Bergamot, ext.

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

21 June 1988 - 4 December 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to an equivalent of OECD guideline 421 and under GLP conditions. Klimisch 2 reliability has been assigned in accordance with (ECHA Practical Guide #6) due to the read-across purpose of this study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)BR

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Females: 187-232 g
- Housing: Individually
- Diet (e.g. ad libitum): Ad libitum, Certified Rodent Chow
- Water (e.g. ad libitum): Ad libitum, filtered local water (chlorine added)
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 35-65
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test article dissolved in corn oil at concentrations of 0, 50, 100 and 200 mg/mL.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly

VEHICLE
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg, adjusted daily on the basis of individual body weights
- Lot/batch no. (if required): APR0789B, APR1489A and 80299

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 7 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

7 days prior to cohabitation.
After cohabitation until day 4 of lactation or day 25 of presumed gestation.

Frequency of treatment:

Once daily

Details on study schedule:

Not relevant

Remarks:

Doses / Concentrations:
250, 500, 1000 mg coriander oil/kg bw/day, equivalent to 183, 365, 829 mg linalool/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on toxicity studies that were conducted earlier

Positive control:

Not necessary

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked:
Viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Two times during acclimation period and daily during dosage period

BODY WEIGHT: Yes
- Time schedule for examinations: Daily during dosage period

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Oestrous cyclicity (parental animals):

Not performed

Sperm parameters (parental animals):

Not relevant, males were not dosed

Litter observations:

STANDARDISATION OF LITTERS
Not relevant, termination of study on day 4 postpartum

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: All surviving animals on day 4 or 5 of lactation. Dams that did not deliver litter on day 25 of presumed gestation, were sacrificed that day.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including gross lesions and placement of implantation sites.

HISTOPATHOLOGY
Tissues of dams (gross lesions, ovaries) were preserved for possible future evaluation.

Postmortem examinations (offspring):

SACRIFICE
No data

GROSS NECROPSY
- Pups that died were subjected to postmortem examinations and examined for the cause of death.
- Gross necropsy consisted of external examination for gross lesions.

HISTOPATHOLOGY / ORGAN WEIGTHS
Tissue of litter (gross lesions) were preserved for possible future evaluation.

Statistics:

Parametric
- Bartlett's test: For homogeneity of variance
- Dunnett's test
- Covariance analysis T-test
Nonparametric
- Kruskal-Wallis
- Dunn's test
- Fisher's Exact Test
Test for proportion data
- Variance test for homogeneity of the binomial distribution

Reproductive indices:

Pregnant rats/rats mated

Offspring viability indices:

Pups surviving 4 days/liveborn pups

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Excess in salivation was noted at all dose groups being significant for middle and high dosage group. Significant number of rats in the 1000 mg/kg/day group had urine-stained abdominal fur during premating period and 1-2 rats in this group showed ataxia and/or decreased motor activity infrequently during the premating and/or gestation periods. However, excess salivation was not considered to be evidence for strong toxicity and was therefore considered as non-adverse.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Biologically remarkable decreases in body weight gain and feed consumption occurred for the 1000 mg/kg/day dosage group rats during premating (significant after first dosage). During gestation, biologically remarkable increase in weight and feed consumption occurred for each group given the test article. Statistically significant increases in body weight gain occurred for the low and high dosage group rats and statistically significant increases in absolute and relative feed consumption values occurred for each group given the test article. These effects decreased in severity during lactation.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: decreased food consumption, body weight

Dose descriptor:

NOAEL

Remarks:

linalool

Effect level:

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

Sex:

female

Basis for effect level:

other: decreased food consumption, body weight

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:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Pup mortality was significantly increased in litters of dams administered 1000 mg/kg/day coriander oil. When comparing implantation averages to delivered litter size in the 1000 mg/kg/day, the litter size was more decreased as compared to other groups. This indicates more resorptions in the high dosage group.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased litter size; increased pup mortality

Dose descriptor:

NOAEL

Remarks:

linalool

Generation:

F1

Effect level:

365 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased litter size; increased pup mortality

Reproductive effects observed:

not specified

Not relevant

Conclusions:

The maternal NOEL for B10 was below 250 mg/kg/day, based on clinical signs, such as salivation and altered body weight gains and feed consumption. These changes were not considered to be evidence for strong toxicity, hence the NOAEL was set higher at 500 mg/kg/day. The NOEL for B10 was 500 mg/kg/day administered to dams. The highest-dosage (1000 mg/kg/day) group had reduced delivered litter sizes, indicating in utero deaths, and siginifcant incidences of pup mortality in the first four days postpartum. No adverse effects regarding mating, fertility or duration of gestation or parturition occurred in any treatment group including the high-dose at 1000 mg/kg/day. Clear adverse effects on reproductive performance and pup development occurred at 1000 mg/kg/day, that also resulted in significant maternal clinical signs, significant inhibition of average maternal weight gain before mating and significant increases in maternal weight gain and feed consumption during gestation. In the absence of significant toxicity to the dams, B10 did not affect the reproductive performance or the developmental parameters of pups. The effects observed on reproduction and development are not, therefore, uniquely reprotoxic or developmentally toxic effects but general toxic effects.

