspecified
- Vehicle:
- not specified
- Details on study design:
- No data
- Details on maternal toxic effects:
- Details on maternal toxic effects:
See results tables - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: Body weight gain
- Description (incidence and severity):
- decreased bodyweight gain
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
See results tables - Dose descriptor:
- LOAEL
- Effect level:
- 2 363 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- skeletal malformations
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- skeletal: rib
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 2 363 mg/kg bw/day
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- not specified
- Relevant for humans:
- not specified
- Conclusions:
- The developmental toxicity of cornmint oil was assessed using read across from the source substance d-limonene. Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.
- Executive summary:
The developmental toxicity of cornmint oil was assessed using read across from the source substance d-limonene. In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation. Bodyweights of pregnant mice were recorded during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, sensory functions, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.
A significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day. However, no anomalies were observed in the general behavior of dams during the period of gestation. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control. In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded ossifications were restored to normal during postnatal development. A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.
Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal skeletal formation in fetuses at 2363 mg/kg bw/day.
Referenceopen allclose all
Maternal toxicity:
- Survival of dams: no deaths
- Body weight of dams: no compound-related changes compared to control (only positive control treated mice showed decreased body weight gain)
Fetotoxicity :
- Death : no dead fetuses in dosage groups (3 deaths in positive control)
- Average fetus weight: no change in treated groups compared to controls
- Abnormalities/malfunctions (no. of fetuses affected/no. of litters affected) (sham control, pos. control, 1.85, 8.59, 39.9, 185.5 mg/kg bw)
- Skeletal findings:
sternebrae (incomplete oss.): 80/22, 94/18, 92/20, 93/22, 101/19, 92/19
sternebrae (missing): 14/6, 11/19, 11/8, 17/5, 11/4, 0/22
skull (incomplete closure): 41/16, 114/19, 46/15, 63/16, 67/20, 49/17
- Soft tissue abnormalities:
pos. control: 7 pups with meningoencephalocele and spina bifida
10.15 mg/kg: 1 pup: petechiae, 1 pup: anophthalmia
47.05 mg/kg: 2 pups anophthalmia, 2 pups: gastroschisis,1 pup hydrocephalus
All other findings were completely in the range of spontaneous abnormalities found in negative controls.
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights P and F1: no
- Histopathology P and F1:
P: urogenital tract, number of implantation and resorption sites
F1: All fetuses were examined grossly, one-third of fetuses of each litter underwent detailed visceral examinations employing 10x magnification, two-third were examined for skeletal defects
Table 1: Effect of d-limonene on prenatal development of rabbit fetuses
Dose (mg/kg bw) |
Control |
250 |
500 |
1000 |
No. of pregnant animals |
10 |
10 |
10 |
18 |
No. of dead clams |
0 |
0 |
0 |
6 |
(%) |
|
|
|
33 |
No. of examined clams |
10 |
10 |
10 |
10 |
No. of implantations |
96 |
94 |
85 |
91 |
(mean ± S.E.) |
9.50 ± 0.25 |
9.40 ± 0.21 |
8.50 ± 0.33 |
9.10 ± 0.25 |
No. of resorbed fetuses |
5 |
4 |
4 |
8 |
No. of dead fetuses |
3 |
5 |
0 |
3 |
No. of live fetuses |
88 |
85 |
81 |
80 |
Sex ratio (Male/Female) |
0.73 (37/51) |
1.13 (45/50) |
0.62 (31/50) |
1.11 (38/42) |
Fetus body weight (g) |
|
|
|
|
Male (mean ± S.E.) |
44.39 ± 1.33 |
48.09 ± 1.07 * |
44.76 ± 1.51 |
43.22 ± 0.96 |
Female (mean ± S.E.) |
45.64 ± 1.00 |
47.45 ± 1.08 |
46.14 ± 1.21 |
45.13 ± 1.10 |
Placental weight (g) |
|
|
|
|
Male (mean ± S.E.) |
5.76 ± 0.17 |
5.84 ± 0.17 |
5.95 ± 0.29 |
5.77 ± 0.19 |
Female (mean ± S.E.) |
5.87 ± 0.19 |
5.70 ± 0.15 |
6.16 ± 0.18 |
5.87 ± 0.23 |
* Significantly different from the control at 5% level
Table 2: Prenatal examinations of rabbit fetuses
Dose (mg/kg bw) |
Control |
250 |
500 |
1000 |
External examination |
|
|
|
|
No. of examined fetuses |
91 |
90 |
81 |
83 |
No. of malforrned fetuses |
0 |
0 |
0 |
0 |
Visceral examination |
|
|
|
|
No. of examined fetuses |
88 |
85 |
81 |
80 |
No. of malformed fetuses |
|
|
|
|
Atrial septal defect (%) |
0 |
2 (2.4) |
0 |
0 |
No. of minor abnormality |
|
|
|
|
Incomplete lobulation of lungs (%) |
11 (12.5) |
16 (18.8) |
19 (23.5) |
19 (23.8) |
Enlargement of foramen ovale (%) |
2 (2.3) |
2 (2.4) |
5 (6.2) |
4 (5.0) |
Skeletal examination |
|
|
|
|
No. of examined fetuses |
86 |
87 |
81 |
80 |
No. of malformed fetuses |
0 |
0 |
0 |
0 |
No. of variation |
|
|
|
|
Left lumbar rib (%) |
18 (20.9) |
26 (29.9) |
14 (17.3) |
25 (31.3) |
Right lumbar rib (%) |
16 (18.6) |
22 (25.3) |
14 (17.3) |
22( 27.5) |
Ossification pattern |
|
|
|
|
Retarded ossification of 5th sternebrae (%) |
11 (12.8) |
14 (16.1) |
8 (9.9) |
18 (22.5) |
Retarded ossification of middle phalanx of fore limbs (%) |
2 (2.3) |
3 (3.4) |
0 |
6 (7.4) |
Table 3: Absolute organ weights of rabbit offsprings
|
Male |
Female |
||||||
|
Control |
250 |
500 |
1000 |
Control |
250 |
500 |
1000 |
No. of offsprings |
13 |
12 |
8 |
13 |
10 |
13 |
16 |
9 |
Final body weight (g) |
893.0 ± 45.3 |
1021.2 ± 45.4 ** |
931.9 ± 55.5 |
957.3 ± 52.4 |
1005.5 ± 53.7 |
1093.1 ± 46.6 |
860.0 ± 31.6 * |
1071.7 ± 58.1 |
Liver (g) |
36.49 ± 2.52 |
45.08 ± 1.52 * |
38.67 ± 3.10 |
34.91 ± 2.76 |
41.79 ± 3.39 |
13.95 ± 3.18 |
34.68 ± 1.90 |
48.30 ± 6.14 |
Lungs (g) |
5.53 ± 0.35 |
6.25 ± 0.26 |
5.98 ± 0.36 |
5.46 ± 0.18 |
5.91 ± 0.25 |
5.94 ± 0.28 |
5.72 ± 0.18 |
5.93 ± 0.45 |
Heart (g) |
2.63 ± 0.17 |
3.50 ± 0.16 ** |
2.86 ± 0.17 |
3.04 ± 0.19 |
3.19 ± 0.17 |
3.38 ± 0.21 |
2.72 ± 0.12 * |
3.34 ± 0.20 |
Spleen (g) |
0.71 ± 0.05 |
0.77 ± 0.04 |
0.71 ± 0.08 |
0.81 ± 0.04 |
0.64 ± 0.06 |
0.74 ± 0.05 |
0.74 ± 0.04 |
0.78 ± 0.07 |
Thymus (g) |
2.31 ± 0.20 |
2.51 ± 0.23 |
2.11 ± 0.38 |
1.96 ± 0.11 |
2.40 ± 0.30 |
2.77 ± 0.19 |
1.671.14 * |
2.36 ± 0.31 |
Kidneys (g) |
7.67 ± 0.42 |
9.80 ± 0.46 ** |
8.29 ± 0.29 |
8.58 ± 0.52 |
8.82 ± 0.46 |
8.40 ± 0.30 |
7.86 ± 0.37 |
9.56 ± 0.55 |
Thyroids (mg) |
75.89 ± 8.35 |
102.68 ± 4.18* |
86.74 ± 10.97 |
80.93 ± 7.41 |
82.78 ± 8.00 |
89.90 ± 4.11 |
75.75 ± 5.43 |
96.30 ± 9.67 |
Adrenals (mg) |
69.65 ± 6.02 |
9.1.93 ± 1.06 ** |
78.79 ± 5.89 |
71.36 ± 6.00 |
82.23 ± 4.37 |
94.24 ± 5.07 |
87.64 ± 4.18 |
105.39 ± 15.11 |
Testes or Ovaries (mg) |
180.42 ± 17.15 |
272.86 ± 16.46 ** |
185.62 ± 23.78 |
162.84 ± 20.59 |
46.31 ± 7.90 |
46.10 ± 2.80 |
43.53 ± 2.69 |
47.77 ± 3.59 |
* Significantly different from the control at 5% level
** Significantly different from the control at 1% level
Table 4: Relative organ weights per 100 g of rabbit offsprings
