Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

Endpoint summary

Description of key information

A 13 week repeated dose study was performed to determine the cumulative toxicity of d-carvone in F344/N rats: 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 (NTP, 1982).

A 90-day repeated dose toxicity test was performed with d-carvone according to OECD 408 and compliant with GLP in Wistar Crl : (WI) BR rats. The NOAEL is 5 mg/kg bw/day as a result of significant increase of the relative liver and kidney weight (Schoenmaker, 1996)(Woutersen & Bos-Kuijpers, 2003).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

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

Specific details on test material used for the study:

d-CARVONE
CAS NO.2244-16-8

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

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.

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.

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.

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

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

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The histopathological results were re-evaluated by Woutersen & Bos-kuijpers, 2003.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Carvone

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Crl : (WI) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
not specified

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
not specified

DIET PREPARATION
not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

not specified

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a 14-day range finding study with 0, 50, 200 and 1000 mg/kg bw/day test substance

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: condition and behaviour were monitored daily.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopy was carried out prior to treatment and at the end of the study on all rats.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study blood samples were taken from the retro-orbital sinus.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked :RBC, Hb, HCT, MCV, MCHb, MCHC, Platelet count, Red cell distribution width, WBC, Differential WBC, PT and PTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study blood samples were taken from the retro-orbital sinus.
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked: Serum separated from the same samples was examined for ALAT, ASAT, bilirubin, creatinine, glucose, urea, total protein, albumin, ALP, Na, K, Cl, Ca and P.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the study all rats were subject to a necropsy including gross examination and weighing of adrenals, brain, heart, kidneys, liver, ovaries, prostate, spleen, testes and thymus. Samples of a full range of tissues were preserved for microscopic examination.
HISTOPATHOLOGY: Yes.
Slides were prepared from all tissues of the control and high dose groups and examined microscopically. Based on the findings of this examination kidneys were examined from the remaining two treatment groups.

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

No mortality occurred.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

180 mg/kg bw/day: Slightly reduced body weight gain of males this was matched by a reduced food intake.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

180 mg/kg bw/day dose group: a reduced food intake was observed.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

180 mg/kg bw/day dose group: Reduction of prothrombin time (PT) in males and a significant increase in partial thromboplastin time (PTT) for females was observed.
(since reduction of prothrombin time (PT) was present in the results of the 14-day study this may not be a chance finding)

Description (incidence and severity):

180 mg/kg bw/day dose group: Males had a significant reduction in ASAT and a reduction was also seen in females however the difference was not significant.
Apart from a non-dose-related reduction (approx 10 %) in serum calcium in all treated female groups there were no treatment-related differences between the groups in the results of clinical chemistry analyses.

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):

180 and 30 mg/kg bw/day dose groups:
Liver: The relative liver weights of both males and females were significantly higher than those of controls at the 180 mg/kg bw/day dose. Furthermore, relative liver weights of the females dosed with 30 mg/kg bw/day were al significantly higher than the controls.

Kidneys: Mean relative kidney weights were significantly increased compared with controls for both sexes at the 180 and 30 mg/kg bw/day dose groups.

Thymus and spleen: Although some statistically significant differences were seen between controls and treated animals in thymus and spleen weights these were confined to females and were not dose-related and are thus considered unlikely to be due to treatment.

Gross pathological findings:

not specified

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed no histopathological changes related to treatment apart from those in the kidney which consisted of tubular necrosis, exclusively in male rats, and basophilic tubules in both sexes.
A re-evaluation of the kidney slides was performed by Woutersen and Bos-Kuijpers, 2003.
Kidney slides from both sexes for all groups were re-evaluated:
The re-evaluation revealed slight to severe tubular changes in the kidneys of the males rats dosed with 30 and 180 mg/kg bw/day carvone. The observed changes were: severe proteinaceous (hyalin) dropletswithin the proximal tubular cells, accompanied by epithelial cell necrosis and the occurrence of granular casts in the outer medulla, and proteinaceous casts and regenerating tubules.

Furthermore, staining with antibody against α2u-globulin revealed by accumulation of α2u -globulin in the proximal tubular cells of the males dosed with 180 mg/kg bw/day. It was concluded that the renal histopathological changes in the kidney of male rats dosed with 30 and 180 mg/kg bw/day are the result of accumulation of α2u-globulin in the proximal tubular cells, α2u-globulin nephropathy. α2u -globulin is not present in higher mammals, therefore the α2u-globulin-related effects are not considered relevant for man.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

LOAEL

Effect level:

30 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

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No substance related adverse effects were observed.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 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:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