The maternal and developmental NOAELs were established to be 500 mg/kg/day. This corresponds with a NOAEL of 365 mg linalool/kg bw/day. It can be concluded that linalool does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.

Executive summary:

A reproductive and developmental toxicity screening test (similar to OECD 421) was performed. Female rats were orally (gavage) administered 0, 250, 500 and 1000 mg/kg/day of coriander oil (containing 72.9% linalool). Males were excluded from the test system. Females were dosed throughout the 7-day premating period, mating, gestation and lactation (post-natal day 4). The rats were observed for clinical signs, weight, feed consumption and were necropsied and examined for gross lesions. Litter (F1) were examined for number, viability, weight, sex ratio and external morphology of the pups. Delivered pups were additionally examined for viability, clinical signs and body weight during a 4-day postparturition period.

Excess in salivation was noted in all groups being significant for middle and high dosage group. A significant number of rats in the 1000 mg/kg/day group had urine-stained abdominal fur during premating period and 1-2 rats in this group showed ataxia and/or decreased motor activity infrequently during the premating and/or gestation periods.

Biologically remarkable decreases in body weight gain and feed consumption occurred for the 1000 mg/kg/day dosage group rats during premating (significant after first dosage). During gestation, biologically remarkable increase in weight and feed consumption occurred for each group given the test article. Statistically significant increases in body weight gain occurred for the low and high dosage group rats and statistically significant increases in absolute and relative feed consumption values occurred for each group given the test article. These effects decreased in severity during lactation.

No adverse effects on mating, fertility or the durations of gestation or parturition occurred for female rats given dosages of linalool as high as 1000 mg/kg/day.

Pup mortality was significantly increased for litters of dams given 1000 mg/kg/day coriander oil. When comparing implantation averages to delivered litter size in the 1000 mg/kg/day, the litter size was more decreased as compared to other groups. This indicates more resorptions in the high dosage group.

The maternal NOEL for B10 was below 250 mg/kg/day, based on clinical signs, such as salivation and altered body weight gains and feed consumption. These changes were not considered to be evidence for strong toxicity, hence the NOAEL was set higher at 500 mg/kg/day. The NOEL for B10 was 500 mg/kg/day administered to dams. The highest-dosage (1000 mg/kg/day) group had reduced delivered litter sizes, indicating in utero deaths, and siginifcant incidences of pup mortality in the first four days postpartum. No adverse effects regarding mating, fertility or duration of gestation or parturition occurred in any treatment group including the high-dose at 1000 mg/kg/day. Clear adverse effects on reproductive performance and pup development occurred at 1000 mg/kg/day, that also resulted in significant maternal clinical signs, significant inhibition of average maternal weight gain before mating and significant increases in maternal weight gain and feed consumption during gestation. In the absence of significant toxicity to the dams, B10 did not affect the reproductive performance or the developmental parameters of pups. The effects observed on reproduction and development are not, therefore, uniquely reprotoxic or developmentally toxic effects but general toxic effects.

The maternal and developmental NOAELs were established to be 500 mg/kg/day. This corresponds with a NOAEL of 365 mg linalool/kg bw/day. It can be concluded that linalool does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

365 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

One reliable study (Klimisch 2) is available for this endpoint, which is considered the most relevant study with regard to the NOAEL. Three other studies (Klimisch 4; for another constituent) are available that support these findings. Therefore the quality of the database is considered sufficient to fill this endpoint.

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 repro-/developmental toxicity studies are available with Bergamot oil itself. As an alternative, a constituent approach was chosen based on the availability of repro-/developmental toxicity studies in different species for the major constituents of Bergamot oil: d-limonene, linalyl acetate and linalool. The selected study provides the most critical NOAEL for the reproduction endpoint.

Effects on developmental toxicity

Description of key information

Toxicity to reproduction:
- Reproscreening study in rats (similar to OECD421, read-across): NOAELmaternal and developmental = 365 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No repro-/developmental toxicity studies are available with Bergamot oil itself. As an alternative, a constituent approach was chosen based on the availability of repro-/developmental toxicity studies in different species for the major constituents of Bergamot oil: d-Limonene (CAS nr 5989-27-5), Linalyl acetate (CAS nr. 115-95-7) and Linalool (CAS nr. 78-70-6) . Using these data results in a coverage of >80% of the composition of Bergamot oil,which ensures that the same data requirements are covered as for monoconstituent substances.

