Farnesol

Endpoint summary

Description of key information

The key information supporting the repeat dose toxicity of the test item included a regulatory subchronic repeated dose toxicity study with reproductive toxicity screen by oral administration (inclusion levels in the feed of 1500, 4000 and 12000 ppm) of the read-across substance, Nerolidol, in rats (OECD 422) conducted to GLP. In this study No Observed Adverse Effect Levels (NOAELs) were established following repeated daily treatment to the parent animals. Read-across is appropriate as the test substance and nerolidol are very similar in chemical structure, differing only by the interchange of a hydroxyl group and double bond on carbon positions 1 and 2, and very similar in physico-chemical properties. The oral and dermal acute and repeat dose toxicity data show that the toxicity of farnesol and nerolidol are similar in order of magnitude when comparing dose levels expressed in terms of mg/kg bodyweight. Given the similarities in chemical and biological effects, nerolidol is considered to be a suitable read-across substance. A supporting study of 28 days duration in rats by daily oral gavage administration at 500 or 1000 mg/kg/day also incorporated a recovery period and was extended to assess hepatotoxicity by analysing drug metabolising enzyme activities.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis is based on different compounds with qualitatively similar properties using the analogue approach, following Scenario 2 of the Read-Across Assessment Framework (ECHA 2017).

2. SOURCE AND TARGET CHEMICAL(S)
Target substance: Farnesol (CAS: 4602-84-0; EC: 225-004-1)
Source substance: Nerolidol (CAS: 7212-44-4; EC: 230-597-5)

3. ANALOGUE APPROACH JUSTIFICATION
For the prediction of human health endpoints, similar qualitative effects were observed in the acute dermal toxicity study and repeat dose oral toxicity study for farnesol and nerolidol. Limited information is available to assess the reproduction / developmental toxicity of farnesol, however given the similarities in toxicokinetic profile as demonstrated in other toxicity studies, no difference in effects from the source substance are predicted. Both the target and source substances undergo a similar metabolic pathway. The bioavailability of nerolidol may be greater than farnesol (as observed experimentally), therefore read-across from nerolidol may represent a worst-case approach.

4. DATA MATRIX
See "Read-across justification" in Section 13.2.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test)

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

In total, females were treated for 57 days and males treated for 36 days

Frequency of treatment:

Treated diets were fed ad libitum

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gain males and females at 12000 ppm

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced intake in males and females at 12000 ppm

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slightly increased GGT levels in males and females treated at 12000 ppm

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver weights in males and females treated at 12000 ppm and in females treated at 4000 ppm

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dark brown discolouration of the liver in males and females treated at 12000 ppm

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes in the liver of females only treated at 12000 or 4000ppm

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs of ill health or reaction to treatment observed at 12000, 4000or 1500 ppm and there were no treatment related mortalities among the F0 parent animals.

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gain in males weeks 0-1 treated at 12000 ppm (45% below controls); Decreased body weight gain in females treated at 12000 ppm in weeks 0-1 (73% below controls), gestation period (37% below controls) and during lactation (40 % below controls).

FOOD CONSUMPTION AND COMPOUND INTAKE
Reduced food intake in males treated at 12000 ppm in weeks 0-2 (10-18% below controls); Reduced food intake in females treated at 12000 ppm in weeks 0-1 (25% below controls), gestation period (20% below controls) and post natal days 1-4 (32% below controls).
The dietary inclusion levels of 1500, 4000 and 12000 ppm correspond to 102, 266 and 758 mg/kg/day in males; 105, 279 and 705 mg/kg/day in non-pregnant females; 120, 340 and 824 mg/kg/day in pregnant females; 193, 468 and 1194 mg/kg/day in lactating females.

HAEMATOLOGY
There were no changes in the haematology profile that were considered to be related to treatment.

CLINICAL CHEMISTRY
There was a sight increase in Gamma-Glutamyltransferase values in males and females treated at 12000 ppm.

