Cyclohexane, 1-methyl-4-(1-methylethyl)-, tetradehydro deriv.

Endpoint summary

Description of key information

Weight of evidence for Depanol I was obtained from subchronic and chronic toxicity testing with d-limonene in rats and subchronic toxicity testing in mice. The three studies were conducted according to older standards, however in combination they covered all needed study parameters. In addition, other studies are available for d-limonene demonstrating its safety.  In all studies, NOAEL was above 500 mg/kg bw, taking into account that the kidney findings in male rats were not of relevance for humans. Supporting information was also available from two 6-month dog studies, confirming that the nephropathology was specific to male rats. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Based on a read across evaluation, the registered substance has a comparable physicochemical and toxicological profile to d-limonene. The study was in Japanese and less details were available, however it was conducted according to valid methods and included haematology, serum biochemistry, urinalysis, organ weights and histopatholgoy. In combination with the other weight of evidence, the data are considered relevant, reliable and adequate for classification.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Duration extended to 6 months (equivalent in rats to chronic testing); histopathology limited to 5 animals/group & sex.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: SD-JCL (SPF)

Sex:

male/female

Route of administration:

oral: unspecified

Vehicle:

other: 1% Tween 80 solution

Details on oral exposure:

Administered volume: 5 mL/kg bw

Duration of treatment / exposure:

6 months

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 277, 554 and 1385 mg/kg
Basis:

No. of animals per sex per dose:

10 or 9 (See Any other information on material and methods incl. tables)

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- Reported after 1, 2, 3, 4 and 6 months

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly
- Reported after 1, 2, 3, 4 and 6 months

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly
- Reported after 1, 2, 3, 4 and 6 months

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: terminal
- Anaesthetic used for blood collection: Yes ,ether
- How many animals: all available
- Parameters examined: total erythrocyte count (RBC), total leucocyte count (WBC), hematocrit (HCT),mean cell hemoglobin (Hb), Differential count (lymphocytes, neutrophils, monocytes, eosinophils, basophils)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: terminal
- Anaesthetic used for blood collection: Yes ,ether
- How many animals: all available
- Parameters examined: alkaline phosphatase (ALP), aspartate aminotransferase (AST or sGOT), alanine aminotransferase (ALT or sGPT), total protein, albumin/globulin ratio (A/G), total cholesterol, blood sugar, bilirubin, blood urea nitrogen (BUN).

URINALYSIS: Yes
- Time schedule for collection of blood: terminal
- Anaesthetic used for blood collection: Yes ,ether
- How many animals: 5 animals/group
- Parameters examined: PH, protein, occult blood, ketone bodies, glucose, bilirubin, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsy of all available animals
- Organ weights: absolute and relative to body weight of pitiutary gland, thyroids, thymus, lungs, heart, spleen, kidneys, liver, adrenals, testes.

HISTOPATHOLOGY: Yes
- How many animals: 5 animals/group
- pitiutary gland, thyroids, thymus, lungs, heart, spleen, kidneys, liver, adrenals, testes, stomach, duodenum, pancreas, lymph nodes, ovaries.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased at 1385 mg/kg bw in males & females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Little or no change at 1385 mg/kg in males & females.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Little or no change at 1385 mg/kg in males and females.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No changes were observed in the histopathological examination, except granular casts in the kidney of some male rats.

Details on results:

BODY WEIGHT AND WEIGHT GAIN
A slight decrease was caused in the body weight at high dose levels (1385 mg/kg).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Little or no change was observed in food consumptions at high dose levels (1385 mg/kg).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Little or no change was observed in water consumptions at high dose levels (1385 mg/kg).

HAEMATOLOGY
No significant changes were noted in the hematological examinations.

CLINICAL CHEMISTRY
No significant changes were noted in the biochemical examinations.

URINALYSIS
No significant changes were noted in the urinalysis.

ORGAN WEIGHTS
Little or no effects were observed on the organ weight and the relative organ weight.

HISTOPATHOLOGY: NON-NEOPLASTIC
No changes were observed in the histopathological examination, except granular casts in the kidney of some male rats.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LOAEL

Effect level:

1 385 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight decreases, food and water consumption decreases, histopathological kideny effects (males)

Critical effects observed:

not specified

Conclusions:

A slight decrease was caused in the body weight, but little or no change in water and food consumptions at high dose levels (1385 mg/kg).
The NOAEL was 554 mg/kg bw in males and females; the LOAEL was 1385 mg/kg bw in males and females.

Executive summary:

To determine the safety of d-limonene the chronic toxicity was studied by oral gavage in groups of 9 -10 male and female rats for six months and the methods and results were as follows. Male and female rats were divided into respective 4 groups, three of which were given orally d-limonene at daily doses of 277, 554 and 1385 mg/kg, and one of which was given orally the vehicle (1% Tween 80 solution) in the corresponding volume. A slight decrease was caused in the body weight, but little or no change in water and food consumptions at high dose levels (1385mg/kg). Little or no effects were observed on the organ weight and the relative organ weight. No significant changes were also noted in the urinalysis, hematological and biochemical examinations. No changes were observed in the histopathological examination, except granular casts in the kidney of some male rats. The latter effects were considered male rat specific, with no human relevance. The NOAEL was 554 mg/kg bw in males and females; the LOAEL was 1385 mg/kg bw in males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Studies were considered as Weight of Evidence, as they were performed according to older standards, however when taken toghether they cover all necessary study parameters up to chronic toxicity testing.

