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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1981 - November 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose for cross-reference:
read-across source
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs observed in the 750 and 1500 mg/kg bw dose group: hypoactivity, coat unkempt, piloerection, dehydration, excessive salivation, wet yellow stained anogenital region, impaired righting reflex, decreased grasping reflex, decreased limb tone, hypothermia, ataxia and prostration.
Mortality:
mortality observed, treatment-related
Description (incidence):
1500 mg/kg bw: all animals died during the first week.
750 mg/kg bw: 9/10 males and 10/10 females died before study termination.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights of the males rats in the 187 and 375 mg/kg bw dose groups were lower than the body weights of the control group 7 and 11%, respectively. Female body weights were not significantly different compared to the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- The relative liver weights of the males and females of the 93, 187, and 375 mg/kg bw dose groups were increased. These increases in weight were statistically significantly compared with controls (t-test, P< 0.05). The weight increase was dose-related and there was no dose without effect.
- The relative kidney weights of the males and females of the 375 mg/kg bw dose group were increased. Furthermore, the relative kidney weights of the males in the 187 dose group were increased. A slight, but not significant increase in kidney weight was observed in the kidneys of the females at 187 mg/kg bw/day and in both sexes at 93 mg/kg bw/day.
- The relative weight of the right testis and lung/bronchi of the males in the 187 and 375 mg/kg bw dose groups were increased.
- The relative brain weight of females of the 375 mg/kg bw dose group was decreased.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology was performed on vehicle controls, rats receiving 375 mg/kg bw test substance and all animals dying or killed before study termination.

- Kidney: 1500 mg/kg bw dose group: kidney degeneration was reported for 27 males. 750 mg/kg bw dose group: kidney degeneration was reported for 7 males. 375 mg/kg bw dose group: one male had renal dilatation and three has tubular protein casts in the kidney but otherwise no degenerative changes.

- Liver: 375 mg/kg bw dose group: in 3 males cytoplasmic vacuolation in the liver was observed. 1500 mg/kg bw dose group: similar changes in liver were observed. 750 mg/kg bw dose group: no changes in the liver were observed.

- Reproductive organs:
375 mg/kg bw dose group: There was depressed sperm motility and a mild decrease in sperm concentration, in the assessments made at the end of the study but not on any other occasion.
750 mg/kg bw dose group: Testicular degeneration was seen in this group. This was characterised by moderate to marked loss of germinal epithelium in the seminiferous tubules and an absence of the sperm in the epididymal ducts. Syncytial giant cells of the spermatids were present in the seminiferous tubules of some rats.
No effects were seen on testes or sperm parameters or any aspect of reproductive function at lower doses.

- Thymic cortex: In the male and female rats of the 750 mg/kg bw dose group, atrophy of the thymic cortex was observed.

- Stomach: 750 mg/kg bw dose group: Gastric mucosal ulceration involving both glandular and non-glandular regions was recorded at necropsy and confirmed by microscopic examination in 3 males and in 2 females.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
< 93 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
93 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
93 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney

Cited from EFSA Journal 2014;12(7):3806:

Liver data

Dose (mg/kg)

 

Liver/Bodyweight ratio (%)

Mean (SD)

Liver/Bodyweight ratio (%)

Median (Min, Max)

Dose group / control Means Difference (95% CI)

 

p-value *

 

 

Control - Males

3.50 (0.200)

3.42 ( 3.32, 3.89)

 

 

93 mg/kg - Males

3.99 (0.200)

3.98 ( 3.73, 4.31)

0.49 (0.251, 0.723)

<0.001

187 mg/kg - Males

4.13 (0.200)

4.10 ( 3.80, 4.54)

0.63 (0.393, 0.865)

<0.001

375 mg/kg - Males

4.86 (0.180)

4.83 ( 4.65, 5.19)

1.36 (1.119, 1.590)

<0.001

Control - Females

2.96 (0.170)

2.99 ( 2.68, 3.24)

 

 

93 mg/kg - Females

3.39 (0.240)

3.30 ( 3.17, 3.97)

0.43 (0.126, 0.726)

0.003

187 mg/kg - Females

3.47 (0.140)

3.43 ( 3.29, 3.75)

0.51 (0.210, 0.809)

<0.001

375 mg/kg - Females

4.19 (0.370)

4.07 ( 3.76, 4.89)

1.23 (0.927, 1.527)

<0.001

* Tukey’s multiple comparisons of means (compared to the control group)

Kidney data

Dose (mg/kg)

 

Kidney/Bodyweight ratio (%)

Mean (SD)

Kidney/Bodyweight ratio (%)

Median (Min, Max)

Dose group / control Means Difference (95% CI)

 

p-value *

 

 