URINALYSIS
There were no changes in the urinalysis profile that were considered to be related to treatment.

NEUROBEHAVIOUR
Detailed clinical examinations in an open field, detailed observations in a functional observational battery and measurements of motor activity did not reveal any indications of test substance-induced effects in any treatment group.

ORGAN WEIGHTS
Statistically signifcant absolute and relative increased liver weights in males and females treated at 12000 ppm and also in females treated at 4000 ppm.

GROSS PATHOLOGY
The liver was discoloured in males and females treated at 12000 ppm, however, this finding is not considered to be adverse.

HISTOPATHOLOGY: NON-NEOPLASTIC
Minimal or slight central hepatocellular hypertrophy was observed in females treated at 12000 ppm. In addition a minimal fatty change was noted in females treated at 12000 or 4000 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

266 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

105 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

266 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The NOAEL for general, systemic toxicity of the test substance is 1500 ppm (equivalent to 105 mg/kg/day) for the F0 parental females based on reduction in food consumption and body weights/body weight gain, clinical pathological changes, increased liver weights and central hepatocellular hypertrophy and central fatty change. The NOAEL for F0 parental males was found to be 4000 ppm (equivalent to 266 mg/kg/day) based on reduction in food consumption and body weight gain, clinical pathological changes and increased liver weights.

Executive summary:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats treatment with Nerolidol showed general systemic toxicity in the F0 males at 12000 ppm and in the F0 females at 12000 and 4000 ppm (Schneider 2010). Salient clinical findings were distinct decreases in food consumption and body weights/ body weight gain which were most severe in females during gestation and lactation. Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal any indications of test substance-induced effects in any of the treated rats. In clinical pathology the main effects of the compound administration were related to enzyme induction in the liver: This was true for the increased GGT activity in rats of both sexes treated at 12000 ppm. Pathology revealed the liver to be the target organ of toxicity. The terminal body weight was significantly decreased in females treated at 12000 ppm. The statistically significant absolute and relative increased liver weights in males treated at 12000 ppm as well as in females treated at 4000 ppm and 12000 ppm are related to treatment. The increase of the relative liver weight in females treated at 1500 ppm was only slight (+8%) and there were no histopathological correlates. Although a substance-related effect cannot be ruled out, the effects are non-adverse in nature. Macroscopically, the liver was discolored in males and females treated at 12000 ppm. The males showed a slightly brown discoloration, in females the livers were dark brown. For the discoloration there was no histopathological correlate. The discoloration of the liver is considered treatment-related but non-adverse. Histopathologically, a minimal or slight central hepatocellular hypertrophy was observed in females treated at 12000 ppm that correlated with the increased liver weights. The hypertrophy is assessed treatment-related and adaptive. In addition, a minimal central fatty change was noted in five females treated at 4000 ppm as well as in four females treated at 12000 ppm. Although the central fatty change occurred only in some females and the severity was only minimal, a treatment-related effect cannot be ruled out but is assessed as non-adverse. In the thyroid glands the number of males and females with minimal follicular hypertrophy/ hyperplasia was increased in animals treated at 12000 ppm. In affected animals the number of small follicles was minimally increased or the follicular epithelium was minimal higher. It was a very weak effect that might be related to treatment but is assessed as non adverse.

Consequently, the NOAEL for general, systemic toxicity of the test substance is 1500 ppm (equivalent to105 mg/kg/day) for the F0 parental females based on reduction in food consumption and body weights/body weight gain, clinical pathological changes, increased liver weights and central hepatocellular hypertrophy and central fatty change. The NOAEL for F0 parental males was found to be 4000 ppm (equivalent to 266 mg/kg/day) based on reduction in food consumption and body weight gain, clinical pathological changes and increased liver weights. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 422 and OPPTS 870.3650. This data may be used as key data, as nerolidol is a suitable read-across source to the target substance based on similar chemical structure and hazard data.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-12 weeks
- Weight at study initiation: Males 279.3-311.0g; Females 182.8-218.0g
- Housing: Individually housed in Makrolon type M III cages; pregnant females were provided with nesting material (Cellulose wadding); for enrichment wooden gnawing blocks were added to the cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24oC
- Humidity (%): 30-70%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: October 2009 To: December 2009