Data for d-Limonene and Linalool are available and further summarized below. The data for Linalool also cover Linalyl acetate, as this substance is quickly metabolized to Linalool.

 

Selected limit value for derivation of the DNEL for Bergamot oil

The following NOAELs en LOAELs were available for the major constituents d-limonene and linalool.

Constituent	Study	Animal	Type of study	NOAEL (mg/kg bw/day)	LO(A)EL (mg/kg bw/day)	Critical effect LOAEL
d-Limonene	Kodama, 1977	Mice	developmental toxicity study	591 (maternal and fetal toxicity)	2363 (maternal and fetal toxicity)	Decreased bodyweight gain in dams, increased incidences of abnormal skeletal formation in fetuses and decreased body weight gain in male offspring.
	Tsuji, 1975	Rats	developmental toxicity study	591 (maternal and fetal toxicity)	2869 (maternal and fetal toxicity)	Maternal: deaths and decreased bodyweight gain Fetus: significantly delayed ossification, significantly decreased bodyweight gain (male offspring) and organ weights
	Kodama, 1977	Rabbits	developmental toxicity study	>1000 (fetal ) 250 (maternal)	NA (fetal) 1000 (maternal)	decreased bodyweight gain (maternal)
Linalool	Hoberman, 1989	Rats	reproductive and developmental toxicity screening test	365 (maternal and fetal toxicity)	729 (maternal and fetal toxicity)	Maternal:clinical signs Fetus: decreased litter size and increased pup mortality

Table 1. Relevant NOAELs/LOAELs for DNEL derivation.

The most critical NOAEL for repro-/developmental toxicity is 365 mg/kg bw/day, established in a reproductive and developmental toxicity screening test in rats with Coriander oil (containing 72.9% Linalool).

Repro-/developmental toxicity of d-Limonene

Three developmental toxicity studies were available for limonene. The studies were performed in different species (mice, rats and rabbits), and are described below.

Study in mice

In a prenatal developmental toxicity study (Kodama, 1977), d-Limonene was administered to groups of pregnant ICR mice (20 animals/dose: 15 animals for teratogenicity study, 5 animals for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day (via oral route) for 6 days during gestation day 7 to 12. A significant decrease in bodyweight gain was observed in pregnant mice administered with 2363 mg/kg bw/day, compared to controls. No abnormalities were observed in general behavior of dams during gestation. Significantly increased incidence of lumbar rib variations and fused ribs in fetuses administered with 2363 mg/kg bw/day, compared to controls. Examination of skeletal development in fetuses revealed significantly retarded ossification of proximal and middle phalanx of fore limb and significantly retarded ossification of hind limb metatarsal bone and proximal phalanx in the 2363 mg/kg bw/day dose group, compared to controls. These retarded ossifications were restored to normal during postnatal development. No significant skeletal malformations and variations were observed in mouse offspring. A significant decreased bodyweight gain was observed in male offspring of dams administered with 2363 mg/kg bw/day. No differences were observed in weaning rate, sensory function, organ weight and histological findings of testis and ovaries compared to controls. Under the test conditions, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on significantly decreased bodyweight gain in dams and significantly increased incidences of abnormal skeletal development in fetuses at 2363 mg/kg bw/day.

Study in rats

In the developmental toxicity study in rats (Tsuji, 1975), d-Limonene was administered to pregnant Wistar rats (20 animals/dose: 15 animals for teratogenicity study, 5 animals for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day (via oral route, suspended with 1% gum-arabic solution) for 7 days during gestation day 9-15. At 2869 mg/kg bw/day, maternal bodyweight decreased (significant at GD 16) and several mothers (40%) died during the treatment period. At the 591 mg/kg bw/day dose level no maternal effects were observed. Significantly delayed ossification of fetal metacarpal bone and proximal phalanx was found in the 2869 mg/kg bw/day dose group, compared to the control group. This effect was restored to normal within several weeks after birth. A decreased tendency of bodyweight was noted in postnatal male offspring of dams administered with 2869 mg/kg bw/day, compared to the control group. Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on maternal deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on significantly delayed ossification in fetuses, significantly decreased bodyweight gain (male offspring) and organ weights at 2869 mg/kg bw/day

Study in rabbits

In the developmental toxicity study with rabbits (Kodama, 1977), groups of pregnant Japanese white rabbits were administered d-Limonene at dose levels of 250, 500 and 1000 mg/kg bw/day (via oral route) for 13 days during gestation day 6 to 18. Maternal toxicity and fetal developmental effects were evaluated. Treatment with the highest dose level (1000 mg/kg bw/day) of d-limonene resulted in death of 6/18 dams (33% mortality). Body weight gain and food consumption were temporarily decreased in dams administered 500 and 1000 mg/kg. No abnormalities were observed in general behaviour of dams administered 250 and 500 mg/kg of d-limonene during gestation. External examination of fetuses showed no abnormalities. Visceral and skeletal examinations revealed some abnormalities in rabbit fetuses such as incomplete lobulation of the lungs, enlargement of the foramen ovale, retarded ossification of the middle phalanx of fore limbs and the 5th sternebrae. These effects did not appear to be significant and dose-dependent. No skeletal malformations were observed during postnatal examination of rabbit offspring. Under the test conditions, the NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on decreased bodyweight gain and food consumption and maternal death. The NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day.

