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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
L-Carvone and D-Carvone, as enantiomers, are structurally similar compounds with the same molecular weight and molecular formula. The only structural difference is the opposite specific rotation. For example, the chirality is known to influence what olfactory receptors that L- and D-carvone interact with giving different odours/tastes. However, based on the available data, there is no information indicating that one of the isomers is clearly more toxic than the other (see 5. Data matrix). In addition, L-Carvone has a long history of safe use as a flavoring in a variety of foods and beverages, as well as in toothpaste, mouth wash and air fresheners (WHO, 1999). No reliable data on the repeated dose toxicity of L-Carvone is available. The enantiomer D-Carvone has a NOAEL of 375 mg/kg bw/day in a 13-week gavage study (Similar to OECD 408, NTP, 1990; Carvone CLH report, 2012). In addition, no compound-related clinical signs and no increase in neoplastic lesions were seen in two-year gavage study with D-Carvone resulting in a peer-reviewed LOAEL of 375 mg/kg bw/day (Similar to OECD 451, NTP, 1990; Carvone CLH report, 2012). L-Carvone is predicted to have a comparable level of repeated dose toxicity to D-Carvone, so using a read-across approach, a NOAEL of 375 mg/kg bw/day is predicted for L-carvone. In addition, published toxicokinetic data demonstrates that L- and D-Carvone have no differences in metabolism, which further supports the read-across application (Engel, 2001). On the basis of the metabolism and other toxicological properties (see 5. Data matrix), the information from the source chemical is reliable and read across from D-carvone to L-Carvone for repeated dose toxicity is proposed.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990

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:
no
Remarks:
Indicated in the report that the experimental and tabulated data were examined for accuracy, consistency, completeness, and compliance with Good Laboratory Practice regulations.
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:
5-isopropenyl-2-methylcyclohex-2-en-1-one
Test material form:
other: colourless to pale yellow liquid
Details on test material:
- Name of test material (as cited in study report): d-CARVONE
- Physical state: colourless to pale yellow liquid
- Analytical purity: 96.5%
- Lot/batch No.: Lot no. K-332
- Stability under test conditions: The d-carvone/corn oil solutions were stable under simulated dosing conditions for at least 3 hours.
- Storage condition of test material: The d-carvone/corn oil solutions were stored at room temperature under nitogen for a maximum of 1 week

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, (Portage, MI), USA.
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 20.3 ± 0.6 to 23.6 ± 0.5 (males); 18.2 ± 0.4 to 18.9 ± 0.4 (females)
- Housing: 5 animals per cage (polycarbonate (Hazleton Systems, inc., Aberdeen, MD) with Reemay spun-bonded polyester filters (Snow Filtration, Cincinnati, OH); Beta Chips bedding (Northeastern Products, Inc., Warrensburg, NY). Cages were rotated on the racks throughout the studies.
- Diet (e.g. ad libitum): NIH 07 Rat and Mouse Ration (Zeigler Hros., Inc., Gardners, PA); available ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): average 23.38°C (74.1°F)
- Humidity (%): average 53.6%
- Air changes (per hr): 6-12 per hr
- Photoperiod (hrs dark / hrs light): Fluorescent light 12 hrs/day

IN-LIFE DATES: From: To:11/6/81 (necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Feed blends of d-carvone were found to be unstable under the feed blending and simulated dosing conditions. Corn oil was selected as the
vehicle. The stability of d-carvone in corn oil at 0.5% (5 mg/g) stored at room temperature or at 5°C for 21 days was determined. 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.

Dose preparation: The appropriate weight of d-carvone was dissolved in corn oil to a specified volume in a graduated cylinder.