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets were prepared four times during the study. The maximum period for which each test diet was fed was less than 48 days.
- Mixing appropriate amounts with (Type of food): Kliba maintenance diet mouse/rat "GLP" meal
- Storage temperature of food: Not stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in the diet for a period of 48 days at room temperature were carried out prior to the start of the study. Homogeneity and concentration control analyses were carried out during the premating and the gestation periods. Duplicate samples were kept in reserve until after report finalization.

Duration of treatment / exposure:

In total, females were treated for 57 days and males treated for 36 days

Frequency of treatment:

Treated diets were fed ad libitum

Dose / conc.:

1 500 ppm

Dose / conc.:

4 000 ppm

Dose / conc.:

12 000 ppm

No. of animals per sex per dose:

10 males and 10 females per group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Not given
- Rationale for animal assignment (if not random): Randomised according to bodyweight

Positive control:

No positive control included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily; the parturition and lactation behaviour of the dams was also generally evaluated in the mornings in combination with the daily clinical inspection.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for male and female parental animals with the following exceptions for female animals; during the mating period parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD7, 14 and 20; Females with litter were weighed on the day after parturition (PND1) and on PND4.

FOOD CONSUMPTION AND ACHIEVED INTAKE: Yes
- Time schedule for examinations: Weekly for male and female parental animals with the exception of: during the mating period (male and femal F0 animals); F0 females with evidence of sperm food consumption recorded on Gestation days 0-7, 7-14 and 14-20; F0 females giving birth to a litter food consumption was determined for post natal days 1-4.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


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 data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After completetion of the treatment period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per group
- Parameters examined: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, platelets, differntial blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After completion of the treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females per group
- Parameters examined: ALT, AST, ALP, GGT, Na, K, Cl, inorganic phosphate, Ca, urea, creatinine, glucose, bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, Mg

URINALYSIS: Yes
- Time schedule for collection of urine: After completion of the treatment period
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters examined: volume, colour, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood.

FUNCTIONAL OBSERVATION BATTERY (FOB) EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period
- Dose groups that were examined: 5 males and 5 females in each group
- Battery of functions tested:
- Home cage observations - posture; tremors; convulsions; abnormal movements; impairment of gait; other
- Open field observations - behaviour when removed from cage; fur; skin; salivation; nose discharge; lacrimation; eyes/pupil size; posture; palpebral closure; respiration; tremors; convulsions; abnormal movements/stereotypy; impairment of gait; activity/arousal level; faeces excreted during observation period; urine excreted during observation period; number of rearings
- Sensory motor tests - approach response; touch response; visual placing response; pupillary reflex; pinna reflex; startle response; righting response; behaviour during handling; vocalisation; tail pinch; grip strength; landing foot splay test

MOTOR ACTIVITY MEASUREMENT
- Time schedule for examinations: At the end of the administration period
- Dose groups that were examined: 5 males and 5 females in each group
- Method: Examinations performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, USA

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All F0 parental animals were necropsied and assessed by gross pathology
- The following organ weights were determined for each animal: adrenal, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, testes, seminal vesicles, spleen, thymus, thyroid, uterus
HISTOPATHOLOGY: Yes
- A full list of tissues were preserved from all F0 parental animals
- All tissues from all animals in the control and high dose level groups were examined in the first instance with selected tissues examined from animals in the intermediate and low dose groups.