|
Male |
Female |
||||||
|
Control |
250 |
500 |
1000 |
Control |
250 |
500 |
1000 |
No. of offsprings |
13 |
12 |
8 |
13 |
10 |
13 |
16 |
9 |
Final body weight (g) |
893.0 ± 45.3 |
1021.2 ± 45.4 ** |
931.9 ± 55.5 |
957.3 ± 52.4 |
1005.5 ± 53.7 |
1093.1 ± 46.6 |
860.0 ± 31.6 * |
1071.7 ± 58.1 |
Liver (g/100 g) |
4.08 ± 0.25 |
3.99 ± 0.22 * |
4.16 ± 0.26 |
3.52 ± 0.10 |
4.25 ± 0.17 |
4.03 ± 0.21 |
4.02 ± 0.16 |
4.04 ± 0.32 |
Lungs (g/100 g) |
0.61 ± 0.03 |
0.55 ± 0.03 |
0.65 ± 0.04 |
0.58 ± 0.03 |
0.62 ± 0.02 |
0.55 ± 0.02 * |
0.67 ± 0.03 |
0.51 ± 0.02 ** |
Heart (g/100 g) |
0.29 ± 0.01 |
0.31 ± 0.01 |
0.31 ± 0.01 |
0.31 ± 0.02 |
0.33 ± 0.01 |
0.31 ± 0.01 |
0.32 ± 0.01 |
0.29 ± 0.01 * |
Spleen (g/100 g) |
0.08 ± 0.01 |
0.07 ± 0.01 |
0.08 ± 0.01 |
0.08 ± 0 |
0.07 ± 0.01 |
0.07 ± 0.01 |
0.09 ± 0 |
0.07 ± 0.01 |
Thymus (g/100 g) |
0.25 ± 0.02 |
0.22 ± 0.02 |
0.25 ± 0.03 |
0.20 ± 0.01 * |
0.24 ± 0.03 |
0.25 ± 0.01 |
0.195 ± 0.01 |
0.20 ± 0.02 |
Kidneys (g/100 g) |
0.85 ± 0.04 |
0.86 ± 0.04 |
0.90 ± 0.03 |
0.88 ± 0.02 |
0.91 ± 0.02 |
0.80 ± 0.01 |
0.91 ± 0.01 |
0.83 ± 0.03 * |
Thyroids (mg/100 g) |
8.21 ± 0.69 |
9.10 ± 0.51 |
9.21 ± 0.87 |
8.18 ± 0.42 |
8.40 ± 0.56 |
8.41 ± 0.45 |
8.81 ± 0.57 |
8.13 ± 0.41 |
Adrenals (mg/100 g) |
7.82 ± 0.60 |
8.37 ± 0.40 |
8.58 ± 0.77 |
7.18 ± 0.28 |
8.61 ± 0.51 |
8.79 ± 0.57 |
9.15 ± 0.42 |
9.04 ± 0.14 |
Testes or Ovaries (mg/100 g) |
23.68 ± 1.31 |
19.68 ± 0.94 * |
19.48 ± 1.69 |
16.35 ± 1.56 |
5.15 ± 1.19 |
4.33 ± 0.32 |
5.19 ± 0.43 |
3.84 ± 0.32 |
* Significantly different from the control at 5% level
** Significantly different from the control at 1% level
Table 5: Effects of d-limonene on gross differentiations of rabbit offsprings
|
Control |
250 |
500 |
1000 |
No. of examined offsprings |
23 |
25 |
24 |
22 |
Days of gross differentiation after birth Opening of the ear-shell |
|
|
|
|
6th day (%) |
0 |
0 |
1 (4.2) |
0 |
7th day (%) |
23 (100) |
25 (100) |
23 (95.8) |
22 (100) |
Coating with the hair |
|
|||
2nd day (%) |
7 (30.4) |
0 |
0 |
0 |
3rd day (%) |
16 (69.6) |
25 (100) |
24 (100) |
22 (100) |
Odontiasis |
|
|||
At birth (%) |
23 (100) |
25 (100) |
24 (100) |
22 (100) |
Opening of the eyelids |
|
|||
9th day (%) |
0 |
0 |
0 |
3 (13.6) |
10th day (%) |
11 (47.8) |
4 (16.0) |
13 (54.2) |
5 (22.7) |
11th day (%) |
4 (17.4) |
15 (60.0) |
10 (41.7) |
12 (54.5) |
12th day (%) |
3 (13.0) |
5 (20.0) |
1 (4.2) |
2 (9.1) |
13th day (%) |
5 (21.7) |
1 (4.0) |
0 |
0 |
Table 6: Effects of d-limonene on postnatal development of rabbit offsprings
|
Control |
250 |
500 |
1000 |
No of dams |
3 |
3 |
3 |
3 |
No. of still-birth (Male/Female) |
1 (1/0) |
0 |
0 |
1 (0/1) |
No. of offsprings (Male/Female) At birth |
28 (15/13) |
27 (14/13) |
26 (8/18) |
27 (15/12) |
1st week |
28 (15/13) |
27 (14/13) |
25 (8/17) |
26 (14/12) |
2nd week |
26 (14/12) |
27 (14/13) |
25 (8/17) |
26 (14/12) |
3rd week |
24 (14/10) |
27 (14/13) |
25 (8/17) |
25 (14/11) |
4th week |
23 (13/10) |
26 (13/13) |
25 (8/17) |
22 (13/ 9) |
5th week |
23 (13/10) |
25 (12/13) |
25 (8/17) |
22 (13/ 9) |
6th week |
23 (13/10) |
25 (12/13) |
25 (8/17) |
22 (13/ 9) |
7th week |
23 (13/10) |
25 (12/13) |
24 (8/16) |
22 (13/ 9) |
Weanling rate (%) |
79.3 (81.2/76.9) |
92.6 (85.7/100) |
92.3 (100/88.9) |
78.6 (86.7/69.2) |
Table 7: Postnatal examinations of rabbit offsprings
|
Control |
250 |
500 |
1000 |
No. of dams |
3 |
3 |
3 |
3 |
No. of examined offsprings |
23 |
25 |
24 |
22 |
Sensory function |
Normal |
Normal |
Normal |
Normal |
External examination No. of malformed offsprings |
0 |
0 |
0 |
0 |
Visceral examination No. of malformed offsprings |
0 |
0 |
0 |
0 |
No. of minor abnormality Incomplete lobulation of lungs (%) |
2 (8.7) |
1 (4.0) |
0 |
0 |
Accessory spleen (%) |
2 (8.7) |
0 |
0 |
0 |
Protrusion of gall bladder (%) |
1 (4.3) |
1 (4.0) |
0 |
0 |
Skeletal examination No. of malformed offsprings |
0 |
0 |
0 |
0 |
No. of variation Left lumbar rib (%) |
4 (17.4) |
4 (16.0) |
4 (16.7) |
4 (18.2) |
Right lumbar rib (%) |
2 (8.7) |
6 (24.0) |
6 (25.0) |
4 (18.2) |
Translocation of caudal vertebrae (%) |
1 (4.3) |
0 |
1 (4.2) |
0 |
Ossification pattern Retarded ossification of 5th sternebrae (%) |
0 |
2 (8.0) |
0 |
1 (4.5) |
Accessory ossification center of 5th sternebrae (%) |
1 (4.3) |
2 (8.0) |
0 |
3 (13.6) |
Maternal examinations:
- Significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day.
- No anomalies were observed in the general behavior of dams during the period of gestation.
Fetal examinations:
- An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control.
- In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded ossifications were restored to normal during postnatal development.
- A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.
Table 1: Effects of d-limonene on development of mouse fetuses.
|
Control |
591 |
2363 |
No. of mothers |
15 |
15 |
15 |
No. of implantations |
162 |
179 |
154 |
(mean ± S.E.) |
(10.80 ± 0.20) |
(11.93 ± 0.13) |
(10.27 ± 0.21) |
No. of dead fetuses |
9 |
1.1 |
6 |
(mean ± S.E.) |
(0.60 ± 0.05) |
(0.73 ± 0.10) |
(0.40 ± 0.05) |
No. of resorbed fetuses |
18 |
20 |
20 |
(mean ± S.E.) |
(1.20 ± 0.05) |
(1.33 ± 0.10) |
(1.33 ± 0.10) |
No. of live fetuses |
135 |
148 |
125 |
Sex ratio (Male/Female) |
1.33 |
0.83 |
1.13 |
Fetuses Body weight (g) Male (mean ± S.E.) |
1.34 ± 0.02 |
1.24 ± 0.01 |
1.28 ± 0.02 |
Female (mean ± S.E.) |
1.28 ± 0.02 |
1.22 ± 0.01 |
1.19 ± 0.02 |
Placental weight (mg) Male (mean ± S.E.) |
94 (2 ± 2.0) |
86 ( 0 ± 1.7) |
89 (3 ± 1.7) |
Female (mean ± S.E.) |
87 (8 ± 2.6) |
81 (5 ± 1.9) |
83 (5 ± 2.2) |
External observation No. of fetuses examined |
135 |
148 |
128 |
No. of fetuses malformed |
0 |
4 |
0 |
Cleft palate |
0 |
4 |
0 |
Incidence (%) |
0 |
2.7 |
0 |
Visceral observation No. of fetuses examined |
71 |
76 |
68 |
No. of fetuses malformed |
4 |
4 |
3 |
Enlargement of foramen ovale |
4 |
4 |
3 |
Incidence (%) |
5.6 |
5.3 |
4.4 |
Table 2: Effects of d-limonene on skeletal development of mouse fetuses
Dose (mg/kg bw) |
Control |
591 |
2363 |
No. of fetuses examined |
64 |
72 |
61 |
Variation Lumbar rib (%) |
17 (26.6) |
12 (16.7) |
28 (46.7) * |
Cervical rib (%) |
1 (1.6) |
(5.6) |
1 (1.7) |
Fused rib (%) |
0 (0) |
0 (1) |
5 (8.3) * |
Crooked rib (%) |
2 (3.1) |
0 (1) |
0 (0) |
Asymmetry of sternebrae (%) |
2 (1.7) |
7 (9.7) |
7 (11.7) |
Fused sternebrae |
0 (0) |
1 (1.4) |
1 (1.7) |
No. of ossification Sternebrae |
5.99 ± 0.01 |
5.97 ± 0.02 |
5.94 ± 0.03 |
Fore limb Metacarpal bone |
8.00 ± 0 |
7.97 ± 0.03 |
8.00 ± 0 |
Proximal phalanx |
7.50 ± 0.13 |
7.67 ± 0.16 |
6.87 ± 0.30 * |
Middle phalanx |
1.16 ± 0.23 |
2.14 ± 0.29 ** |
2.18 ± 0.29 ** |
Distal phalanx |
9.08 ± 0.32 |
9.72 ± 0.20 |
9.13 ± 0.36 |
Hind limb Metatarsal bone |
10.00 ± 0 |
9.92 ± 0.05 |
9.80 ± 0.09 * |
Proximal phalanx |
8.12 ± 0.23 |
8.03 ± 0.23 |
7.23 ± 0.39 * |
Middle phalanx |
0.09 ± 0.09 |
0.33 ± 0.18 |
0.20 ± 0.11 |
Distal phalanx |
9.47 ± 0.24 |
9.72 ± 0.20 |
9.30 ± 0.31 |
Caudal vertebrae |
7.12 ± 0.95 |
6.50 ± 0.21 |
7.00 ± 0.25 |
* Significantly different from the control at 5% level.