Control - Males

0.31 (0.020)

0.30 ( 0.28, 0.34)

 

 

93 mg/kg - Males

0.32 (0.020)

0.32 ( 0.28, 0.34)

0.01 (-0.008, 0.033)

0.384

187 mg/kg - Males

0.34 (0.010)

0.33 ( 0.32, 0.36)

0.03 (0.009, 0.051)

0.002

375 mg/kg - Males

0.38 (0.010)

0.38 ( 0.36, 0.41)

0.07 (0.054, 0.096)

<0.001

Control - Females

0.32 (0.020)

0.32 ( 0.29, 0.35)

 

 

93 mg/kg - Females

0.34 (0.020)

0.33 ( 0.31, 0.38)

0.02 (-0.011, 0.044)

0.38

187 mg/kg - Females

0.34 (0.020)

0.34 ( 0.32, 0.37)

0.02 (-0.006, 0.048)

0.179

375 mg/kg - Females

0.36 (0.030)

0.37 ( 0.33, 0.43)

0.04 (0.017, 0.071)

<0.001

 

 

 

 

 

* Tukey’s multiple comparisons of means (compared to the control group)

 

Conclusions:
Under the conditions of the test the NOAEL was determined to be <93 mg/kg bw/day based on the decreased male body weights and increased liver and kidney weights in males as well as females.
Executive summary:

A 13 week repeated dose study was performed to determine the cumulative toxicity of d-carvone. F344/N rats were exposed to 0 (corn oil), 93, 187, 375, 750, and 1500 mg/kg bw. 10 animals per sex were dosed with 187 and 750 mg/kg bw d-carvone. For the remaining dose groups 30 animals per sex were used. Animals were dosed via oral gavage, 5 doses/week for 13 weeks. In the 1500 mg/kg bw dose group, all animals died during the first week. In the 750 mg/kg bw dose group, 9/10 males and 10/10 females died before study termination. Final mean body weights of the males rats in the 187 and 375 mg/kg bw dose groups were lower than the body weights of the control group 7 and 11%, respectively. Female body weights were not significantly different compared to the control group.  Effects on liver, kidney, testis, lung/bronchi and brain weight were observed. The relative liver weights of the males and females of the 93, 187, and 375 mg/kg bw dose groups were increased. These increases in weight were statistically significantly compared with controls (t-test, P< 0.05). The weight increase was dose-related and there was no dose without effect. The relative kidney weights of the males and females of the 375 mg/kg bw dose group were increased. Furthermore, the relative kidney weights of the males in the 187 dose group were increased. Under the conditions of the test the NOAEL was determined to be <93 mg/kg bw/day based on the decreased male body weights and increased liver and kidney weights in males as well as females.

Data source

Referenceopen allclose all

Reference Type:
review article or handbook
Title:
Initial risk assessment provided by the rapporteur Member State Sweden for the existing active substance SPEARMINT OIL of the fourth stage of the review programme referred to in Article 8(2) of Council Directive 91/414/EEC.
Author:
KEMI (kemikalieinspektionen, Swedisch Chemicals Agency)
Year:
2008
Bibliographic source:
DAR, Volume 3, Annex B, Part 2, B.6
Reference Type:
review article or handbook
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(S)-2-methyl-5-(1-methylvinyl)cyclohex-2-en-1-one
EC Number:
218-827-2
EC Name:
(S)-2-methyl-5-(1-methylvinyl)cyclohex-2-en-1-one
Cas Number:
2244-16-8
Molecular formula:
C10H14O
IUPAC Name:
(S)-2-methyl-5-(1-methylvinyl)cyclohex-2-en-1-one
Test material form:
liquid
Details on test material:
- Analytical purity: approximately 96%
Specific details on test material used for the study:
d-CARVONE
CAS NO.2244-16-8

Test animals

Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
F 344/N rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 - 5 weeks
- Diet: ad libitum
- Water: ad libitum
- Housing: 5 animals per cage

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Preparation: Appropriate weight of d-carvone was dissolved in corn oil to a specified volume in a graduated cylinder. Mixture was stored under nitrogen.
- Maximum Storage Time: 1 week
- Storage Conditions: Room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Because the feed blends of d-carvone were found to be unstable under the feed blending and simulated dosing conditions and because d-carvone is insoluble in water, corn oil gavage was selected as the route of administration for these studies.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies performed by high-performance liquid chromatography (Varian 5000) indicated that d-carvone was stable as a bulk chemical when stored in the dark in glass containers with Teflon-lined caps at temperatures up to 25° C for 2 weeks. Samples kept at 60° C had about 3% decomposition.