Other examinations:

MALE REPRODUCTION DATA
- Male mating index (number of males with confirmed mating/number of males placed with females x 100)
- Male fertility index (number of males with proven fertility/number of males placed wih females x 100)

FEMALE REPRODUCTION AND DELIVERY DATA
- Female mating index (number of females mated/number of females placed with males x 100)
- Female fertility index (number of females pregnant/number of females mated x 100)
- Gestation index (number of females with live pups on the day of birth/number of females pregnant x 100)
- Live birth index (number of liveborn pups at birth/total number of pups born x 100)
- Postimplantation loss (number of implantations-number of pups delivered/number of implantations x 100)
LITTER/PUP DATA
- Pup number and status at delivery
- Pup viability/mortality
- Sex ratio
- Pup clinical observations
- Pup body weight data
- Pup necropsy observations

Statistics:

Appropriate statistical methods were applied to all types of data generated.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gain males and females at 12000 ppm

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced intake in males and females at 12000 ppm

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slightly increased GGT levels in males and females treated at 12000 ppm

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver weights in males and females treated at 12000 ppm and in females treated at 4000 ppm

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dark brown discolouration of the liver in males and females treated at 12000 ppm

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes in the liver of females only treated at 12000 or 4000ppm

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs of ill health or reaction to treatment observed at 12000, 4000or 1500 ppm and there were no treatment related mortalities among the F0 parent animals.

BODY WEIGHT AND WEIGHT GAIN
Decreased body weight gain in males weeks 0-1 treated at 12000 ppm (45% below controls); Decreased body weight gain in females treated at 12000 ppm in weeks 0-1 (73% below controls), gestation period (37% below controls) and during lactation (40 % below controls).

FOOD CONSUMPTION AND COMPOUND INTAKE
Reduced food intake in males treated at 12000 ppm in weeks 0-2 (10-18% below controls); Reduced food intake in females treated at 12000 ppm in weeks 0-1 (25% below controls), gestation period (20% below controls) and post natal days 1-4 (32% below controls).
The dietary inclusion levels of 1500, 4000 and 12000 ppm correspond to 102, 266 and 758 mg/kg/day in males; 105, 279 and 705 mg/kg/day in non-pregnant females; 120, 340 and 824 mg/kg/day in pregnant females; 193, 468 and 1194 mg/kg/day in lactating females.

HAEMATOLOGY
There were no changes in the haematology profile that were considered to be related to treatment.

CLINICAL CHEMISTRY
There was a sight increase in Gamma-Glutamyltransferase values in males and females treated at 12000 ppm.

URINALYSIS
There were no changes in the urinalysis profile that were considered to be related to treatment.

NEUROBEHAVIOUR
Detailed clinical examinations in an open field, detailed observations in a functional observational battery and measurements of motor activity did not reveal any indications of test substance-induced effects in any treatment group.

ORGAN WEIGHTS
Statistically signifcant absolute and relative increased liver weights in males and females treated at 12000 ppm and also in females treated at 4000 ppm.

GROSS PATHOLOGY
The liver was discoloured in males and females treated at 12000 ppm, however, this finding is not considered to be adverse.

HISTOPATHOLOGY: NON-NEOPLASTIC
Minimal or slight central hepatocellular hypertrophy was observed in females treated at 12000 ppm. In addition a minimal fatty change was noted in females treated at 12000 or 4000 ppm.

Key result

Dose descriptor:

NOAEL

Effect level:

266 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

105 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

266 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The NOAEL for general, systemic toxicity of the test substance is 1500 ppm (equivalent to 105 mg/kg/day) for the F0 parental females based on reduction in food consumption and body weights/body weight gain, clinical pathological changes, increased liver weights and central hepatocellular hypertrophy and central fatty change. The NOAEL for F0 parental males was found to be 4000 ppm (equivalent to 266 mg/kg/day) based on reduction in food consumption and body weight gain, clinical pathological changes and increased liver weights.