** Significantly different from the control at 1% level.
Table 3: Effects of d-limonene on postnatal development of mouse offsprings
|
|
d-Limonene (mg/kg bw) |
|
|
Control |
591 |
2363 |
No. of mothers |
5 |
5 |
5 |
No. of implantations |
51 |
52 |
50 |
(mean ± S.E.) |
(10.80 ± 0.33) |
(10.41 ± 0.96) |
(10.03 ± 0.63) |
No. of offsprings |
50 |
46 |
39 |
No. of dead offsprings at birth |
0 |
0 |
0 |
Sensory function |
Normal |
Normal |
Normal |
No. of live offsprings At birth |
50 |
46 |
39 |
1st week |
50 |
46 |
39 |
2nd week |
50 |
46 |
39 |
3rd week |
50 |
46 |
39 |
4th week |
50 |
46 |
39 |
5th week |
50 |
46 |
39 |
6th week |
50 |
46 |
39 |
7th week |
50 |
46 |
39 |
Weanling rate (%) |
100 |
100 |
100 |
Table 4: Effects of d-limonene on gross differentiation of mouse offsprings
|
|
d-Limonene (mg/kg bw) |
|
Gross differentiation |
Control |
591 |
2363 |
Opening of the ear-shell |
3.5 ± 0.07 |
3.6 ± 0.08 |
4.1 ± 0.08 |
Coating with the hair |
5.0 ± 0.00 |
4.11 ± 0.08 |
5.2 ± 0.06 |
Odontiasis |
9.8 ± 0.07 |
9.3 ± 0.07 |
9. 1 ± 0.04 |
Opening of the eyelid |
13.3 ± 0.08 |
12.9 ± 0.06 |
13.6 ± 0.09 |
Descending of the testis |
23.0 ± 0.20 |
23.3 ± 0.11 |
25.0 ± 0.27 |
Opening of the vaginal orifice |
29.5 ± 0.26 |
30.5 ± 0.16 |
30.6 ± 0.15 |
Table 5: Absolute organ weights of postnatal mouse offsprings born to mothers given d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings |
Final BW (g) |
Thyroids (mg) |
Thymus (g) |
Lungs (g) |
Heart (g) |
Spleen (g) |
Kidneys (g) |
Liver (g) |
Adrenals (g) |
Testes (g) or Ovaries (mg) |
Female |
Control |
27 |
34.2 ± 0.50 |
5.43 ± 0.18 |
75.72 ± 4.14 |
199.26 ± 4.70 |
158.22 ± 2.13 |
131.54 ± 9.96 |
0.63 ± 0.02 |
2.06 ± 0.09 |
8.39 ± 0.49 |
217.52 ± 1.65 |
591 |
27 |
34.9 ± 0.44 |
6.04 ± 0.68 |
65.26 ± 4.26 |
203.76 ± 2.95 |
166.66 ± 7.58 |
121.66 ± 4.74 |
0.63 ± 0.02 |
2.08 ± 0.07 |
8.29 ± 0.67 |
209.76 ± 9.23 |
|
2363 |
23 |
32.2 ± 0.77 |
5.41 ± 0.62 |
64.08 ± 3.64 |
189.60 ± 6.69 |
158.96 ± 3.12 |
125.52 ± 3.04 |
0.58 ± 0.02 |
2.14 ± 0.04 |
8.53 ± 0.86 |
200.20 ± 0.39 |
|
Male |
Control |
23 |
27.3 ± 0.50 |
4.80 ± 0.22 |
81.47 ± 4.38 |
172.80 ± 7.20 |
118.98 ± 3.91 |
121.76 ± 6.48 |
0.37 ± 0.01 |
1.37 ± 0.02 |
11.71 ± 0.40 |
13.38 ± 0.68 |
591 |
19 |
28.8 ± 0.45 |
4.29 ± 0.20 |
69.44 ± 5.52 |
174.26 ± 5.66 |
129.96 ± 3.62 |
112.68 ± 2.79 |
0.38 ± 0.01 |
1.37 ± 0.04 |
11.36 ± 0.43 |
16.98 ± 1.71 |
|
2363 |
16 |
28.1 ± 0.34 |
3.53 ± 0.41 * |
63.95 ± 9.72 |
171.75 ± 5.70 |
135.15 ± 6.89 |
116.15 ± 5.78 |
0.38 ± 0.01 |
1.46 ± 0.03 * |
11.61 ± 0.30 * |
17.93 ± 1.30 * |
* Significantly different from the control at 5% level.
Table 6: Relative organ weights per 100 g body weights of postnatal mouse offsprings born to mothers given d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings |
Final BW (g) |
Thyroids(mg/100 g) |
Thymus(mg/100 g) |
Lungs(mg/100 g) |
Heart(mg/100 g) |
Spleen(mg/100 g) |
Kidneys(g/100 g) |
Liver(g/100 g) |
Adrenals(mg/100 g) |
Testes or Ovaries (mg/100 g) |
Female |
Control |
27 |
34.2 ± 0.50 |
15.80 ± 0.44 |
220.36 ± 10.67 |
580.50 ± 13.59 |
461.18 ± 8.65 |
386.55 ± 39.35 |
1.83 ± 0.07 |
6.01 ± 0.26 |
24.48 ± 1.55 |
634.84 ± 19.06 |
591 |
27 |
34.9 ± 0.44 |
17.20 ± 1.93 |
185.29 ± 8.48 * |
581.21 ± 9.42 |
474.60 ± 717.10 |
346.85 ± 12.29 |
1.80 ± 0.05 |
5.93 ± 0.14 |
23.55 ± 1.48 |
596.69 ± 14.95 |
|
2363 |
23 |
32.2 ± 0.77 |
16.55 ± 2.64 |
191.59 ± 6.25 |
568.43 ± 13.67 |
477.39 ± 11.90 |
376.91 ± 10.23 |
1.73 ± 0.03 |
6.44 ± 0.29 |
25.74 ± 2.82 |
602.30 ± 34.26 |
|
Male |
Control |
23 |
27.3 ± 0.50 |
17.13 ± 18.47 |
291.98 ± 34.66 |
619.50 ± 8.54 |
425.00 ± 32.37 |
437.67 |
1.31 ± 0.04 |
4.89 ± 0.08 |
41.89 ± 1.41 |
47.85 ± 2.42 |
591 |
19 |
28.8 ± 0.45 |
15.06 ± 0.61 * |
242.35 ± 13.57 |
611.98 ± 17.12 |
456.02 ± 4.93 * |
397.44 ± 20.40 |
1.33 ± 0.02 |
4.81 ± 0.11 |
39.96 ± 1.73 |
59.59 ± 5.78 |
|
2363 |
16 |
28.1 ± 0.34 |
12.60 ± 1.44 * |
229.38 ± 35.46 |
612.74 ± 20.32 |
482.14 ± 24.21 * |
414.30 ± 17.34 |
1.37 ± 0.04 |
5.23 ± 0.09 * |
41.45 ± 1.27 |
64.33 ± 5.38 * |
* Significantly different from the control at 5% level.
Table 7: Summaried data on postnatal development of mouse offsprings
|
|
d-limonene (mg/kg bw) |
|
|
Control |
591 |
2363 |
External observation |
|
||
No. of offsprings examined |
50 |
46 |
39 |
No. of offsprings malformed |
0 |
0 |
0 |
Visceral observation |
|
||
No. of offsprings examined |
50 |
46 |
39 |
No. of offsprings malformed |
0 |
0 |
0 |
Skeletal observation |
|
||
No. of offsprings examined |
50 |
46 |
39 |
No. of offsprings malformed |
0 |
0 |
0 |
Skeletal variation |
|
||
Lumbar rib (%) |
17 (34.0) |
23 (50.0) |
20 (51.3) |
Fusion of 13th and lumbar rib (%) |
0 |
0 |
1 (2.6) |
Fusion of lumbar vertebra (%) |
1 (2.0) |
1 (2.2) |
1 (2.6) |
Crooked tail (%) |
0 |
0 |
1 (2.6) |
Maternal examinations:
- At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed.
Fetal examinations:
- Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth.
- A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group.
- Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg bw/day.