The stability of d-carvone in corn oil at 0.5% (5mg/g) stored at room temperature or at 5° C for 21 days was determined. The corn oil solutions were extracted with methanol and analyzed by high-performance liquid chromatography with a Brownlee RP-18 column and ultraviolet detection at 229 nm. The d-carvone corn oil solutions were found to be stable for at least 21 days when stored in the dark at room temperature or at 5"C. The corn oil solutions were also stable under simulated dosing conditions for at least 3 hours.

The appropriate amount of d-carvone was mixed (w/v) with corn oil to give the desired concentrations. Periodic analyses of formulated d-carvone corn oil dose mixtures were conducted at the study laboratory and the analytical chemistry laboratory. Dose mixtures were analyzed before the start of, and once during, the 13-week studies. During the 13-week studies, concentrations of d-carvone in corn oil were determined by gas chromatography with a 10% Carbowax column and flame ionization detection. During the 13-week studies, all dose mixtures were found to be within 10% of the target concentrations by the study laboratory. The referee laboratory analyzed one dose mixture.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 doses/ week for 13 weeks.
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: mg/kg bw
Dose / conc.:
93 other: mg/kg bw
Dose / conc.:
187 other: mg/kg bw
Dose / conc.:
375 other: mg/kg bw
Dose / conc.:
750 other: mg/kg bw
Dose / conc.:
1 500 other: mg/kg bw
No. of animals per sex per dose:
10 animals per sex per dose for the 187 and 750 mg/kg dose groups. 30 animals per sex per dose for the 0, 93, 375, and 1500 mg/kg dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on toxicity observed in the 16 day (12 doses) study with 0,150, 328, 723, 1590, and 3,500 mg/kg test substance.

Positive control:
no postive control included.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed two times per day.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded at the beginning of the study, once per week, and at the end of the study.

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No
Sacrifice and pathology:
HISTOPATHOLOGY: Necropsy was performed on all animals. Full macroscopic examination and weights of brain, lungs, heart, thymus, liver, right testis and right kidney recorded.
Other examinations:
Semen was collected from the animals in the 0, 93, and 375 mg/kg bw dose groups during week 2, 4, 6, and 8. Sperm concentration, motility and morphology were monitored. The same examination was conducted on all survivors at scheduled necropsy.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs observed in the 750 and 1500 mg/kg bw dose group: hypoactivity, coat unkempt, piloerection, dehydration, excessive salivation, wet yellow stained anogenital region, impaired righting reflex, decreased grasping reflex, decreased limb tone, hypothermia, ataxia and prostration.
Mortality:
mortality observed, treatment-related
Description (incidence):
1500 mg/kg bw: all animals died during the first week.
750 mg/kg bw: 9/10 males and 10/10 females died before study termination.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights of the males rats in the 187 and 375 mg/kg bw dose groups were lower than the body weights of the control group 7 and 11%, respectively. Female body weights were not significantly different compared to the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- The relative liver weights of the males and females of the 93, 187, and 375 mg/kg bw dose groups were increased. These increases in weight were statistically significantly compared with controls (t-test, P< 0.05). The weight increase was dose-related and there was no dose without effect.
- The relative kidney weights of the males and females of the 375 mg/kg bw dose group were increased. Furthermore, the relative kidney weights of the males in the 187 dose group were increased. A slight, but not significant increase in kidney weight was observed in the kidneys of the females at 187 mg/kg bw/day and in both sexes at 93 mg/kg bw/day.
- The relative weight of the right testis and lung/bronchi of the males in the 187 and 375 mg/kg bw dose groups were increased.
- The relative brain weight of females of the 375 mg/kg bw dose group was decreased.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology was performed on vehicle controls, rats receiving 375 mg/kg bw test substance and all animals dying or killed before study termination.

- Kidney: 1500 mg/kg bw dose group: kidney degeneration was reported for 27 males. 750 mg/kg bw dose group: kidney degeneration was reported for 7 males. 375 mg/kg bw dose group: one male had renal dilatation and three has tubular protein casts in the kidney but otherwise no degenerative changes.

- Liver: 375 mg/kg bw dose group: in 3 males cytoplasmic vacuolation in the liver was observed. 1500 mg/kg bw dose group: similar changes in liver were observed. 750 mg/kg bw dose group: no changes in the liver were observed.

- Reproductive organs:
375 mg/kg bw dose group: There was depressed sperm motility and a mild decrease in sperm concentration, in the assessments made at the end of the study but not on any other occasion.
750 mg/kg bw dose group: Testicular degeneration was seen in this group. This was characterised by moderate to marked loss of germinal epithelium in the seminiferous tubules and an absence of the sperm in the epididymal ducts. Syncytial giant cells of the spermatids were present in the seminiferous tubules of some rats.
No effects were seen on testes or sperm parameters or any aspect of reproductive function at lower doses.