VEHICLE
- Justification for use and choice of vehicle (if other than water): d-carvone is insoluble in water, so corn oil was selected as the vehicle.
- Concentration in vehicle: 0, 93, 187, 375, 750, 1,500 mg/kg
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose mixtures were analyzed before the start of, and once during the study. During the study, concentrations of d- carvone in corn oil were determined by gas chromatography with a 10% Carbowax column and flame ionization detection. During the study, all dose mixtures were found to be within ± 10% of the target concentrations by the study laboratory.
Duration of treatment / exposure:
5 days per week
Frequency of treatment:
13 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
93, 187, 375, 750, 1,500 mg/kg
Basis:
other: nominal conc
Remarks:
Doses / Concentrations:
93, 185, 365, 742, 1480 mg/kg
Basis:
other: actual conc (31-07-81)
Remarks:
Doses / Concentrations:
87, 176, 363, 762, 1540 mg/kg
Basis:
other: actual conc (11-09-81)
No. of animals per sex per dose:
10 male and 10 female (0, 93, 187, 375, 750 mg/kg)
30 male and 10 female (1,500 mg/kg)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Animals assigned to groups according to a table of random numbers

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed two times per day

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal weights were recorded at the beginning of the studies, once per week thereafter, and at
the end of the studies.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes (see below). Histopathologic examinations were conducted on vehicle controls, animals killed at 2, 4, 6, and 8
weeks, animals that received 750 mg/kg and animals that died before the end of the studies.
Statistics:
Organ weight to body ratios: Dunnett's test (Dunnett, 1955); P<0.05, P<0.0l

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
See details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See details on results
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Hypoactivity, ataxia, and hypersensitivity to touch were seen at 1,500 mg/kg. The female that received 1,500 mg/kg and survived to
the end of the study had body tremors, hypersensitivity to touch, and impaired hind limb function during the study.

All 30 male and 9/10 female mice that received 1,500 mg/kg and 1/10 males that received 93 mg/kg died before the end of the studies. All other animals survived until the end of the study (Table 4).

BODY WEIGHT AND WEIGHT GAIN
Final mean body weights of dosed and vehicle control male mice were similar. The final mean body weight of the female survivor at
1,500 mg/kg was 12% lower than that of vehicle controls (Table 4).


ORGAN WEIGHTS
The relative liver weights for mice that received 750 mg/kg were significantly greater than those for vehicle controls (males P<0.01; females P<0.05) (Table 5).

HISTOPATHOLOGY: NON-NEOPLASTIC
No test substance-related lesions were observed.



Effect levels

Dose descriptor:
NOAEL
Effect level:
375 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Study report attachments:

Table 4 Survival and Mean Body Weights (NTP TR 381)

Table 5 Organ weight to body weight ratios (NTP TR 381)

Read-across justification - Full report is attached.

Analogue approach justification

According to Annex XI 1.5, a read-across approach can be used to fill the data gap when certain criteria are fulfilled. The fulfilled of the criteria are discussed below. The information from the REACH technical guidance document R.6 is used for this assessment as well as ECHA’s Practical guide 6 on category and read-across approaches (ECHA REACH TGD; ECHA, 2009).

Quality of the experimental data of the analogues

See data matrix for an overview of the experimental repeated-dose toxicity data.

A 13-week and a 2-year study of the source chemical D-Carvone has been conducted by the U.S. Department of Health and Human Services (DHHS) National Toxicology Program (NTP, 1990). The 13-week study is equivalent or similar to OECD guideline 408 and the 2 year study is equivalent or similar to OECD guideline 451. Both of the studies were examined for accuracy consistency completeness and compliance with Good Laboratory Practice regulations by the NTP. Both the 13-week and 2-year study are assigned a Klimisch score of 2.

In addition, metabolic differences were investigated by a metabolism of ingestion correlated amounts (MICA) experiment in humans (Wolfgang Engel, 2001). This data further supports the read across approach, as no differences were noted. Therefore these data have limited uncertainty and can be used in an analogue approach.

Toxicokinetics

The source and target chemical indicate similarity in toxicokinetic behaviour based on the similar physicochemical properties: the molecular weight (150g/mol), physical form (liquids), vapour pressures (low), boiling point 231°C as is shown in the data matrix.