Executive summary:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats treatment with Nerolidol showed general systemic toxicity in the F0 males at 12000 ppm and in the F0 females at 12000 and 4000 ppm. Salient clinical findings were distinct decreases in food consumption and body weights/ body weight gain which were most severe in females during gestation and lactation. Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal any indications of test substance-induced effects in any of the treated rats. In clinical pathology the main effects of the compound administration were related to enzyme induction in the liver: This was true for the increased GGT activity in rats of both sexes treated at 12000 ppm. Pathology revealed the liver to be the target organ of toxicity. The terminal body weight was significantly decreased in females treated at 12000 ppm. The statistically significant absolute and relative increased liver weights in males treated at 12000 ppm as well as in females treated at 4000 ppm and 12000 ppm are related to treatment. The increase of the relative liver weight in females treated at 1500 ppm was only slight (+8%) and there were no histopathological correlates. Although a substance-related effect cannot be ruled out, the effects are non-adverse in nature. Macroscopically, the liver was discolored in males and females treated at 12000 ppm. The males showed a slightly brown discoloration, in females the livers were dark brown. For the discoloration there was no histopathological correlate. The discoloration of the liver is considered treatment-related but non-adverse. Histopathologically, a minimal or slight central hepatocellular hypertrophy was observed in females treated at 12000 ppm that correlated with the increased liver weights. The hypertrophy is assessed treatment-related and adaptive. In addition, a minimal central fatty change was noted in five females treated at 4000 ppm as well as in four females treated at 12000 ppm. Although the central fatty change occurred only in some females and the severity was only minimal, a treatment-related effect cannot be ruled out but is assessed as non-adverse. In regard to thyroid glands, the number of males and females with minimal follicular hypertrophy/ hyperplasia was increased in animals treated at 12000 ppm. In affected animals the number of small follicles was minimally increased or the follicular epithelium was minimal higher. It was a very weak effect that might be related to treatment but is assessed as non adverse.

Consequently, the NOAEL for general, systemic toxicity of the test substance is 1500 ppm (equivalent to105 mg/kg/day) for the F0 parental females based on reduction in food consumption and body weights/body weight gain, clinical pathological changes, increased liver weights and central hepatocellular hypertrophy and central fatty change. The NOAEL for F0 parental males was found to be 4000 ppm (equivalent to 266 mg/kg/day) based on reduction in food consumption and body weight gain, and increased liver weights. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 422 and OPPTS 870.3650.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not of guideline design including just two treatment groups and a control, and was not conducted to GLP.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study design was similar to an OECD407 28-day repeated dose toxicity study although only two treated groups were included together with a standard vehicle control group. The numbers of animals per group complied with guideline requirements for studies of this type along with the experimental conditions. Half the animals from each group were terminated after 28 days of oral dosing whilst the remaining animals were terminated after a further 28-day period without treatment to assess recovery from any toxicities.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were individually housed in stainless steel cages suspended over absorbent cage boards, and were held in a temperature-controlled room maintained on a 12 h light/dark cycle. Rats were permitted free access to drinking water (supplied by automatic watering system) at all times during the study; rats were also allowed free access to Purina Certified Rodent Diet 5002 (PMI Nutrition International Inc., Brentwood, MO) throughout the study, except during an overnight fast prior to scheduled necropsies.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose levels of 500 or 1000 mg/kg/day were selected by the National Cancer Institute on the basis of a previous preclinical 28-day toxicity study without recovery

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not stated
- Concentration in vehicle: Not stated
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): Not stated
- Purity: Not stated

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days followed by a 28-day recovery period

Frequency of treatment:

Animals were dosed daily for 28 consecutive days

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 male and 20 female rats per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels of 500 and 1000 mg/kg/day were selected by the National Cancer Institute on the basis of a previous preclinical 28-day toxicity study
- Rationale for animal assignment (if not random): animals randomly assigned to groups
- Rationale for selecting satellite groups: not stated
- Post-exposure recovery period in satellite groups: 28-day recovery period