- Thymic cortex: In the male and female rats of the 750 mg/kg bw dose group, atrophy of the thymic cortex was observed.

- Stomach: 750 mg/kg bw dose group: Gastric mucosal ulceration involving both glandular and non-glandular regions was recorded at necropsy and confirmed by microscopic examination in 3 males and in 2 females.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
< 93 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
93 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
93 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Cited from EFSA Journal 2014;12(7):3806:

Liver data

Dose (mg/kg)

 

Liver/Bodyweight ratio (%)

Mean (SD)

Liver/Bodyweight ratio (%)

Median (Min, Max)

Dose group / control Means Difference (95% CI)

 

p-value *

 

 

Control - Males

3.50 (0.200)

3.42 ( 3.32, 3.89)

 

 

93 mg/kg - Males

3.99 (0.200)

3.98 ( 3.73, 4.31)

0.49 (0.251, 0.723)

<0.001

187 mg/kg - Males

4.13 (0.200)

4.10 ( 3.80, 4.54)

0.63 (0.393, 0.865)

<0.001

375 mg/kg - Males

4.86 (0.180)

4.83 ( 4.65, 5.19)

1.36 (1.119, 1.590)

<0.001

Control - Females

2.96 (0.170)

2.99 ( 2.68, 3.24)

 

 

93 mg/kg - Females

3.39 (0.240)

3.30 ( 3.17, 3.97)

0.43 (0.126, 0.726)

0.003

187 mg/kg - Females

3.47 (0.140)

3.43 ( 3.29, 3.75)

0.51 (0.210, 0.809)

<0.001

375 mg/kg - Females

4.19 (0.370)

4.07 ( 3.76, 4.89)

1.23 (0.927, 1.527)

<0.001

* Tukey’s multiple comparisons of means (compared to the control group)

Kidney data

Dose (mg/kg)

 

Kidney/Bodyweight ratio (%)

Mean (SD)

Kidney/Bodyweight ratio (%)

Median (Min, Max)

Dose group / control Means Difference (95% CI)

 

p-value *

 

 

Control - Males

0.31 (0.020)

0.30 ( 0.28, 0.34)

 

 

93 mg/kg - Males

0.32 (0.020)

0.32 ( 0.28, 0.34)

0.01 (-0.008, 0.033)

0.384

187 mg/kg - Males

0.34 (0.010)

0.33 ( 0.32, 0.36)

0.03 (0.009, 0.051)

0.002

375 mg/kg - Males

0.38 (0.010)

0.38 ( 0.36, 0.41)

0.07 (0.054, 0.096)

<0.001

Control - Females

0.32 (0.020)

0.32 ( 0.29, 0.35)

 

 

93 mg/kg - Females

0.34 (0.020)

0.33 ( 0.31, 0.38)

0.02 (-0.011, 0.044)

0.38

187 mg/kg - Females

0.34 (0.020)

0.34 ( 0.32, 0.37)

0.02 (-0.006, 0.048)

0.179

375 mg/kg - Females

0.36 (0.030)

0.37 ( 0.33, 0.43)

0.04 (0.017, 0.071)

<0.001

 

 

 

 

 

* Tukey’s multiple comparisons of means (compared to the control group)

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test the NOAEL was determined to be <93 mg/kg bw/day based on the decreased male body weights and increased liver and kidney weights in males as well as females.
Executive summary:

A 13 week repeated dose study was performed to determine the cumulative toxicity of d-carvone. F344/N rats were exposed to 0 (corn oil), 93, 187, 375, 750, and 1500 mg/kg bw. 10 animals per sex were dosed with 187 and 750 mg/kg bw d-carvone. For the remaining dose groups 30 animals per sex were used. Animals were dosed via oral gavage, 5 doses/week for 13 weeks. In the 1500 mg/kg bw dose group, all animals died during the first week. In the 750 mg/kg bw dose group, 9/10 males and 10/10 females died before study termination. Final mean body weights of the males rats in the 187 and 375 mg/kg bw dose groups were lower than the body weights of the control group 7 and 11%, respectively. Female body weights were not significantly different compared to the control group.  Effects on liver, kidney, testis, lung/bronchi and brain weight were observed. The relative liver weights of the males and females of the 93, 187, and 375 mg/kg bw dose groups were increased. These increases in weight were statistically significantly compared with controls (t-test, P< 0.05). The weight increase was dose-related and there was no dose without effect. The relative kidney weights of the males and females of the 375 mg/kg bw dose group were increased. Furthermore, the relative kidney weights of the males in the 187 dose group were increased. Under the conditions of the test the NOAEL was determined to be <93 mg/kg bw/day based on the decreased male body weights and increased liver and kidney weights in males as well as females.