Repro-/developmental toxicity of Linalool

A reproductive and developmental toxicity screening test (similar to OECD 421) was performed for Coriander oil containing 72.9% linalool (Hoberman, 1989). Coriander oil was adminsteredorally via gavage once daily to female rats at dosages of 0, 250, 500 or 1000 mg/kg/day. Females were dosed throughout the 7-day premating period and during mating, gestation, delivery and lactation until post-natal day 4. No maternal mortality was observed during the study. Excess in salivation was noted in all groups and was found significant in the middle and high dosage group compared to controls. A significant number of rats in the 1000 mg/kg/day group had urine-stained abdominal fur during the premating period and 1-2 rats in this group showed ataxia and/or decreased motor activity infrequently during the premating and/or gestation periods.

Decreases in body weight gain and feed consumption occurred for the 1000 mg/kg/day dosage group rats during premating with significant weight loss in this group after the first dosage. During gestation, an increase in weight gain and feed consumption occurred in each group given the test article. Statistically significant increases in body weight gain occurred for the low and high dosage group rats and statistically significant increases in absolute and relative feed consumption values occurred for each group given the test article, as compared with control animals. These effects remained present but decreased in severity during lactation.

No adverse effects on mating, fertility, implantation averages occurred in female rats at dosages of coriander oil up to 1000 mg/kg/day. Administration of 1000 mg/kg/day coriander oil to the dams resulted in decreased litter size and a statistically significant increase in pup mortality. This indicates a higher number of in utero deaths in the high dosage group. No effects on durations of gestation, pup sex ratios, pup body weights and pup gross morphology were observed.

 

Based on the results of the study, the maternal NOEL for coriander oil was below 250 mg/kg/day, based on clinical signs including salivation and altered body weights and feed consumption. These changes were not considered to be evidence for strong adverse toxicity, hence the NOAEL for maternal toxicity was set higher at 500 mg/kg/day. No adverse effects on mating, fertility, implantation averages, durations of gestation, pup sex ratios, pup body weights and pup gross morphology occurred in any treatment group including the highest-dose group that received 1000 mg/kg/day. Clear adverse effects on reproductive performance and pup development occurred at 1000 mg/kg/day, that also resulted in significant maternal clinical signs, significant inhibition of average maternal weight gain before mating and significant increases in maternal weight gain and feed consumption during gestation. In the absence of significant toxicity in the dams, Coriander oil did not affect the reproductive performance of dams or development of the offspring. The maternal and fetal NOAELs were established to be 500 mg/kg/day, based on maternal clinical signs, decreased litter size and increased pup mortality. This corresponds to a NOAEL of 365 mg Linalool/kg bw/day, based on 72.9% Linalool in Coriander oil. The maternal and fetal LOAELs were established to be 1000 mg/kg/day. This corresponds to a LOAEL of 729 mg Linalool/kg bw/day.

 

Justification for selection of Effect on developmental toxicity: via oral route:
No repro-/developmental toxicity studies are available with Bergamot oil itself. As an alternative, a constituent approach was chosen based on the availability of repro-/developmental toxicity studies in different species for the major constituents of Bergamot oil: d-limonene, linalyl acetate and linalool. The study selected under 'Effects on fertility' provides the most critical NOAEL for the reproduction endpoint.

Justification for classification or non-classification

Evaluation of toxicity to reproduction of Bergamot oil is based on the major constituents of Bergamot oil: d-Limonene, Linalyl acetate and Linalool in a constituent approach. The available reproductive and developmental toxicity screening test with coriander oil (containing 72.9% linalool) in rats provides the most critical NOAEL of 365 mg/kg bw/day.

As stated in the study with Coriander oil, no effects on reproductive performance of female rats or the development of offspring was observed in the absence of maternal toxicity. It was concluded that Coriander oil is not intrinsically hazardous to the reproductive performance of female rats or the development of offspring. As outlined in section 3.7.3.3.1 of 1272/2008/EC (CLP) 'Classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and substances shall not be so classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects’. 

Based on this criterium and given the evaluation of the other constituents, Bergamot oil does not have to be classified with regard to toxicity to reproduction.