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days 29 and 57
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Hematologic parameters (erythrocyte count and morphology, hemoglobin, mean corpuscular volume, platelet count, absolute and relative reticulocyte count, total white blood cell count, and absolute and relative differential white blood cell counts) were quantitated using the ADVIA System 120 Hematology Analyzer (Bayer Corp., Tarrytown, NY). Hematocrit,mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were calculated on the basis of the ADVIA data. Coagulation parameters (prothrombin time, fibrinogen and activated partial thromboplastin time) were quantitated using a MLA Electra 900 Automatic Coagulation Timer (Hemoliance, Raritan, NJ).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days 29 and 57
- Animals fasted: Yes
- How many animals: 10 males and 10 females per group
- Serum chemistry parameters (sodium, potassium, chloride, calcium, inorganic phosphorus, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, total bilirubin, blood urea nitrogen, creatinine, glucose, total protein, albumin, cholesterol, and triglycerides) were evaluated using a Beckman Synchron CX5 analyzer (Beckman Instruments Inc., Brea, CA). Serum globulin, albumin:globulin ratio and blood urea nitrogen:creatinine ratio were calculated from the automated data.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsy with tissue collection
- Prior to removal, the liver was perfused in situ via the portal vein with approximately 10 ml of saline. After weighing, portions of the liver and kidneys were snap-frozen in liquid nitrogen and were stored frozen at approximately −70 ◦C; frozen tissues were used for quantitation of drug metabolizing enzyme activities
(see below). Approximately 45 other tissues were collected from each animal at necropsy; these tissues and remaining portions of liver and kidney were fixed in 10% neutral buffered formalin for histopathologic evaluation.

HISTOPATHOLOGY: Yes
- Tissues collected at the end of the treatment period from all rats in the high dose and control groups were processed by routine histologic methods and evaluated histopathologically. Histopathologic evaluations in low dose rats euthanized on day 29, and from rats in recovery groups (euthanized on day 57) were limited to gross lesions and identified target tissues.

Other examinations:

From all animals killed at the scheduled sacrifices liver and kidney samples were taken and subcellular fractions prepared for the analysis of the following drug metabolising enzyme activities:
Rat cytochrome (CYP) 1A1/2
Rat CYP2B1/2
Rat CYP2A1-3
Rat CYP2C11/12
Rat CYP3A1/2
Rat CYP4A1-3
Rat CYP2D2
Rat CYP2E1
Rat CYP19 (aromatase)
UDP-glucuronosyltransferase (UGT)
Glutathione S-transferase (GST)
Glutathione reductase
NADPH/quinone oxidoreductase
Sulfotransferase

Statistics:

Body weight, body weight gain, food consumption, clinical pathology, organ weight and metabolizing enzymatic activity data were compared by analysis of variance (ANOVA) followed by the post hoc analysis via Dunnett’s test for comparing multiple treatment groups to a single control. A minimum significance level of p < 0.05 was used in all comparisons. In all comparisons, values that were below the quantitative limits of the assay were excluded from the analyses.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increases in absolute and/or realtive liver and kidney weights in males and/or females at 500 or 1000 mg/kg/day in comparison with vehicle controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities or clinical signs of ill-health that were attributed to treatment

BODY WEIGHT AND WEIGHT GAIN
There were no effects on bodyweight or weight gain that were attributed to treatment

FOOD CONSUMPTION
There were no effects on food intake that were attributed to treatment

HAEMATOLOGY
There were no statistically significant effects on any haematology or coagulation parameters evaluated

CLINICAL CHEMISTRY
Small but statistically significant alterations were seen in several clinical chemistry endpoints, including triglyceride levels (31% increase in high dose females, 43% decrease in high dose males); urea nitrogen levels (21% decrease in high dose females only); glucose levels (7 and 8% decreases in low and high dose females only); aspartate transaminase activity (15% decrease in high dose females only); and alkaline phosphatase activity (52% increase in high dose males only). All alterations in clinical chemistry parameters were reversed during the 28-day recovery period (data not shown). The modest magnitude of these changes in clinical chemistry, their limitation to a single affected sex, their apparent lack of linkage to any identifiable toxicity and most importantly, their reversal following treatment cessation suggest that these differences are of limited toxicological significance.