Table 1: Body weight changes in pregnant rats treated orally with d-limonene
Dose (mg/kg bw) |
Gestational days |
Gain |
||||
0 |
9 |
12 |
16 |
20 |
||
Control |
214.80 ± 32.55 |
248.70 ± 28.92 |
265.60 ± 30.53 |
289.85 ± 35.01 |
325.30 ± 44.01 |
105.50 ± 29.67 |
591 |
221.25 ± 39.58 |
254.65 ± 41.16 |
264.80 ± 39.94 |
290.10 ± 38.37 |
325.60 ± 52.75 |
103.95 ± 19.38 |
2869 |
214.75 ± 4.57 |
258.75 ± 32.76
|
248.92 ± 26.27 |
263.17 ± 22.96 * |
305.00 ± 27.07 |
90.25 ± 22.38 |
* Significantly different from the control, P <0.05
Table 2: Effects of d-limonene on rat fetuses
Dose (mg/kg bw) |
Control |
591 |
2869 |
No. of mothers |
15 |
15 |
15 |
Mortality of mothers (%) |
0 |
0 |
40 |
No. of total implants |
12.73 ± 2.96 |
12.18 ± 3.65 |
10.44 ± 3.71 |
No. of dead fetuses |
0 |
0 |
0 |
No. of resorbed fetuses |
1.00 ± 1.10 |
1.47 ± 2.42 |
0.89 ± 0.73 |
No. of live fetuses |
176 |
162 |
87 |
Sex ratio (Male/Female) |
0.69 |
1.22 |
0.85 |
Fetuses body weight (g) Male |
3.71 ± 0.45 |
3.53 ± 0.35 |
3.73 ± 0.52 |
Female |
3.46 ± 0.44 |
3.38 ± 0.45 |
3.63 ± 0.40 |
Placental weight(g) Male |
0.49 ± 0.07 |
0.49 ± 0.10 |
0.48 ± 0.06 |
Female |
0.47 ± 0.07 |
0.46 ± 0.06 |
0.44 ± 0.05 |
Malformation External |
0 |
0 |
0 |
Visceral |
1 |
0 |
0 |
Table 3: Effects of d-limonene on skeletal development of rat fetuses
Dose (mg/kg bw) |
Control |
591 |
2869 |
No. of examined fetuses |
83 |
84 |
42 |
Variation Shortness of 13th rid |
1 |
0 |
0 |
Lumbar rid |
0 |
1 |
2 |
Asymmetry of sternebrae |
0 |
0 |
1 |
Ossification Delayed ossification of parietal bone |
2 |
0 |
0 |
Non-ossification of occipital bone
|
0 |
4 |
1 |
Non-ossification of parietal bone |
0 |
3 |
0 |
No. of ossified metacarpal bone |
7.69 ± 0.72 |
7.49 ± 0.84 |
6.97 ± 0.96 * |
No. of ossified proximal phalanx (Forelimb) |
2.48 ± 1.71 |
2.25 ± 1.81 |
0.55 ± 1.28 * |
No. of ossified metatarsal bone
|
7.98 ± 0.56 |
8.01 ± 011 |
8.00 ± 0 |
No. of ossified sternebraea |
5.47 ± 0.98 |
5.60 ± 0.71 |
5.52 ± 0.73 |
No. of ossified caudal vertebrae |
3.76 ± 0.64 |
3.80 ± 0.57 |
3.95 ± 0.68 |
* Significantly different from the control, P <0.05
Table 4: Body weight changes of postnatal rat offsprings born to mothers treated orally with d-limonene
Postnatal weeks |
Males |
Females |
||||
Dose (mg/kg bw) |
Dose (mg/kg bw) |
|||||
Control |
591 |
2869 |
Control |
591 |
2869 |
|
0 |
5.19 ± 0.55 |
5.46 ± 0.52 |
4.79 ± 0.46 |
4.93 ± 0.62 |
5.09 ± 0.68 |
4.89 ± 0.66 |
1 |
13.06 ± 1.50 |
12.49 ± 0.99 |
10.62 ± 1.54 * |
12.82 ± 1.59 |
12.22 ± 1.07 |
10.66 ± 1.84 |
2 |
26.06 ± 3.12 |
24.86 ± 2.74 |
22.67 ± 5.05 * |
25.88 ± 3.35 |
24.31 ± 2.42 |
22.72 ±.3.92 |
3 |
41.91 ± 5.89 |
39.55 ± 5.14 |
40.77 ± 5.16 |
41.08 ± 5.59 |
38.56 ± 4.37 |
38.39 ± 4.96 |
4 |
73.12 ± 9.89 |
71.33 ± 8.87 |
67.67 ± 8.02 |
69.66 ± 9.43 |
67.38 ± 6.71 |
66.01 ± 9.29 |
5 |
122.32 ± 12.25 |
116.47 ± 12.78 |
112.77 ± 12.65 * |
109.57 ± 10.61 |
107.58 ± 7.95 |
107.10 ± 13.34 |
6 |
176.04 ± 15.80 |
164.68 ± 16.69 |
163.11 ± 17 .85 * |
141.39 ± 10.54 |
140.11 ± 9.40 |
139.45 ± 14.22 |
7 |
235.52 ± 17.72 |
222.43 ± 18.57 |
213.64 ± 20.10 * |
173.06 ± 8.89 |
169.65 ± 11.13 |
167.84 ± 15.86 |
* Significantly different from the control, P <0.05
Table 5: Effects of d-limonene on postnatal development of the rats
Dose (mg/ kg bw) |
Days of postnatal development |
|||||
Opening of the ear-shell |
Coating with |
Odontiasis |
Opening of the eyelid |
Descending of the testis |
Opening of the vaginal orifice |
|
Control |
2.55 ± 0.76 |
5.51 ± 0.91 |
10.1 ± 0.96 |
14.83 ± 0.55 |
22.5 ± 1.30 |
35.6 ± 2.50 |
591 |
2.09 ± 0.82 |
6.00 ± 0 |
10.4 ± 0.71 |
15.00 ± 0.76 |
21.6 ± 1.39 |
35.5 ± 1.75 |
2869 |
2.41 ± 0.49 |
8.50 ±0.50 |
10.4 ± 1.85 |
15.14 ± 0.75 |
21.27 ± 0.57 |
35.93 ± 2.20 |
Table 6: Effects of d-limonene on development of rat offsprings
Dose (mg/kg) |
Control |
591 |
2869 |
No. of mothers |
5 |
5 |
5 |
Mortality of mothers |
0 |
0 |
40 |
No. of offspring from birth to the 7th week 0 |
61 |
65 |
33 |
1 |
53 |
63 |
30 |
2 |
53 |
63 |
30 |
3 |
53 |
63 |
28 |
4 |
53 |
63 |
28 |
5 |
53 |
63 |
28 |
6 |
53 |
63 |
28 |
7 |
53 |
63 |
28 |
External abnormality |
0 |
0 |
0 |
No. of total implants |
13.6 ± 3.1 |
14.8 ± 1.7 |
13.7 ± 1.7 |
No. of dead fetuses at birth |
4 |
4 |
5 |
Parturient rate |
95 |
92 |
93 |
Weaning rate |
89 |
97 |
85 |
Table 7: Absolute organ weights of postnatal rat offsprings born to mothers treated orally with d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings. |
Final BW (g) |
Pituitary (mg) |
Thyroids (mg) |
Thymus (g) |
Lungs (g) |
Heart (g) |
Spleen (g) |
Kidneys (g) |
Liver (g) |
Adrenals (g) |
Testes (g) or Ovaries (mg) |
Male |
Control |
27 |
235.5 ± 17.7 |
10.04 ± 1.95 |
14.06 ± 2.52 |
0.77 ± 0.08 |
1.15 ± 0.10 |
0.82 ± 0.08 |
0.75 ± 0.11 |
2.18 ± 0.36 |
11.55 ± 1.14 |
38.85 ± 7.37 |
2.15 ± 0.17 |
591 |
29 |
222.4 ± 18.6 |
9.58 ± 4.84 |
13.12 ± 2.77 |
0.72 ± 0.09 |
1.10 ± 0.11 |
0.81 ± 0.08 |
0.69 ± 0.08 |
2.10 ± 0.20 |
11.24 ± 1 .49 |
36.77 ± 6.93 |
2.13 ± 0.28 |
|
2869 |
11 |
213.6 ± 20.1 |
9.69 ± 0.65 |
14.41 ± 3.60 |
0.66 ± 0.08 * |
1.19 ± 0.17 |
0.80 ± 0.07 |
0.63 ± 0.07 * |
2.23 ± 0.38 |
11.64 ± 1.64 |
43.23 ± 10.91 |
2.18 ± 0.14 |
|
Female |
Control |
25 |
173.1 ± 8.9 |
11.18 ± 3.15 |
12.26 ± 1.32 |
0.59 ± 0.08 |
0.97 ± 0.11 |
0.66 ± 0.07 |
0.52 ± 0.06 |
1.72 ± 0.22 |
8.88 ± 0.85 |
45.51 ± 8.01 |
77.24 ± 22.01 |
591 |
34 |
169.7 ± 11.7 |
10.05 ± 3.34 |
11.57 ± 1.62 |
0.54 ± 0.07 |
0.96 ± 0.07 |
0.67 ± 0.05 |
0.50 ± 0.06 |
1.60 ± 0.15 * |
8.16 ± 0.85 |
46.56 ± 8.45 |
85.84 ± 42.52 * |
|
2869 |
17 |
167.8 ± 15.9 |
9.95 ± 1.87 |
12.31 ± 1.80 |
0.51 ± 0.07 * |
0.94 ± 0.10 |
0.62 ± 0.06 |
0.44 ± 0.04 * |
1.62 ± 0.17 * |
8.50 ± 0.58 |
47.15 ± 6.63 |
63.15 ± 7.99 |
* Significantly different from the control, P <0.05
Table 8: Relative organ weights per 100 g body weights of postnatal rat offsprings born to mothers treated orally with d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings. |
Final BW (g) |
Pituitary (mg/100 g) |
Thyroids (mg/100 g) |
Thymus (mg/100 g) |
Lungs (mg/100 g) |
Heart (mg/100 g) |
Spleen (mg/100 g) |
Kidneys (g/100 g) |
Liver (g/100 g) |
Adrenals (mg/100 g) |
Testes or Ovaries (mg/100 g) |
Male |
Control |
27 |
235.5 ± 17.7 |
4.31 ± 0.78 |
5.98 ± 0.99 |
0.33 ± 0.04 |
0.48 ± 0.04 |
0.36 ± 0.03 |
0.31 ± 0.05 |
0.93 ± 0.08 |
4.89 ± 0.36 |
16.49 ± 2.71 |
0.90 ± 0.04 |
591 |
29 |
222.4 ± 18.6 |
4.02 ± 0.50 |
5.93 ± 0.86 |
0.33 ± 0.04 |
0.49 ± 0.04 |
0.37 ± 0.04 |
0.33 ± 0.09 |
0.95 ± 0.07 |
5.10 ± 0.37 |
16.39 ± 2.53 |
0.95 ± 0.08 * |
|
2869 |
11 |
213.6 ± 20.1 |
4.23 ± 0.43 |
5.93 ± 0.66 |
0.28 ± 0.03 * |
0.51 ± 0.05 |
0.35 ± 0.02 |
0.27 ± 0.02 * |
0.96 ± 0.06 |
5.03 ± 0.27 |
17.23 ± 2.26 |
0.95 ± 0.09 |
|
Female |
Control |
25 |
173.1 ± 8.9 |
6.09 ± 1.08 |
7.08 ± 0.85 |
0.34 ± 0.05 |
0.54 ± 0.04 |
0.38 ± 0.04 |
0.30 ± 0.04 |
0.99 ± 0.12 |
5.14 ± 0.42 |
26.38 ± 4.71 |
47.94 ± 9.78 |
591 |
34 |
169.7 ± 11.7 |
5.82 ± 0.81 |
6.85 ± 0.96 |
0.32 ± 0.04 |
0.54 ± 0.12 |
0.40 ± 0.03 |
0.30 ± 0.03 |
0.95 ± 0.07 |
4.83 ± 0.37 |
27.61 ± 3.76 |
46.74 ± 10.76 |
|
2869 |
17 |
167.8 ± 15.9 |
6.27 ± 1.90 |
7.31 ± 0.90 |
0.31 ± 0.03 * |
0.57 ± 0.08 |
0.37 ± 0.03 |
0.27 ± 0.04 * |
0.94 ± 0.06 |
5.14 ± 0.33 |
26.75 ± 2.93 |
36.89 ± 4.25 * |
* Significantly different from the control, P <0.05
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 ossificationα in: |
||||
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
Maternal examinations:
- At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed.