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

Additional information

No repeated dose toxicity studies are available for Depanol I, however extensive Weight of Evidence was available from subchronic and chronic toxicity studies of d-limonene in rats and mice, as well as dogs. The studies given were limited to the subchronic to chronic studies at high dose levels. Justification for read across based on the ECHA guidance is provided in Section 13.

- A 13-week oral gavage subchronic toxicity study was conducted with d-limonene in corn oil in rats dosed at 0, 150, 300, 600, 1200 or 2400 mg/kg bw/day, 5 days/week (NTP/Jameson, 1990). 9/10 and 5/10 at 2400 mg/kg bw/day died within the first week of the study. The final mean body weights of male rats receiving the three highest doses (600,1200, and 2400 mg/kg bw/day) were reported to be 6%, 12%, and 23% lower than that of the controls, respectively. In addition, the final body weight of the one surviving female rat at the highest dose was 11% lower compared to that of controls. Rough hair coats, lethargy, and excessive lacrimation were observed for all animals at the two highest dose levels (1200 and 2400 mg/kg bw/day). A dose-related increase in severity of nephropathy was noted, characterized by degeneration of epithelium in the convoluted tubules, granular casts with tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets were observed in the epithelium of the proximal convoluted tubules in all groups of male rats, including vehicle controls. These effects are nowadays known and recognized as male rat specific and not relevant for humans. A NOAEL of 1200 mg/kg bw/day was selected for female rats based on low survival at the highest dose; a NOAEL of 600 mg/kg bw/day was selected for male rats based on low survival and decreased body weights/clinical observation at the 1200 and 2400 mg/kg bw/day.

- A 13-week oral gavage subchronic toxicity study was also conducted with d-limonene in corn oil in mice dosed at 0, 125, 250, 500, 1000, or 2000 mg/kg, 5 days per week. 1/10 males and 2/10 females at 2000 mg/kg and 1/10 females at 500 mg/kg died before the end of the studies. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. The final mean body weights of mice that received 1000 or 2000 mg/kg were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg. Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.

- A 6 -month oral gavage chronic toxicity study was further conducted with d-limonene in 1% Tween 80 solution in rats dosed at 277, 554 and 1385 mg/kg (Tsuji et al. 1975). A slight decrease was caused in the body weight, but little or no change in water and food consumptions at high dose levels (1385 mg/kg). Little or no effects were observed on the organ weight and the relative organ weight. No significant changes were also noted in the urinalysis, hematological and biochemical examinations. No changes were observed in the histopathological examination, except granular casts in the kidney of some male rats. The latter effects were considered male rat specific, with no human relevance. The NOAEL was 554 mg/kg bw in males and females; the LOAEL was 1385 mg/kg bw in males and females.

Further subacute, subchronic and chronic toxicity studies were available for d-limonene in rodents and non-rodents (e.g. dogs), which confirmed that the kidney effects were male rat specific up to low doses,without relevance to humans, and demonstrating that the class of d-limonene substances are generally recognised as safe (see attached publication: FEMA Gras list 2011).

Further supporting information was provided by two 6 -months repeated dose toxicity studies in dogs, adding information in a non-rodent species to understand better the nephrotoxicity seen in (especially) male rats (Adams et al., 2011):

-D-limonene administered by oral gavage to groups of five male and five female adult beagle dogs at doses of 0, 100, or 1000 mg /kg bw/day in divided doses twice a day led to diarrhea and emesis in both dose groups. At the termination of the experiment, a 35% increase in serum cholesterol and a 2-fold increase in serum alkaline phosphatase levels in high-dose dogs of both sexes were noted. No significant changes were reported in the body weights or consumption of feed in either treatment group. No histological abnormalities were noted in any major organ or tissue. No hyaline droplets or histopathological changes were reported in the kidneys. A small increase (not statistically significant) in relative kidney weight was reported in the low-dose group, but a significant increase in relative kidney weight was reported in the high-dose group.

- Groups of male and female beagle dogs (3/sex/group) orally administered d-limonene at dose levels of approximately 340, 1000 and 3000 mg/kg bw/day led to frequent vomiting at dose levels exceeding 1000 and 3000 mg/kg bw/day for female and male dogs, respectively, as well as a decrease in body weight in some animals. Total cholesterol and blood sugar levels were decreased in both male and female dogs at a dose level of 3000 mg/kg bw/day; however, it was not reported if these effects were statistically significant. There were protein casts noted in the renal tubules of female and male dogs orally administered d-limonene at dose levels of 340 and 1000 mg/kg bw/day, respectively; however, no histological changes were observed in the other organs examined. Male NOAEL was 1000 mg/kg bw/day, whereas female NOAEL was 340 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
D-limonene was used as source chemical. The study with longest duration (6 months) was selected, however other studies (90-day rat and mice studies) were available for source chemical d-limonene). Lowest NOAEL was 500 mg/kg bw/day, taking into account that the male rat specific nephropathy was not relevant for humans.

Justification for classification or non-classification

Based on the available data, Depanol I does not have to be classified for repeated dose toxicity according the EU labelling regulations Commision Directive 93/21/EEC or CLP regulation No. 1272/2008 of 16 December 2008.