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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data from secondary source

Data source

Referenceopen allclose all

Reference Type:
secondary source
Title:
Scientific Opinion on the safety assessment of carvone, considering all sources of exposure
Author:
EFSA Scientific Committee / European Food Safety Authority (EFSA)
Year:
2014
Bibliographic source:
EFSA Journal 2014;12(7):3806
Reference Type:
secondary source
Title:
Proposal for Harmonised Classification and Labelling – Carvone
Author:
European Chemicals Agency (ECHA)
Year:
2012
Bibliographic source:
Proposal for Harmonised Classification and Labelling – Carvone,2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
2-Generation reproduction study of d-carvone was performed on Wistar Crl : (WI) BR rats.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: D-Carvone- IUPAC name: (5S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one- Molecular formula: C10H14O- Molecular weight: 150.22 g/mol- Substance type: Organic
Specific details on test material used for the study:
- Name of test material: d-Carvone- IUPAC name: (5S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one- Molecular formula: C10H14O- Molecular weight: 150.22 g/mole- Smiles :C1[C@H](CC=C(C1=O)C)C(C)=C- Inchl: 1S/C10H14O/c1-7(2)9-5-4-8(3)10(11)6-9/h4,9H,1,5-6H2,2-3H3/t9-/m0/s1- Substance type: Organic- Physical state: Colorless to pale-yellow liquid

Test animals

Species:
rat
Strain:
other: Wistar Crl : (WI) BR rats
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Details on exposurePREPARATION OF DOSING SOLUTIONS:The test material diluted with corn oil.DIET PREPARATION- Rate of preparation of diet (frequency):No data available- Mixing appropriate amounts with (Type of food )- Storage temperature of food: No data availableVEHICLE- Justification for use and choice of vehicle (if other than water): test material mixed with corn oil as it is not soluble in water - Concentration in vehicle: f0: 0, 3, 10 or 30 mg/kg/dayF1: 0, 10, 30 and 90 mg/kg/day.- Amount of vehicle (if gavage): No data available- Lot/batch no. (if required): No data available- Purity: No data available
Details on mating procedure:
- M/F ratio per cage:1:1 - Length of cohabitation: - Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy - After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. - Further matings after two unsuccessful attempts: [no / yes (explain)] - After successful mating each pregnant female was caged (how): - Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.for 10 weeks - Selection of parents from F1 generation when pups were [...] days of age. - Age at mating of the mated animals in the study: [...] weeks
Doses / concentrations
Remarks:
F0: 0, 3, 10 or 30 mg/kg/dayF1: 0, 10, 30 and 90 mg/kg/day.
No. of animals per sex per dose:
Total:2000 mg/kg bw/day:25 male and 25 female 3 mg/kg bw/day:25 male and 25 female10 mg/kg bw/day:25 male and 25 female30 mg/kg bw/day:25 male and 25 female
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: daily - Cage side observations checked in table [No.?] were included. DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: daily BODY WEIGHT: Yes - Time schedule for examinations:weekly FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly - 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 - Time schedule for examinations: OTHER:
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:sperm was assessed for motility and morphology plus one testis and epididymis was frozen for enumeration of homogenisation-resistant spermatids and cauda epididymal sperm reserves.
Litter observations:
STANDARDISATION OF LITTERS- Performed on day 4 postpartum: yes- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. PARAMETERS EXAMINEDThe following parameters were examined in [F1 ] offspring:Each litter was examined for number of live and dead pups as soon as possible after birth. The individual weights of all live pups were recorded at day 1, 4, 7, 14 and 21. The sex of all pups was recorded, as was the number with any physical or behavioural abnormality. For the F1 weanlings selected for mating the day of vaginal opening or balanopreputial separation was recordedGROSS EXAMINATION OF DEAD PUPS:[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Postmortem examinations (parental animals):
SACRIFICE - Male animals:. F0 males were killed as soon as possible after successful mating while F1 males were only killed after successful delivery of the dams - Maternal animals: Females from both generations were killed at day 21 post partum. GROSS NECROPSY: yes All parental animals were subject to a necropsy and some tissues weighed (adrenals, brain, epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus with oviducts and cervix) also a range of tissues (adrenals, brain, coagulating glands, epididymides, identification marks, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus with oviducts and cervix, vagina) preserved for histological examination. HISTOPATHOLOGY / ORGAN WEIGHTS The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICEAll pups not selected for mating were taken for necropsy at day 21 post partum or shortly thereafter; organ weights were recorded for brain, spleen and thymus. -
Reproductive indices:
Reproductive parameters recorded were percentage mating, fertility index, conception rate, gestation index, duration of gestation
Offspring viability indices:
percentage live offspring by sex, percentage of post-natal loss, percentage of breeding loss, percentage live at weaning by sex, viability index, weaning index.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
Animals were found dead or killed in extremis due to bad health or delivery difficulties. The deaths are not considered to be treatment-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The reduction in body weight and body weight gain in females of the F0 generation treated with 30 mg/kg bw/day is considered not oftoxicological significance
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Males of the F0 generation given 30 mg/kg bw/day had increased relative kidney weights
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology of the male kidneys showed changes typical of 2u -globulin nephropathy at all doses. No other histopathological changes were reported and no similar changes were observed in females
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no differences between treated and control animals in the results of the sperm morphology and motility measurements
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no differences between treated and control animals in any of the indices of reproductive performance