Fetal examinations:
- Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth.
- A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group.
- Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg bw/day.
Table 1: Body weight changes in pregnant rats treated orally with d-limonene
Dose (mg/kg bw) |
Gestational days |
Gain |
||||
0 |
9 |
12 |
16 |
20 |
||
Control |
214.80 ± 32.55 |
248.70 ± 28.92 |
265.60 ± 30.53 |
289.85 ± 35.01 |
325.30 ± 44.01 |
105.50 ± 29.67 |
591 |
221.25 ± 39.58 |
254.65 ± 41.16 |
264.80 ± 39.94 |
290.10 ± 38.37 |
325.60 ± 52.75 |
103.95 ± 19.38 |
2869 |
214.75 ± 4.57 |
258.75 ± 32.76
|
248.92 ± 26.27 |
263.17 ± 22.96 * |
305.00 ± 27.07 |
90.25 ± 22.38 |
* Significantly different from the control, P <0.05
Table 2: Effects of d-limonene on rat fetuses
Dose (mg/kg bw) |
Control |
591 |
2869 |
No. of mothers |
15 |
15 |
15 |
Mortality of mothers (%) |
0 |
0 |
40 |
No. of total implants |
12.73 ± 2.96 |
12.18 ± 3.65 |
10.44 ± 3.71 |
No. of dead fetuses |
0 |
0 |
0 |
No. of resorbed fetuses |
1.00 ± 1.10 |
1.47 ± 2.42 |
0.89 ± 0.73 |
No. of live fetuses |
176 |
162 |
87 |
Sex ratio (Male/Female) |
0.69 |
1.22 |
0.85 |
Fetuses body weight (g) Male |
3.71 ± 0.45 |
3.53 ± 0.35 |
3.73 ± 0.52 |
Female |
3.46 ± 0.44 |
3.38 ± 0.45 |
3.63 ± 0.40 |
Placental weight(g) Male |
0.49 ± 0.07 |
0.49 ± 0.10 |
0.48 ± 0.06 |
Female |
0.47 ± 0.07 |
0.46 ± 0.06 |
0.44 ± 0.05 |
Malformation External |
0 |
0 |
0 |
Visceral |
1 |
0 |
0 |
Table 3: Effects of d-limonene on skeletal development of rat fetuses
Dose (mg/kg bw) |
Control |
591 |
2869 |
No. of examined fetuses |
83 |
84 |
42 |
Variation Shortness of 13th rid |
1 |
0 |
0 |
Lumbar rid |
0 |
1 |
2 |
Asymmetry of sternebrae |
0 |
0 |
1 |
Ossification Delayed ossification of parietal bone |
2 |
0 |
0 |
Non-ossification of occipital bone
|
0 |
4 |
1 |
Non-ossification of parietal bone |
0 |
3 |
0 |
No. of ossified metacarpal bone |
7.69 ± 0.72 |
7.49 ± 0.84 |
6.97 ± 0.96 * |
No. of ossified proximal phalanx (Forelimb) |
2.48 ± 1.71 |
2.25 ± 1.81 |
0.55 ± 1.28 * |
No. of ossified metatarsal bone
|
7.98 ± 0.56 |
8.01 ± 011 |
8.00 ± 0 |
No. of ossified sternebraea |
5.47 ± 0.98 |
5.60 ± 0.71 |
5.52 ± 0.73 |
No. of ossified caudal vertebrae |
3.76 ± 0.64 |
3.80 ± 0.57 |
3.95 ± 0.68 |
* Significantly different from the control, P <0.05
Table 4: Body weight changes of postnatal rat offsprings born to mothers treated orally with d-limonene
Postnatal weeks |
Males |
Females |
||||
Dose (mg/kg bw) |
Dose (mg/kg bw) |
|||||
Control |
591 |
2869 |
Control |
591 |
2869 |
|
0 |
5.19 ± 0.55 |
5.46 ± 0.52 |
4.79 ± 0.46 |
4.93 ± 0.62 |
5.09 ± 0.68 |
4.89 ± 0.66 |
1 |
13.06 ± 1.50 |
12.49 ± 0.99 |
10.62 ± 1.54 * |
12.82 ± 1.59 |
12.22 ± 1.07 |
10.66 ± 1.84 |
2 |
26.06 ± 3.12 |
24.86 ± 2.74 |
22.67 ± 5.05 * |
25.88 ± 3.35 |
24.31 ± 2.42 |
22.72 ±.3.92 |
3 |
41.91 ± 5.89 |
39.55 ± 5.14 |
40.77 ± 5.16 |
41.08 ± 5.59 |
38.56 ± 4.37 |
38.39 ± 4.96 |
4 |
73.12 ± 9.89 |
71.33 ± 8.87 |
67.67 ± 8.02 |
69.66 ± 9.43 |
67.38 ± 6.71 |
66.01 ± 9.29 |
5 |
122.32 ± 12.25 |
116.47 ± 12.78 |
112.77 ± 12.65 * |
109.57 ± 10.61 |
107.58 ± 7.95 |
107.10 ± 13.34 |
6 |
176.04 ± 15.80 |
164.68 ± 16.69 |
163.11 ± 17 .85 * |
141.39 ± 10.54 |
140.11 ± 9.40 |
139.45 ± 14.22 |
7 |
235.52 ± 17.72 |
222.43 ± 18.57 |
213.64 ± 20.10 * |
173.06 ± 8.89 |
169.65 ± 11.13 |
167.84 ± 15.86 |
* Significantly different from the control, P <0.05
Table 5: Effects of d-limonene on postnatal development of the rats
Dose (mg/ kg bw) |
Days of postnatal development |
|||||
Opening of the ear-shell |
Coating with |
Odontiasis |
Opening of the eyelid |
Descending of the testis |
Opening of the vaginal orifice |
|
Control |
2.55 ± 0.76 |
5.51 ± 0.91 |
10.1 ± 0.96 |
14.83 ± 0.55 |
22.5 ± 1.30 |
35.6 ± 2.50 |
591 |
2.09 ± 0.82 |
6.00 ± 0 |
10.4 ± 0.71 |
15.00 ± 0.76 |
21.6 ± 1.39 |
35.5 ± 1.75 |
2869 |
2.41 ± 0.49 |
8.50 ±0.50 |
10.4 ± 1.85 |
15.14 ± 0.75 |
21.27 ± 0.57 |
35.93 ± 2.20 |
Table 6: Effects of d-limonene on development of rat offsprings
Dose (mg/kg) |
Control |
591 |
2869 |
No. of mothers |
5 |
5 |
5 |
Mortality of mothers |
0 |
0 |
40 |
No. of offspring from birth to the 7th week 0 |
61 |
65 |
33 |
1 |
53 |
63 |
30 |
2 |
53 |
63 |
30 |
3 |
53 |
63 |
28 |
4 |
53 |
63 |
28 |
5 |
53 |
63 |
28 |
6 |
53 |
63 |
28 |
7 |
53 |
63 |
28 |
External abnormality |
0 |
0 |
0 |
No. of total implants |
13.6 ± 3.1 |
14.8 ± 1.7 |
13.7 ± 1.7 |
No. of dead fetuses at birth |
4 |
4 |
5 |
Parturient rate |
95 |
92 |
93 |
Weaning rate |
89 |
97 |
85 |
Table 7: Absolute organ weights of postnatal rat offsprings born to mothers treated orally with d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings. |
Final BW (g) |
Pituitary (mg) |
Thyroids (mg) |
Thymus (g) |
Lungs (g) |
Heart (g) |
Spleen (g) |
Kidneys (g) |
Liver (g) |
Adrenals (g) |
Testes (g) or Ovaries (mg) |
Male |
Control |
27 |
235.5 ± 17.7 |
10.04 ± 1.95 |
14.06 ± 2.52 |
0.77 ± 0.08 |
1.15 ± 0.10 |
0.82 ± 0.08 |
0.75 ± 0.11 |
2.18 ± 0.36 |
11.55 ± 1.14 |
38.85 ± 7.37 |
2.15 ± 0.17 |
591 |
29 |
222.4 ± 18.6 |
9.58 ± 4.84 |
13.12 ± 2.77 |
0.72 ± 0.09 |
1.10 ± 0.11 |
0.81 ± 0.08 |
0.69 ± 0.08 |
2.10 ± 0.20 |
11.24 ± 1 .49 |
36.77 ± 6.93 |
2.13 ± 0.28 |
|
2869 |
11 |
213.6 ± 20.1 |
9.69 ± 0.65 |
14.41 ± 3.60 |
0.66 ± 0.08 * |
1.19 ± 0.17 |
0.80 ± 0.07 |
0.63 ± 0.07 * |
2.23 ± 0.38 |
11.64 ± 1.64 |
43.23 ± 10.91 |
2.18 ± 0.14 |
|
Female |
Control |
25 |
173.1 ± 8.9 |
11.18 ± 3.15 |
12.26 ± 1.32 |
0.59 ± 0.08 |
0.97 ± 0.11 |
0.66 ± 0.07 |
0.52 ± 0.06 |
1.72 ± 0.22 |
8.88 ± 0.85 |
45.51 ± 8.01 |
77.24 ± 22.01 |
591 |
34 |
169.7 ± 11.7 |
10.05 ± 3.34 |
11.57 ± 1.62 |
0.54 ± 0.07 |
0.96 ± 0.07 |
0.67 ± 0.05 |
0.50 ± 0.06 |
1.60 ± 0.15 * |
8.16 ± 0.85 |
46.56 ± 8.45 |
85.84 ± 42.52 * |
|
2869 |
17 |
167.8 ± 15.9 |
9.95 ± 1.87 |
12.31 ± 1.80 |
0.51 ± 0.07 * |
0.94 ± 0.10 |
0.62 ± 0.06 |
0.44 ± 0.04 * |
1.62 ± 0.17 * |
8.50 ± 0.58 |
47.15 ± 6.63 |
63.15 ± 7.99 |
* Significantly different from the control, P <0.05
Table 8: Relative organ weights per 100 g body weights of postnatal rat offsprings born to mothers treated orally with d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings. |
Final BW (g) |
Pituitary (mg/100 g) |