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	2.95 (0.300)	2.95 ( 2.39, 3.60)
5 mg/kg - Males	2.83 (0.110)	2.81 ( 2.63, 3.00)	-0.12 (-0.407, 0.161)	0.652
30 mg/kg - Males	2.94 (0.270)	2.93 ( 2.58, 3.38)	-0.01 (-0.299, 0.270)	0.999
180 mg/kg - Males	3.24 (0.220)	3.19 ( 2.97, 3.67)	0.29 (0.005, 0.573)	0.045
Control - Females	2.75 (0.220)	2.70 ( 2.33, 3.10)
5 mg/kg - Females	2.78 (0.260)	2.72 ( 2.55, 3.40)	0.03 (-0.251, 0.324)	0.986
30 mg/kg - Females	3.09 (0.280)	3.05 ( 2.59, 3.49)	0.34 (0.054, 0.629)	0.015
180 mg/kg - Females	3.23 (0.190)	3.22 ( 2.98, 3.54)	0.48 (0.197, 0.773)	<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	0.58 (0.050)	0.57 ( 0.52, 0.67)
5 mg/kg - Males	0.60 (0.050)	0.59 ( 0.52, 0.67)	0.02 (-0.067, 0.108)	0.921
30 mg/kg - Males	0.66 (0.050)	0.66 ( 0.59, 0.76)	0.08 (-0.000, 0.175)	0.051
180 mg/kg - Males	0.87 (0.110)	0.88 ( 0.64, 1.06)	0.29 (0.205, 0.381)	<0.001
Control - Females	0.56 (0.040)	0.56 ( 0.51, 0.60)
5 mg/kg - Females	0.57 (0.050)	0.55 ( 0.52, 0.65)	0.01 (-0.060, 0.074)	0.993
30 mg/kg - Females	0.64 (0.060)	0.66 ( 0.54, 0.72)	0.08 (0.016, 0.150)	0.01
180 mg/kg - Females	0.68 (0.070)	0.67 ( 0.59, 0.80)	0.12 (0.050, 0.184)	<0.001

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

Kidney and liver data (summary obtained from Proposal for Harmonised Classification and Labelling by National Institute for Public Health and the Environment (RIVM) 2012 version 3.)

Dose groups mg/kg bw/day	0		5		30		180		Dose related
	m	f	m	f	m	f	m	f
Organ Weights relative
-liver	2.95	2.75	2.83	2.78	2.94	3.09 is	3.24 is	3.23 is	dr
-kidneys	0.58	0.56	0.60	0.57	0.66 is	0.64 is	0.87 is	0.68 is	dr
Macroscopy
-Kidney enlarged c	0/10	0/10	0/10	0/10	0/10	0/10	8/10	0/10
Microscopy Kidney
basophilic tubules	5/10	0/10	6/10	0/10	1/10	1/10	0/10	1/10	dr
tubular necrosis	0/10	0/10	0/10	0/10	8/10	0/10	10/10	1/10

is/ds = increased/decreased

c = Besides enlargement, kidneys of the high dose group were discoloured, pale, and granular.

Conclusions:

Under the conditions of the test the NOAEL is 5 mg/kg bw/day as a result of significant increase of the relative liver and kidney weight.

Executive summary:

A 90-day repeated dose toxicity test was performed according to OECD 408 and compliant with GLP. 10 Wistar Crl : (WI) BR rats per sex, per dose, were exposed to carvone via oral gavage. The animals were dosed with 0 (corn oil), 5, 30, and 180 mg/kg bw/day test substance. No mortality occurred during the study. Although some statistically significant differences were seen between controls and treated animals in thymus and spleen weights these were confined to females and were not dose-related and are thus considered unlikely to be due to treatment. The relative liver weights of both males and females were significantly higher than those of controls at the 180 mg/kg bw/day dose. Furthermore, relative liver weights of the females dosed with 30 mg/kg bw/day were al significantly higher than the controls. Mean relative kidney weights were significantly increased compared with controls for both males and females at the 180 and 30 mg/kg bw/day dose groups. Microscopic examination revealed no histopathological changes related to treatment apart from those in the kidney which consisted of tubular necrosis, exclusively in male rats, and basophilic tubules in both sexes.

A re-evaluation of the kidney slides was performed by Woutersen and Bos-Kuijpers, 2003. Kidney slides from both sexes for all groups were re-evaluated. The re-evaluation revealed slight to severe tubular changes in the kidneys of the males rats dosed with 30 and 180 mg/kg bw/day carvone. The observed changes were: severe proteinaceous (hyalin) droplets within the proximal tubular cells, accompanied by epithelial cell necrosis and the occurrence of granular casts in the outer medulla, and proteinaceous casts and regenerating tubules. Furthermore, staining with antibody against α2u-globulin revealed by accumulation of α2u -globulin in the proximal tubular cells of the males dosed with 180 mg/kg bw/day. It was concluded that the renal histopathological changes in the kidney of male rats dosed with 30 and 180 mg/kg bw/day are the result of accumulation of α2u-globulin in the proximal tubular cells, α2u-globulin nephropathy. α2u -globulin is not present in higher mammals, therefore the α2u-globulin-related effects are not considered relevant for man. Under the conditions of the test the NOAEL is 5 mg/kg bw/day, as a result of significant and dose related increase of the relative liver and kidney weight in the 30 and 180 mg/kg bw/day dose groups.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

BMDL10

60 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The 90 days study was performed according to OECD 408 and in compliance with GLP (Schoenmaker, 1996). The histopathological results were re-evaluated by Woutersen & Bos-kuijpers, 2003.