ORGAN WEIGHTS
When compared to the vehicle control group, statistically significant increases in absolute and relative (organ-to-body weight ratio) liver weights were observed in female rats exposed to the low or high dose of farnesol, and in male rats exposed to the high dose only. Relative kidney weights were also increased in high dose males and both absoluteand relative kidney weights were increased in high dose females. All effects on liver and kidney weights were reversible: no statistically significant differences in mean weights of these organs were seen at the end of the recovery period.

GROSS PATHOLOGY
There were no treatment-related gross lesions identified in any animal at the terminal necropsies.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of tissues from rats receiving the high dose (1000 mg/kg/day) for 28 days failed to identify any pattern of histologic alterations that were associated with exposure to the test substance.

OTHER FINDINGS
Daily oral administration of farnesol to rats for 28 days induced statistically significant, dose-related increases in the activity of a number of hepatic drug metabolizing enzymes. Although several parameters were influenced by both low and high dose farnesol administration, most of the statistically significant effects of farnesol on enzyme activity were seen only in rats treated with the high dose (1000 mg/kg/day). The effects of farnesol on the activity of hepatic drug metabolizing enzymes were reversible; none of the statistically significant increases in hepatic enzyme activities that were observed at the end of the exposure period were still present in at the end of the 28-day recovery period

Conclusions:

Oral dose levels of 500 and 1000 mg/kg/day for 28 days were considered to be minimally toxic as a consequence of small changes in clinical chemistry parameters and significant enzyme inducing changes in the liver and kidney in males and females of both treatment groups. A No Observed Adverse Effect Level (NOAEL) was not identified for these changes although complete recovery was observed after 28 days.

Executive summary:

Daily oral administration of 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- at doses of 500 or 1000 mg/kg/day for 28 days was minimally toxic to CD rats. 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- induced no mortality, had no effects on body weight or body weight gain, induced no evidence of toxicity that was identifiable through clinical observation, had no influence on hematology or coagulation parameters, and induced no gross or microscopic alterations in organ structure. Modest, but statistically significant effects on clinical chemistry parameters were observed on day 29; these clinical pathology changes were reversed upon discontinuation of exposure. The primary indicators of 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- toxicity were statistically significant, dose-related increases in liver and kidney weights that were identified at the end of the dosing period. The effects of farnesol on liver and kidney weights were reversible after a 28-day recovery period. Because the observed increases in liver and kidney weights in 2,6,10-Dodecatrien-1-ol, 3,7,11 -trimethyl-treated rats were not associated either with any changes in clinical pathology parameters or any histopathologic alterations, and because these increases were reversible upon discontinuation of farnesol exposure, it is considered likely that farnesol effects on liver and kidney weights are secondary to the induction of drug metabolizing enzymes. The study is considered to be reliable with restrictions (Klimisch 2), as no guideline or GLP-compliance was reported, and the experimental design included only two treatment groups and a control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

105 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

One key study from read-across substance nerolidol is available, which is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 422 and OPPTS 870.3650. A supporting study is available for the target substance, and is considered to be reliable with restrictions (Klimisch 2), as no guideline or GLP-compliance was reported, and the experimental design included only two treatment groups and a control.