Thyroids (mg/100 g) |
Thymus (mg/100 g) |
Lungs (mg/100 g) |
Heart (mg/100 g) |
Spleen (mg/100 g) |
Kidneys (g/100 g) |
Liver (g/100 g) |
Adrenals (mg/100 g) |
Testes or Ovaries (mg/100 g) |
Male |
Control |
27 |
235.5 ± 17.7 |
4.31 ± 0.78 |
5.98 ± 0.99 |
0.33 ± 0.04 |
0.48 ± 0.04 |
0.36 ± 0.03 |
0.31 ± 0.05 |
0.93 ± 0.08 |
4.89 ± 0.36 |
16.49 ± 2.71 |
0.90 ± 0.04 |
591 |
29 |
222.4 ± 18.6 |
4.02 ± 0.50 |
5.93 ± 0.86 |
0.33 ± 0.04 |
0.49 ± 0.04 |
0.37 ± 0.04 |
0.33 ± 0.09 |
0.95 ± 0.07 |
5.10 ± 0.37 |
16.39 ± 2.53 |
0.95 ± 0.08 * |
|
2869 |
11 |
213.6 ± 20.1 |
4.23 ± 0.43 |
5.93 ± 0.66 |
0.28 ± 0.03 * |
0.51 ± 0.05 |
0.35 ± 0.02 |
0.27 ± 0.02 * |
0.96 ± 0.06 |
5.03 ± 0.27 |
17.23 ± 2.26 |
0.95 ± 0.09 |
|
Female |
Control |
25 |
173.1 ± 8.9 |
6.09 ± 1.08 |
7.08 ± 0.85 |
0.34 ± 0.05 |
0.54 ± 0.04 |
0.38 ± 0.04 |
0.30 ± 0.04 |
0.99 ± 0.12 |
5.14 ± 0.42 |
26.38 ± 4.71 |
47.94 ± 9.78 |
591 |
34 |
169.7 ± 11.7 |
5.82 ± 0.81 |
6.85 ± 0.96 |
0.32 ± 0.04 |
0.54 ± 0.12 |
0.40 ± 0.03 |
0.30 ± 0.03 |
0.95 ± 0.07 |
4.83 ± 0.37 |
27.61 ± 3.76 |
46.74 ± 10.76 |
|
2869 |
17 |
167.8 ± 15.9 |
6.27 ± 1.90 |
7.31 ± 0.90 |
0.31 ± 0.03 * |
0.57 ± 0.08 |
0.37 ± 0.03 |
0.27 ± 0.04 * |
0.94 ± 0.06 |
5.14 ± 0.33 |
26.75 ± 2.93 |
36.89 ± 4.25 * |
* Significantly different from the control, P <0.05
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 ossificationα in: |
||||
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
Table 1: Effect of d-limonene on prenatal development of rabbit fetuses
Dose (mg/kg bw) |
Control |
250 |
500 |
1000 |
No. of pregnant animals |
10 |
10 |
10 |
18 |
No. of dead clams |
0 |
0 |
0 |
6 |
(%) |
|
|
|
33 |
No. of examined clams |
10 |
10 |
10 |
10 |
No. of implantations |
96 |
94 |
85 |
91 |
(mean ± S.E.) |
9.50 ± 0.25 |
9.40 ± 0.21 |
8.50 ± 0.33 |
9.10 ± 0.25 |
No. of resorbed fetuses |
5 |
4 |
4 |
8 |
No. of dead fetuses |
3 |
5 |
0 |
3 |
No. of live fetuses |
88 |
85 |
81 |
80 |
Sex ratio (Male/Female) |
0.73 (37/51) |
1.13 (45/50) |
0.62 (31/50) |
1.11 (38/42) |
Fetus body weight (g) |
|
|
|
|
Male (mean ± S.E.) |
44.39 ± 1.33 |
48.09 ± 1.07 * |
44.76 ± 1.51 |
43.22 ± 0.96 |
Female (mean ± S.E.) |
45.64 ± 1.00 |
47.45 ± 1.08 |
46.14 ± 1.21 |
45.13 ± 1.10 |
Placental weight (g) |
|
|
|
|
Male (mean ± S.E.) |
5.76 ± 0.17 |
5.84 ± 0.17 |
5.95 ± 0.29 |
5.77 ± 0.19 |
Female (mean ± S.E.) |
5.87 ± 0.19 |
5.70 ± 0.15 |
6.16 ± 0.18 |
5.87 ± 0.23 |
* Significantly different from the control at 5% level
Table 2: Prenatal examinations of rabbit fetuses
Dose (mg/kg bw) |
Control |
250 |
500 |
1000 |
External examination |
|
|
|
|
No. of examined fetuses |
91 |
90 |
81 |
83 |
No. of malforrned fetuses |
0 |
0 |
0 |
0 |
Visceral examination |
|
|
|
|
No. of examined fetuses |
88 |
85 |
81 |
80 |
No. of malformed fetuses |
|
|
|
|
Atrial septal defect (%) |
0 |
2 (2.4) |
0 |
0 |
No. of minor abnormality |
|
|
|
|
Incomplete lobulation of lungs (%) |
11 (12.5) |
16 (18.8) |
19 (23.5) |
19 (23.8) |
Enlargement of foramen ovale (%) |
2 (2.3) |
2 (2.4) |
5 (6.2) |
4 (5.0) |
Skeletal examination |
|
|
|
|
No. of examined fetuses |
86 |
87 |
81 |
80 |
No. of malformed fetuses |
0 |
0 |
0 |
0 |
No. of variation |
|
|
|
|
Left lumbar rib (%) |
18 (20.9) |
26 (29.9) |
14 (17.3) |
25 (31.3) |
Right lumbar rib (%) |
16 (18.6) |
22 (25.3) |
14 (17.3) |
22( 27.5) |
Ossification pattern |
|
|
|
|
Retarded ossification of 5th sternebrae (%) |
11 (12.8) |
14 (16.1) |
8 (9.9) |
18 (22.5) |
Retarded ossification of middle phalanx of fore limbs (%) |
2 (2.3) |
3 (3.4) |
0 |
6 (7.4) |
Table 3: Absolute organ weights of rabbit offsprings
|
Male |
Female |
||||||
|
Control |
250 |
500 |
1000 |
Control |
250 |
500 |
1000 |
No. of offsprings |
13 |
12 |
8 |
13 |
10 |
13 |
16 |
9 |
Final body weight (g) |
893.0 ± 45.3 |
1021.2 ± 45.4 ** |
931.9 ± 55.5 |
957.3 ± 52.4 |
1005.5 ± 53.7 |
1093.1 ± 46.6 |
860.0 ± 31.6 * |
1071.7 ± 58.1 |
Liver (g) |
36.49 ± 2.52 |
45.08 ± 1.52 * |
38.67 ± 3.10 |
34.91 ± 2.76 |
41.79 ± 3.39 |
13.95 ± 3.18 |
34.68 ± 1.90 |
48.30 ± 6.14 |
Lungs (g) |
5.53 ± 0.35 |
6.25 ± 0.26 |
5.98 ± 0.36 |
5.46 ± 0.18 |
5.91 ± 0.25 |
5.94 ± 0.28 |
5.72 ± 0.18 |
5.93 ± 0.45 |
Heart (g) |
2.63 ± 0.17 |
3.50 ± 0.16 ** |
2.86 ± 0.17 |
3.04 ± 0.19 |
3.19 ± 0.17 |
3.38 ± 0.21 |
2.72 ± 0.12 * |
3.34 ± 0.20 |
Spleen (g) |
0.71 ± 0.05 |
0.77 ± 0.04 |
0.71 ± 0.08 |
0.81 ± 0.04 |
0.64 ± 0.06 |
0.74 ± 0.05 |
0.74 ± 0.04 |
0.78 ± 0.07 |
Thymus (g) |
2.31 ± 0.20 |
2.51 ± 0.23 |
2.11 ± 0.38 |
1.96 ± 0.11 |
2.40 ± 0.30 |
2.77 ± 0.19 |
1.671.14 * |
2.36 ± 0.31 |
Kidneys (g) |
7.67 ± 0.42 |
9.80 ± 0.46 ** |
8.29 ± 0.29 |
8.58 ± 0.52 |
8.82 ± 0.46 |
8.40 ± 0.30 |
7.86 ± 0.37 |
9.56 ± 0.55 |
Thyroids (mg) |
75.89 ± 8.35 |
102.68 ± 4.18* |
86.74 ± 10.97 |
80.93 ± 7.41 |
82.78 ± 8.00 |
89.90 ± 4.11 |
75.75 ± 5.43 |
96.30 ± 9.67 |
Adrenals (mg) |
69.65 ± 6.02 |
9.1.93 ± 1.06 ** |
78.79 ± 5.89 |
71.36 ± 6.00 |
82.23 ± 4.37 |
94.24 ± 5.07 |
87.64 ± 4.18 |
105.39 ± 15.11 |
Testes or Ovaries (mg) |
180.42 ± 17.15 |
272.86 ± 16.46 ** |
185.62 ± 23.78 |
162.84 ± 20.59 |
46.31 ± 7.90 |
46.10 ± 2.80 |
43.53 ± 2.69 |
47.77 ± 3.59 |
* Significantly different from the control at 5% level
** Significantly different from the control at 1% level
Table 4: Relative organ weights per 100 g of rabbit offsprings
|
Male |
Female |
||||||
|
Control |
250 |
500 |
1000 |
Control |
250 |
500 |
1000 |
No. of offsprings |
13 |
12 |
8 |
13 |
10 |
13 |
16 |
9 |
Final body weight (g) |
893.0 ± 45.3 |
1021.2 ± 45.4 ** |
931.9 ± 55.5 |
957.3 ± 52.4 |
1005.5 ± 53.7 |
1093.1 ± 46.6 |
860.0 ± 31.6 * |
1071.7 ± 58.1 |
Liver (g/100 g) |
4.08 ± 0.25 |
3.99 ± 0.22 * |
4.16 ± 0.26 |
3.52 ± 0.10 |
4.25 ± 0.17 |
4.03 ± 0.21 |
4.02 ± 0.16 |
4.04 ± 0.32 |
Lungs (g/100 g) |
0.61 ± 0.03 |
0.55 ± 0.03 |
0.65 ± 0.04 |
0.58 ± 0.03 |
0.62 ± 0.02 |
0.55 ± 0.02 * |
0.67 ± 0.03 |
0.51 ± 0.02 ** |
Heart (g/100 g) |
0.29 ± 0.01 |
0.31 ± 0.01 |
0.31 ± 0.01 |
0.31 ± 0.02 |
0.33 ± 0.01 |
0.31 ± 0.01 |
0.32 ± 0.01 |
0.29 ± 0.01 * |
Spleen (g/100 g) |
0.08 ± 0.01 |
0.07 ± 0.01 |
0.08 ± 0.01 |
0.08 ± 0 |
0.07 ± 0.01 |
0.07 ± 0.01 |
0.09 ± 0 |
0.07 ± 0.01 |
Thymus (g/100 g) |
0.25 ± 0.02 |
0.22 ± 0.02 |
0.25 ± 0.03 |
0.20 ± 0.01 * |
0.24 ± 0.03 |
0.25 ± 0.01 |