The 13-week study was obtained from a reliable source (NTP) and was peer reviewed. Although the 2-year study in rats was preliminary terminated, the National Toxicology Program Board of Scientific Counselors’ Technical Reports Review Subcommittee and associated Panel of Experts concluded that the results obtained with the 13-week study in rats were considered valid (NTP, 1982)

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Both d-carvone and d-limonene resulted in test substance related effects in the male kidneys. The histopathological examination of the kidneys of male rats showed α2µ-globulin in the proximal tubular cells, α2µ-globulin nephropathy. α2µ -globulin is not present in higher mammals, therefore the α2µ-globulin-related effects on the kidneys are not considered relevant for man.

Additional information

BMDL10

Spearmint oil contains 55-85% l-carvone and 0.01-24.9% l-limonene. Since spearmint oil contains more l-carvone and because the obtained data with the read across substance d-carvone are more conservative compared the results obtained with read across substance d-limonene, the endpoint conclusion was based on d-carvone.

 

EFSA Journal, 2014:

The Scientific Committee concluded that, although a BMDL10 based on the result of the relative kidney weights was the most conservative approach, the quality of the data is poor and the confidence interval of a BMDL10 based on the kidney weights would be wide. The committee therefore determined a BMDL10 of 60 mg/kg bw/day for an increase in relative liver weight in the rat based on the relative liver weight in the 90-day studies with d-carvone (NTP, 1982 and Schoenmaker, 1996). With the Hill model, the BMD10 was determined to be 82.3 mg/kg bw/day, with a BMDL10-BMDU10 interval of 60-108.2 mg/kg bw/day. The Scientific Committee selected the lowest BMDL10 (60 mg/kg bw/day) as the reference point for relative liver weight. This BMDL10 was also selected for the endpoint conclusion on spearmint oil.

Schoenmaker, 1995

A 90-day repeated dose toxicity test was performed according to OECD 408 and compliant with GLP. 10 Wistar Crl : (WI) BR rats per sex, per dose, were exposed to Carvone via oral gavage. The animals were dosed with 0 (corn oil), 5, 30, and 180 mg/kg bw/day test substance. No mortality occurred during the study. Although some statistically significant differences were seen between controls and treated animals in thymus and spleen weights these were confined to females and were not dose-related and are thus considered unlikely to be due to treatment. The relative liver weights of both males and females were significantly higher than those of controls at the 180 mg/kg bw/day dose. Furthermore, relative liver weights of the females dosed with 30 mg/kg bw/day were al significantly higher than the controls. Mean relative kidney weights were significantly increased compared with controls for both males and females at the 180 and 30 mg/kg bw/day dose groups. Microscopic examination revealed no histopathological changes related to treatment apart from those in the kidney which consisted of tubular necrosis, exclusively in male rats, and basophilic tubules in both sexes.

A re-evaluation of the kidney slides was performed by Woutersen and Bos-Kuijpers, 2003. Kidney slides from both sexes for all groups were re-evaluated. The re-evaluation revealed slight to severe tubular changes in the kidneys of the males rats dosed with 30 and 180 mg/kg bw/day carvone. The observed changes were: severe proteinaceous (hyalin) droplets within the proximal tubular cells, accompanied by epithelial cell necrosis and the occurrence of granular casts in the outer medulla, and proteinaceous casts and regenerating tubules. Furthermore, staining with antibody against α2u-globulin revealed by accumulation of α2u -globulin in the proximal tubular cells of the males dosed with 180 mg/kg bw/day. It was concluded that the renal histopathological changes in the kidney of male rats dosed with 30 and 180 mg/kg bw/day are the result of accumulation of α2u-globulin in the proximal tubular cells, α2u-globulin nephropathy. α2u -globulin is not present in higher mammals, therefore the α2u-globulin-related effects are not considered relevant for man. Under the conditions of the test the NOAEL is 5 mg/kg bw/day, as a result of significant and dose related increase of the relative liver and kidney weight in the 30 and 180 mg/kg bw/day dose groups.

NTP, 1982

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.

Justification for classification or non-classification

The increased liver and kidney weight in rats dosed with 30 mg/kg bw/day d-carvone, were considered toxicologically relevant. However, no histopathological changes were observed except for tubular necrosis in male rats (caused by accumulation of α2u-globulin and not relevant for man). There is no indication of serious organ dysfunction as a result of d-carvone and the effects observed are not severe enough for classification STOT RE2.

 

Based on a constituent approach with d-carvone it is concluded that spearmint oil is not classified for specific target organ toxicity after repeated exposure, according to Regulation 1272/2008/EC (CLP).