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: No effects on systemic toxicity
Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (estrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: overall reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Females of the 90 mg/kg bw/day group had in increased body weight and body weight gain. The study authors considers this finding not toxicologically relevant
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males of the F1 generation given 30 and 90 mg/kg bw/day had increased mean relative liver and kidney weights.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology of the male kidneys showed changes typical of 2u -globulin nephropathy at all doses. No differences were seen in females and no other histopathological changes were reported. .
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Reproductive performance:
no effects observed

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (estrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: overall no reproductive toxic effects was observed

Target system / organ toxicity (P1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
no toxicologically relevant effect
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Gross pathological findings:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: No overall developmental toxic effects was observed

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Gross pathological findings:
no effects observed
Description (incidence and severity):
no toxicologically relevant effect
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not specified

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
90 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
viability
sexual maturation
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: no overall developmental toxic effects was observed

Target system / organ toxicity (F2)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Summary table of the 2 generation study

 

Dose (mg/kg bw/day)

0

 

3

 

10

 

30

 

90

 

dr

 

Sex

m

f

m

f

m

f

m

f

m

f

 

F0

Mortality  A

2

1

 

1

 

1

 

1

 

 

 

 

Clinical signs

no toxicologically relevant effect

 

 

 

 

 

Body weight (gain)B

 

 

 

 

 

 

 

ds

 

 

 

 

Food consumptionC

 

 

 

 

 

 

 

ds

 

 

 

 

Oestrus cycle

no toxicologically relevant effect

 

 

 

 

 

Sperm parameters

no toxicologically relevant effect

 

 

 

 

 

Organ weights

-kidney

-thyroids

 

 

 

 

 

 

Isr

 

Isr

 

 

 

 

 

 

Pathology

no toxicologically relevant effect

 

 

 

 

- macroscopy

no toxicologically relevant effect

 

 

 

 

- microscopyD

no toxicologically relevant effect

 

 

 

F1 pups

Litter size

no toxicologically relevant effect

 

 

 

 

Survival index

no toxicologically relevant effect

 

 

 

 

Sex ratio

no toxicologically relevant effect

 

 

 

 

Body weight

no toxicologically relevant effect

 

 

 

 

Organ weight

no toxicologically relevant effect

 

 

 

 

Pathology

 

 

 

 

 

- macroscopy

no toxicologically relevant effect

 

 

 

 

- microscopy

(weanlings)

not performed

 

 

 

F1

animals

MortalityA

 

 

1

 

 

 

1

 

2

 

1

 

 

Clinical signs

 

 

 

 

no toxicologically relevant effect

 

 

Body weightE

 

 

 

 

no toxicologically relevant effect

 

 

Food consumption

 

 

 

 

no toxicologically relevant effect

 

 

Mating, fertility,

gestation

 

 

 

 

no toxicologically relevant effect

 

 

Oestrus cycle

 

 

 

 

no toxicologically relevant effect

 

 

Sperm parameters

 

 

 

 

no toxicologically relevant effect

 

 

Organ weights

-liver

-kidney

 

 

 

 

 

 

isr

isar

 

isr

isar

 

dr

dr

 

Pathology

 

 

 

 

 

 

 

 

- macroscopy

 

 

 

 

no toxicologically relevant effect

 

 

- microscopyD

 

 

 

 

no toxicologically relevant effect

 

F2 pups

Litter size

 

 

 

 

no toxicologically relevant effect

 

 

Survival index

 

 

 

 

no toxicologically relevant effect

 

 

Sex ratio

 

 

 

 

no toxicologically relevant effect

 

 

Body weight

 

 

 

 

no toxicologically relevant effect

 

 

Organ weight

 

 

 

 

no toxicologically relevant effect

 

 

Pathology

 

 

 

 

no toxicologically relevant effect

 

 

- macroscopy

 

 

 

 

no toxicologically relevant effect

 

 

- microscopy

(weanlings)

 

 

 

 

not performed

 

dr = dose related; i = increased; d = decreased; is = increased significantly; ds = decreased significantly, a= absolute, r=relative

A Animals were found dead or killed in extremis due to bad health or delivery difficulties. The deaths are not considered to be treatment-related.