0.195 ± 0.01 |
0.20 ± 0.02 |
Kidneys (g/100 g) |
0.85 ± 0.04 |
0.86 ± 0.04 |
0.90 ± 0.03 |
0.88 ± 0.02 |
0.91 ± 0.02 |
0.80 ± 0.01 |
0.91 ± 0.01 |
0.83 ± 0.03 * |
Thyroids (mg/100 g) |
8.21 ± 0.69 |
9.10 ± 0.51 |
9.21 ± 0.87 |
8.18 ± 0.42 |
8.40 ± 0.56 |
8.41 ± 0.45 |
8.81 ± 0.57 |
8.13 ± 0.41 |
Adrenals (mg/100 g) |
7.82 ± 0.60 |
8.37 ± 0.40 |
8.58 ± 0.77 |
7.18 ± 0.28 |
8.61 ± 0.51 |
8.79 ± 0.57 |
9.15 ± 0.42 |
9.04 ± 0.14 |
Testes or Ovaries (mg/100 g) |
23.68 ± 1.31 |
19.68 ± 0.94 * |
19.48 ± 1.69 |
16.35 ± 1.56 |
5.15 ± 1.19 |
4.33 ± 0.32 |
5.19 ± 0.43 |
3.84 ± 0.32 |
* Significantly different from the control at 5% level
** Significantly different from the control at 1% level
Table 5: Effects of d-limonene on gross differentiations of rabbit offsprings
|
Control |
250 |
500 |
1000 |
No. of examined offsprings |
23 |
25 |
24 |
22 |
Days of gross differentiation after birth Opening of the ear-shell |
|
|
|
|
6th day (%) |
0 |
0 |
1 (4.2) |
0 |
7th day (%) |
23 (100) |
25 (100) |
23 (95.8) |
22 (100) |
Coating with the hair |
|
|||
2nd day (%) |
7 (30.4) |
0 |
0 |
0 |
3rd day (%) |
16 (69.6) |
25 (100) |
24 (100) |
22 (100) |
Odontiasis |
|
|||
At birth (%) |
23 (100) |
25 (100) |
24 (100) |
22 (100) |
Opening of the eyelids |
|
|||
9th day (%) |
0 |
0 |
0 |
3 (13.6) |
10th day (%) |
11 (47.8) |
4 (16.0) |
13 (54.2) |
5 (22.7) |
11th day (%) |
4 (17.4) |
15 (60.0) |
10 (41.7) |
12 (54.5) |
12th day (%) |
3 (13.0) |
5 (20.0) |
1 (4.2) |
2 (9.1) |
13th day (%) |
5 (21.7) |
1 (4.0) |
0 |
0 |
Table 6: Effects of d-limonene on postnatal development of rabbit offsprings
|
Control |
250 |
500 |
1000 |
No of dams |
3 |
3 |
3 |
3 |
No. of still-birth (Male/Female) |
1 (1/0) |
0 |
0 |
1 (0/1) |
No. of offsprings (Male/Female) At birth |
28 (15/13) |
27 (14/13) |
26 (8/18) |
27 (15/12) |
1st week |
28 (15/13) |
27 (14/13) |
25 (8/17) |
26 (14/12) |
2nd week |
26 (14/12) |
27 (14/13) |
25 (8/17) |
26 (14/12) |
3rd week |
24 (14/10) |
27 (14/13) |
25 (8/17) |
25 (14/11) |
4th week |
23 (13/10) |
26 (13/13) |
25 (8/17) |
22 (13/ 9) |
5th week |
23 (13/10) |
25 (12/13) |
25 (8/17) |
22 (13/ 9) |
6th week |
23 (13/10) |
25 (12/13) |
25 (8/17) |
22 (13/ 9) |
7th week |
23 (13/10) |
25 (12/13) |
24 (8/16) |
22 (13/ 9) |
Weanling rate (%) |
79.3 (81.2/76.9) |
92.6 (85.7/100) |
92.3 (100/88.9) |
78.6 (86.7/69.2) |
Table 7: Postnatal examinations of rabbit offsprings
|
Control |
250 |
500 |
1000 |
No. of dams |
3 |
3 |
3 |
3 |
No. of examined offsprings |
23 |
25 |
24 |
22 |
Sensory function |
Normal |
Normal |
Normal |
Normal |
External examination No. of malformed offsprings |
0 |
0 |
0 |
0 |
Visceral examination No. of malformed offsprings |
0 |
0 |
0 |
0 |
No. of minor abnormality Incomplete lobulation of lungs (%) |
2 (8.7) |
1 (4.0) |
0 |
0 |
Accessory spleen (%) |
2 (8.7) |
0 |
0 |
0 |
Protrusion of gall bladder (%) |
1 (4.3) |
1 (4.0) |
0 |
0 |
Skeletal examination No. of malformed offsprings |
0 |
0 |
0 |
0 |
No. of variation Left lumbar rib (%) |
4 (17.4) |
4 (16.0) |
4 (16.7) |
4 (18.2) |
Right lumbar rib (%) |
2 (8.7) |
6 (24.0) |
6 (25.0) |
4 (18.2) |
Translocation of caudal vertebrae (%) |
1 (4.3) |
0 |
1 (4.2) |
0 |
Ossification pattern Retarded ossification of 5th sternebrae (%) |
0 |
2 (8.0) |
0 |
1 (4.5) |
Accessory ossification center of 5th sternebrae (%) |
1 (4.3) |
2 (8.0) |
0 |
3 (13.6) |
Maternal examinations:
- Significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day.
- No anomalies were observed in the general behavior of dams during the period of gestation.
Fetal examinations:
- An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing with those of control.
- In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed. However, these retarded ossifications were restored to normal during postnatal development.
- A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.
Table 1: Effects of d-limonene on development of mouse fetuses.
|
Control |
591 |
2363 |
No. of mothers |
15 |
15 |
15 |
No. of implantations |
162 |
179 |
154 |
(mean ± S.E.) |
(10.80 ± 0.20) |
(11.93 ± 0.13) |
(10.27 ± 0.21) |
No. of dead fetuses |
9 |
1.1 |
6 |
(mean ± S.E.) |
(0.60 ± 0.05) |
(0.73 ± 0.10) |
(0.40 ± 0.05) |
No. of resorbed fetuses |
18 |
20 |
20 |
(mean ± S.E.) |
(1.20 ± 0.05) |
(1.33 ± 0.10) |
(1.33 ± 0.10) |
No. of live fetuses |
135 |
148 |
125 |
Sex ratio (Male/Female) |
1.33 |
0.83 |
1.13 |
Fetuses Body weight (g) Male (mean ± S.E.) |
1.34 ± 0.02 |
1.24 ± 0.01 |
1.28 ± 0.02 |
Female (mean ± S.E.) |
1.28 ± 0.02 |
1.22 ± 0.01 |
1.19 ± 0.02 |
Placental weight (mg) Male (mean ± S.E.) |
94 (2 ± 2.0) |
86 ( 0 ± 1.7) |
89 (3 ± 1.7) |
Female (mean ± S.E.) |
87 (8 ± 2.6) |
81 (5 ± 1.9) |
83 (5 ± 2.2) |
External observation No. of fetuses examined |
135 |
148 |
128 |
No. of fetuses malformed |
0 |
4 |
0 |
Cleft palate |
0 |
4 |
0 |
Incidence (%) |
0 |
2.7 |
0 |
Visceral observation No. of fetuses examined |
71 |
76 |
68 |
No. of fetuses malformed |
4 |
4 |
3 |
Enlargement of foramen ovale |
4 |
4 |
3 |
Incidence (%) |
5.6 |
5.3 |
4.4 |
Table 2: Effects of d-limonene on skeletal development of mouse fetuses
Dose (mg/kg bw) |
Control |
591 |
2363 |
No. of fetuses examined |
64 |
72 |
61 |
Variation Lumbar rib (%) |
17 (26.6) |
12 (16.7) |
28 (46.7) * |
Cervical rib (%) |
1 (1.6) |
(5.6) |
1 (1.7) |
Fused rib (%) |
0 (0) |
0 (1) |
5 (8.3) * |
Crooked rib (%) |
2 (3.1) |
0 (1) |
0 (0) |
Asymmetry of sternebrae (%) |
2 (1.7) |
7 (9.7) |
7 (11.7) |
Fused sternebrae |
0 (0) |
1 (1.4) |
1 (1.7) |
No. of ossification Sternebrae |
5.99 ± 0.01 |
5.97 ± 0.02 |
5.94 ± 0.03 |
Fore limb Metacarpal bone |
8.00 ± 0 |
7.97 ± 0.03 |
8.00 ± 0 |
Proximal phalanx |
7.50 ± 0.13 |
7.67 ± 0.16 |
6.87 ± 0.30 * |
Middle phalanx |
1.16 ± 0.23 |
2.14 ± 0.29 ** |
2.18 ± 0.29 ** |
Distal phalanx |
9.08 ± 0.32 |
9.72 ± 0.20 |
9.13 ± 0.36 |
Hind limb Metatarsal bone |
10.00 ± 0 |
9.92 ± 0.05 |
9.80 ± 0.09 * |
Proximal phalanx |
8.12 ± 0.23 |
8.03 ± 0.23 |
7.23 ± 0.39 * |
Middle phalanx |
0.09 ± 0.09 |
0.33 ± 0.18 |
0.20 ± 0.11 |
Distal phalanx |
9.47 ± 0.24 |
9.72 ± 0.20 |
9.30 ± 0.31 |
Caudal vertebrae |
7.12 ± 0.95 |
6.50 ± 0.21 |
7.00 ± 0.25 |
* Significantly different from the control at 5% level.