It can be seen that the water solubility of the source chemicals were slightly soluble in water (less than 100 mg/L), and a moderate log Kow about 2.7, the same as target chemicals which own a different specific rotation. The slight differences in the cited data are within the range of uncertainty range of laboratory tests. For the toxicokinetic behaviour of the target chemicals, these physicochemical differences are expected to be minimal considering absorption via the oral, inhalation and dermal route, because these properties of the chemicals are still within the range for expected ready absorption. Experimental data showed no differences in metabolism between L- and D-Carvone in human (Wolfgang Engel, 2001). For repeated dose toxicity, the kinetic behaviour largely supports the application of read across from D-carvone to L-carvone.

Reactivity

The source chemical D-Carvone has been tested in a 13-week repeated dose toxicity study (NTP, 1990). D-carvone was administered to B6C3F1 mice by gavage at dose levels of 0, 93, 187, 375, 750 mg/kg (10 males and 10 females) and 1,500 mg/kg (30 males and 10 females). Clinical signs noted at 1,500 mg/kg were hypoactivity, ataxia, and hypersensitivity to touch. D-carvone was toxic at 1,500 mg/kg; one female dosed at 1,500 mg/kg survived to the end of the study and had a reduced final mean body weight (12%) and one male that received 93 mg/kg died before the end of the study. The relative liver weights for male and female mice that received 750 mg/kg were significantly greater than those for vehicle controls. There were no other test substance-related effects in body weight, weight gain, organ weight or histopathology. The test results receive reliability 2, and were chosen as the key study. A NOAEL of 375 mg/kg bw/day was determined and this value is also indicated in the CLH report (CLH, 2012).

In addition, no compound-related clinical signs and no increase in neoplastic lesions were seen in two-year gavage study with D-Carvone (50 males/female B6C3F1 mice; 0, 375, 750 mg/kg) resulting in a peer-reviewed LOAEL of 375 mg/kg bw/day. The 2-year study was given a reliability of 2 and is considered a supporting study. The MICA experimental data indicated no differences in metabolism between L-Carvone and D-Carvone, which supports the read across approach.

In conclusion, a NOAEL value of 375 mg/kg bw/day was derived from a repeated dose toxicity study of D-Carvone. In addition, no difference in metabolism between L-Carvone and D-Carvone were noted. A NOAEL value of 375 mg/kg bw/day is predicted for repeated dose toxicity of L-carvone.

Common breakdown products or metabolites

L-Carvone and D-carvone metabolism studied in humans using a metabolism ingestion correlated amounts (MICA) approach indicated metabolic pathways of oxidation of the double bond in the side chain and to a minor extent 1,2- and 1,4+1,2-reduction presented in Figure 1. The major in vivo metabolites were newly identified dihydrocarvonic acid (M1), carvonic acid (M2), and uroterpenolone (M3). Minor metabolites were identified the alcohols carveol (M4) and dihydrocarveol (M5). The author concluded no difference in metabolism between L-Carvone and D- carvone (Wolfgang Engel, 2001).

Similarities in results for toxicological endpoints between the target and source chemicals

As is presented in the data matrix, the acute oral and dermal toxicity data show similar limited acute toxicity for the source and the target chemicals: LD50>2000 mg/kg bw. No eye irritation and skin sensitization were noted for both. No skin irritation was noted for L-Carvone, but mild irritation was noted for D-Carvone. Both chemicals have negative Ames test results; further mutagenicity tests in the dossier (OECD 473, OECD 476) for L-carvone are also negative.

In the 13-week repeated dose toxicity study, a NOAEL of 375 mg/kg bw/day was derived for D-Carvone and in a 2 year study a LOAEL of 375 mg/kg bw/day was derived in 2-year study. A NOAEL of 375 mg/kg bw/day is predicted for L-carvone for repeated dose toxicity.

Data matrix

Substance

L-Carvone

D-Carvone

Structure

See attached (left)

 See attached (right)

CAS No.