B Females of the 30 mg/kg bw/day group showed small but significant decreases in body weight gain during lactation on days

4, 7 and 14, and body weight loss on day 4 during lactation.

C Females of the 30 mg/kg bw/day group showed a significant decrease in food consumption from day 1-4 during lactation.

D Males of all treated groups showed histopathological changes in the kidney consistent with accumulation ofα2u-globulin.

E Females of the 90 mg/kg bw/day group had in increased body weight and body weight gain. The study authors consider this finding not toxicologically relevant. The present reviewers endorse this view

Applicant's summary and conclusion

Conclusions:
The NOAEL for systemic toxicity is 30 mg/kg bw/day. No effects on reproductive parameters were observed. Based on this the NOAEL for reproductive effects for treatment of two generations of rats is 90 mg/kg bw/day. When male and female Wistar Crl : (WI) BR rats were treated with d-carvone (2244-16-8) orally.
Executive summary:

2-Generation reproduction study of d-carvone(2244-16-8) was performed on male and female Wistar Crl : (WI) BR rat according to GLP and to OECD Guideline 416 (OECD, 2001). The test material soluble in corn oil in dose concentration 0, 3, 10 or 30 mg/kg/day and administered by oral gavage for 10 weeks prior to mating until termination.Groups of 25 male and 25 female per dose group were used in F0 generation.For the F1 generation the dose levels were revised to 0, 10, 30 and 90 mg/kg/day. Females were mated individually and remained with the litters during weaning. Pups were not treated during the weaning period. On day 4 after birth all litters of more than 8 pups were culled to approximately 4 males and 4 females. After weaning of the F1 generation, animals were selected and again treated for 10 weeks prior to mating and up to termination. F0 males were killed as soon as possible after successful mating while F1 males were only killed after successful delivery of the dams. Females from both generations were killed at day 21 post partum. All animals were checked at least once daily for clinical signs and condition. Body weight and food consumption measurements were recorded weekly. All parental animals were subject to a necropsy and some tissues weighed (adrenals, brain, epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus with oviducts and cervix) also a range of tissues (adrenals, brain, coagulating glands, epididymides, identification marks, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroid, uterus with oviducts and cervix, vagina) preserved for histological examination. All pups not selected for mating were taken for necropsy at day 21 post partum or shortly thereafter; organ weights were recorded for brain, spleen and thymus.

For males surviving to scheduled necropsy, sperm was assessed for motility and morphology plus one testis and epididymis was frozen for enumeration of homogenisation-resistant spermatids and cauda epididymal sperm reserves. For females, the ovaries and uterus were examined prior to fixation and the number of corpora lutea and implantation sites recorded. Each litter was examined for number of live and dead pups as soon as possible after birth. The individual weights of all live pups were recorded at day 1, 4, 7, 14 and 21. The sex of all pups was recorded, as was the number with any physical or behavioural abnormality. For the F1 weanlings selected for mating the day of vaginal opening or balanopreputial separation was recorded. Reproductive parameters recorded were percentage mating, fertility index, conception rate, gestation index, duration of gestation, percentage live offspring by sex, percentage of post-natal loss, percentage of breeding loss, percentage live at weaning by sex, viability index, weaning index. 

There were no differences between treated and control animals in any of the indices of reproductive performance or in the results of the sperm morphology and motility measurements. Males of the F0 generation given 30 mg/kg bw/day had increased relative kidney weights. Histopathology of the male kidneys showed changes typical of 2u -globulin nephropathy at all doses. No other histopathological changes were reported and no similar changes were observed in females. Males of the F1 generation given 30 and 90 mg/kg bw/day had increased mean relative liver and kidney weights. Histopathology of the male kidneys showed changes typical of 2u -globulin nephropathy at all doses. No differences were seen in females and no other histopathological changes were reported.Hence The NOAEL for systemic toxicity is 30 mg/kg bw/day. No effects on reproductive parameters were observed. Based on this the NOAEL for reproductive effects for treatment of two generations of rats is 90 mg/kg bw/day.When male and femaleWistar Crl : (WI) BR ratswere treated with d-carvone(2244-16-8)orally.