** Significantly different from the control at 1% level.
Table 3: Effects of d-limonene on postnatal development of mouse offsprings
|
|
d-Limonene (mg/kg bw) |
|
|
Control |
591 |
2363 |
No. of mothers |
5 |
5 |
5 |
No. of implantations |
51 |
52 |
50 |
(mean ± S.E.) |
(10.80 ± 0.33) |
(10.41 ± 0.96) |
(10.03 ± 0.63) |
No. of offsprings |
50 |
46 |
39 |
No. of dead offsprings at birth |
0 |
0 |
0 |
Sensory function |
Normal |
Normal |
Normal |
No. of live offsprings At birth |
50 |
46 |
39 |
1st week |
50 |
46 |
39 |
2nd week |
50 |
46 |
39 |
3rd week |
50 |
46 |
39 |
4th week |
50 |
46 |
39 |
5th week |
50 |
46 |
39 |
6th week |
50 |
46 |
39 |
7th week |
50 |
46 |
39 |
Weanling rate (%) |
100 |
100 |
100 |
Table 4: Effects of d-limonene on gross differentiation of mouse offsprings
|
|
d-Limonene (mg/kg bw) |
|
Gross differentiation |
Control |
591 |
2363 |
Opening of the ear-shell |
3.5 ± 0.07 |
3.6 ± 0.08 |
4.1 ± 0.08 |
Coating with the hair |
5.0 ± 0.00 |
4.11 ± 0.08 |
5.2 ± 0.06 |
Odontiasis |
9.8 ± 0.07 |
9.3 ± 0.07 |
9. 1 ± 0.04 |
Opening of the eyelid |
13.3 ± 0.08 |
12.9 ± 0.06 |
13.6 ± 0.09 |
Descending of the testis |
23.0 ± 0.20 |
23.3 ± 0.11 |
25.0 ± 0.27 |
Opening of the vaginal orifice |
29.5 ± 0.26 |
30.5 ± 0.16 |
30.6 ± 0.15 |
Table 5: Absolute organ weights of postnatal mouse offsprings born to mothers given d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings |
Final BW (g) |
Thyroids (mg) |
Thymus (g) |
Lungs (g) |
Heart (g) |
Spleen (g) |
Kidneys (g) |
Liver (g) |
Adrenals (g) |
Testes (g) or Ovaries (mg) |
Female |
Control |
27 |
34.2 ± 0.50 |
5.43 ± 0.18 |
75.72 ± 4.14 |
199.26 ± 4.70 |
158.22 ± 2.13 |
131.54 ± 9.96 |
0.63 ± 0.02 |
2.06 ± 0.09 |
8.39 ± 0.49 |
217.52 ± 1.65 |
591 |
27 |
34.9 ± 0.44 |
6.04 ± 0.68 |
65.26 ± 4.26 |
203.76 ± 2.95 |
166.66 ± 7.58 |
121.66 ± 4.74 |
0.63 ± 0.02 |
2.08 ± 0.07 |
8.29 ± 0.67 |
209.76 ± 9.23 |
|
2363 |
23 |
32.2 ± 0.77 |
5.41 ± 0.62 |
64.08 ± 3.64 |
189.60 ± 6.69 |
158.96 ± 3.12 |
125.52 ± 3.04 |
0.58 ± 0.02 |
2.14 ± 0.04 |
8.53 ± 0.86 |
200.20 ± 0.39 |
|
Male |
Control |
23 |
27.3 ± 0.50 |
4.80 ± 0.22 |
81.47 ± 4.38 |
172.80 ± 7.20 |
118.98 ± 3.91 |
121.76 ± 6.48 |
0.37 ± 0.01 |
1.37 ± 0.02 |
11.71 ± 0.40 |
13.38 ± 0.68 |
591 |
19 |
28.8 ± 0.45 |
4.29 ± 0.20 |
69.44 ± 5.52 |
174.26 ± 5.66 |
129.96 ± 3.62 |
112.68 ± 2.79 |
0.38 ± 0.01 |
1.37 ± 0.04 |
11.36 ± 0.43 |
16.98 ± 1.71 |
|
2363 |
16 |
28.1 ± 0.34 |
3.53 ± 0.41 * |
63.95 ± 9.72 |
171.75 ± 5.70 |
135.15 ± 6.89 |
116.15 ± 5.78 |
0.38 ± 0.01 |
1.46 ± 0.03 * |
11.61 ± 0.30 * |
17.93 ± 1.30 * |
* Significantly different from the control at 5% level.
Table 6: Relative organ weights per 100 g body weights of postnatal mouse offsprings born to mothers given d-limonene
Sex |
Dose (mg/kg bw) |
No. of offsprings |
Final BW (g) |
Thyroids(mg/100 g) |
Thymus(mg/100 g) |
Lungs(mg/100 g) |
Heart(mg/100 g) |
Spleen(mg/100 g) |
Kidneys(g/100 g) |
Liver(g/100 g) |
Adrenals(mg/100 g) |
Testes or Ovaries (mg/100 g) |
Female |
Control |
27 |
34.2 ± 0.50 |
15.80 ± 0.44 |
220.36 ± 10.67 |
580.50 ± 13.59 |
461.18 ± 8.65 |
386.55 ± 39.35 |
1.83 ± 0.07 |
6.01 ± 0.26 |
24.48 ± 1.55 |
634.84 ± 19.06 |
591 |
27 |
34.9 ± 0.44 |
17.20 ± 1.93 |
185.29 ± 8.48 * |
581.21 ± 9.42 |
474.60 ± 717.10 |
346.85 ± 12.29 |
1.80 ± 0.05 |
5.93 ± 0.14 |
23.55 ± 1.48 |
596.69 ± 14.95 |
|
2363 |
23 |
32.2 ± 0.77 |
16.55 ± 2.64 |
191.59 ± 6.25 |
568.43 ± 13.67 |
477.39 ± 11.90 |
376.91 ± 10.23 |
1.73 ± 0.03 |
6.44 ± 0.29 |
25.74 ± 2.82 |
602.30 ± 34.26 |
|
Male |
Control |
23 |
27.3 ± 0.50 |
17.13 ± 18.47 |
291.98 ± 34.66 |
619.50 ± 8.54 |
425.00 ± 32.37 |
437.67 |
1.31 ± 0.04 |
4.89 ± 0.08 |
41.89 ± 1.41 |
47.85 ± 2.42 |
591 |
19 |
28.8 ± 0.45 |
15.06 ± 0.61 * |
242.35 ± 13.57 |
611.98 ± 17.12 |
456.02 ± 4.93 * |
397.44 ± 20.40 |
1.33 ± 0.02 |
4.81 ± 0.11 |
39.96 ± 1.73 |
59.59 ± 5.78 |
|
2363 |
16 |
28.1 ± 0.34 |
12.60 ± 1.44 * |
229.38 ± 35.46 |
612.74 ± 20.32 |
482.14 ± 24.21 * |
414.30 ± 17.34 |
1.37 ± 0.04 |
5.23 ± 0.09 * |
41.45 ± 1.27 |
64.33 ± 5.38 * |
* Significantly different from the control at 5% level.
Table 7: Summaried data on postnatal development of mouse offsprings
|
|
d-limonene (mg/kg bw) |
|
|
Control |
591 |
2363 |
External observation |
|
||
No. of offsprings examined |
50 |
46 |
39 |
No. of offsprings malformed |
0 |
0 |
0 |
Visceral observation |
|
||
No. of offsprings examined |
50 |
46 |
39 |
No. of offsprings malformed |
0 |
0 |
0 |
Skeletal observation |
|
||
No. of offsprings examined |
50 |
46 |
39 |
No. of offsprings malformed |
0 |
0 |
0 |
Skeletal variation |
|
||
Lumbar rib (%) |
17 (34.0) |
23 (50.0) |
20 (51.3) |
Fusion of 13th and lumbar rib (%) |
0 |
0 |
1 (2.6) |
Fusion of lumbar vertebra (%) |
1 (2.0) |
1 (2.2) |
1 (2.6) |
Crooked tail (%) |
0 |
0 |
1 (2.6) |
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no study available
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
No information is available for cornmint or peppermint oil, however, no effects on development and/or reproduction were observed for 71.1% of the constituents. In the table below the available information on constituents is summarized.
Constituent |
Developmental toxicity/Toxicity to reproduction |
NOAEL |
Major constituents (≥10%) |
||
L-menthol |
Developmental toxicity (rat) |
No developmental effects observed 218 mg/kg bw /day |
Menthone |
RA to L-menthol Developmental toxicity (rat) |
No developmental effects observed 218 mg/kg bw /day |
Minor constituents (<10%) |
||
Limonene |
Developmental toxicity (rat, rabbit, mouse) |
No developmental effects observed 591 mg/kg bw/d (rat) >1000 mg/kg bw/d (rabbit) 591 mg/kg bw/d (mouse) |
Alpha-pinene |
RA to Camphene Developmental toxicity (rat) |
No developmental effects observed 250 mg/kg bw /day |
Beta-pinene |
RA to Alpha-pinene/Camphene Developmental toxicity (rat) |
No developmental effects observed 250 mg/kg bw /day |
Myrcene |
One generation toxicity to reproduction (rat) Developmental toxicity (rat) |
300 mg/kg bw/d
500 mg/kg bw/d |
For the remaining constituents also no effects on development or reproduction are expected, as most of the constituents are structurally similar (terpenes, terpenoids). Based on the absence of effects on development and reproduction in the major and some minor constituent, cornmint oil and peppermint oil are not expected to be toxic to the development or reproduction.
Justification for selection of Effect on developmental toxicity: via oral route:
No selection is made as a Weight of Evidence approach with constituent data was followed which is described below.
Justification for classification or non-classification
Based on the available information on constituents, cornmint oil does not have to be classified as toxic to the development or reproduction in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2008/EC.
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