6485-40-1

2244-16-8

Molecular weight

150.22

150.22

Physical state

liquid

liquid

Melting point (ºC)

9.86ºC (QSAR calculate)

<15ºC (CRC handbook)

Boiling point (ºC)

231ºC (CRC handbook)

231ºC (CRC handbook)

Vapour pressure

15.3 Pa at 25 °C

 (QSAR calculate)

1.9 Pa at 20

(reviewed report, 2008)

13.33 Pa at 25 °C, Carvone unspecified isomer ratio (Perry’s Chemical Engineers’ Handbook, 1999)

Partition coefficient (Log Kow)

2.74 (S. Griffin, 1999)

2.71(GESTIS Substance Database)

Water solubility

47.2 mg/l (wanxiang test report, 2012)

27-79 mg/l at 20 °C (reviewed report, 2008)

sl H2O; vs EtOH; s eth, ctc, chl(CRC handbook)

sl H2O; vs EtOH; s eth, chl

(CRC handbook)

Acute oral toxicity(mg/kg bw)

4600-6300 (Quest test report, 1986)

>2000 (reviewed report, 2008)

Acute inhalation toxicity (g/m3)

> 5.66(d/lisomerratio of minimally 4:1)(reviewed report, 2008)

Acute dermal toxicity (mg/kg bw)

>2000 (Symrise test report, 1999)

>4000 (reviewed report, 2008)

Skin irritation/corrosiveness

No irritation(Symrise test report, 1999)

Mildly irritation (reviewed report, 2008)

Eye irritation

Not irritation(Symrise test report, 1999)

No irritation (reviewed report, 2008)

Sensitization

Sensitizing to skin (RIFMtest report, 2007)

Sensitizing to skin(reviewed report, 2008)

Genotoxicity – Ames test

Negative(OECD 471) (wanxiangtest report, 2007)

Negative (NTP, 1990)

Genotoxicity in vitro mouse lymphoma assay

Negative (OECD 476) (wanxiangtest report, 2007)

 

Genotoxicity in vitroChromosomal aberration

Negative (OECD 473) (wanxiangtest report, 2007)

Equivocal

The data quality is not very reliable

(NTP, 1990)

Repeated dose toxicity

Read-across from D-Carvone

NOAEL=375 mg/kg bw/day

NOAEL=375 mg/kg bw/day in 13-week study

(NTP, 1990; CLH report, 2012)

NOAEL=125 mg/kg bw/day in one-year study

The substance is likely to be L-carvone, butthe data quality is not reliable

(E.C. Hagan, 1967; CLH report, 2012)

LOAEL=375 mg/kg bw/day in 2-year study

(NTP, 1990; CLH report, 2012)

Reproductive and developmental toxicity (mg/kg bw/day)

Developmental: Read-across from D-Carvone

NOAEL=200 mg/kg bw/day

Developmental: 200 mg/kg bw/day (highest dose tested) (OECD 414)

(CLH report, 2012)

Applicant's summary and conclusion

Conclusions:
In a 13-week study in mice, D-carvone was toxic at 1,500 mg/kg. At 750 mg/kg, it did not affect body weight, survival, or histopathology,
but relative liver weights were increased. The NOAEL value was 375 mg/kg bw/day. These results are suitable for REACH purposes and using a read-across approach, a NOAEL value of 375 mg/kg bw/day is predicted for L-carvone.
Executive summary:

In a subchronic toxicity study (NTP TR 381), D-carvone was administered to B6C3F1 mice by gavage at dose levels of 0, 93, 187, 375, 750 mg/kg (10 males and 10 females) and 1,500 mg/kg (30 males and 10 females).

Clinical signs noted at 1,500 mg/kg were hypoactivity, ataxia, and hypersensitivity to touch . d-carvone was toxic at 1,500 mg/kg; one female dosed at 1,500 mg/kg survived to the end of the study and had a reduced final mean body weight (12%) and one male that received 93 mg/kg died before the end of the study. The relative liver weights for male and female mice that received 750 mg/kg were significantly greater than those for vehicle controls. There were no other test substance-related effects in body weight, weight gain, organ weight or histopathology.

The NOAEL is 375 mg/kg bw/day.

This subchronic toxicity study in the mouse is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in mice.