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

Additional information

In the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats treatment with the read-across substance, Nerolidol, general systemic toxicity was observed in the F0 males at 12000 ppm and in the F0 females at 12000 and 4000 ppm. Salient clinical findings were distinct decreases in food consumption and body weights/ body weight gain which were most severe in females during gestation and lactation. Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal any indications of test substance-induced effects in any of the treated rats. In clinical pathology the main effects of the compound administration were related to enzyme induction in the liver: This was true for the increased GGT activity in rats of both sexes treated at 12000 ppm. Pathology revealed the liver to be the target organ of toxicity. The terminal body weight was significantly decreased in females treated at 12000 ppm. The statistically significant absolute and relative increased liver weights in males treated at 12000 ppm as well as in females treated at 4000 ppm and 12000 ppm are related to treatment. The increase of the relative liver weight in females treated at 1500 ppm was only slight (+8%) and there were no histopathological correlates. Although a substance-related effect cannot be ruled out, the effects are non-adverse in nature. Macroscopically, the liver was discoloured in males and females treated at 12000 ppm. The males showed a slightly brown discoloration, in females the livers were dark brown. For the discoloration there was no histopathological correlate. The discoloration of the liver is considered treatment-related but non-adverse. Histopathologically, a minimal or slight central hepatocellular hypertrophy was observed in females treated at 12000 ppm that correlated with the increased liver weights. The hypertrophy is assessed treatment-related and adaptive. In addition, a minimal central fatty change was noted in five females treated at 4000 ppm as well as in four females treated at 12000 ppm. Although the central fatty change occurred only in some females and the severity was only minimal, a treatment-related effect cannot be ruled out but is assessed as non-adverse. In the thyroid glands the number of males and females with minimal follicular hypertrophy/ hyperplasia was increased in animals treated at 12000 ppm. In affected animals the number of small follicles was minimally increased or the follicular epithelium was minimal higher. It was a very weak effect that might be related to treatment but is assessed as non adverse. Consequently, the NOAEL for general, systemic toxicity of the test substance is 1500 ppm (equivalent to105 mg/kg/day) for the F0 parental females based on reduction in food consumption and body weights/body weight gain, clinical pathological changes, increased liver weights and central hepatocellular hypertrophy and central fatty change. The NOAEL for F0 parental males was found to be 4000 ppm (equivalent to 266 mg/kg/day) based on reduction in food consumption and body weight gain, clinical pathological changes and increased liver weights. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 422 and OPPTS 870.3650. This data may be used as key data, as nerolidol is a suitable read-across source to the target substance based on similar chemical structure and hazard data.

In the supporting study with the source compound, daily oral administration of 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- at doses of 500 or 1000 mg/kg/day for 28 days was minimally toxic to CD rats. 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- induced no mortality, had no effects on body weight or body weight gain, induced no evidence of toxicity that was identifiable through clinical observation, had no influence on hematology or coagulation parameters, and induced no gross or microscopic alterations in organ structure. Modest, but statistically significant effects on clinical chemistry parameters were observed on day 29; these clinical pathology changes were reversed upon discontinuation of exposure. The primary indicators of 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- toxicity were statistically significant, dose-related increases in liver and kidney weights that were identified at the end of the dosing period. The effects of farnesol on liver and kidney weights were reversible after a 28-day recovery period. Because the observed increases in liver and kidney weights in 2,6,10-Dodecatrien-1-ol, 3,7,11 -trimethyl-treated rats were not associated either with any changes in clinical pathology parameters or any histopathologic alterations, and because these increases were reversible upon discontinuation of farnesol exposure, it is considered likely that farnesol effects on liver and kidney weights are secondary to the induction of drug metabolizing enzymes. The study is considered to be reliable with restrictions (Klimisch 2), as no guideline or GLP-compliance was reported, and the experimental design included only two treatment groups and a control.

Justification for classification or non-classification

The lowest NOAEL for target organ toxicity from both the key and supporting repeat-dose toxicity studies is 105 mg/kg/day, taken from the repeat dose oral exposure study in rats with the read-across substance, Nerolidol. This dose level is outside the classification criteria for Specific Target Organ Toxicity -Repeat Exposure (STOT-RE) Category 2 (10 to 100 mg/kg/day) as described in the CLP Regulation (EC) No 1272/2008 for repeated exposure. Therefore, the test item is not classifiable for this endpoint.

Further to this justification it could be considered that the target organ toxicity (liver) demonstrated in both these studies is an adaptive metabolic response which was shown in the supporting study to be completely reversible at higher dose levels after a 28-day recovery period. However, hepatotoxicants can produce secondary effects over time and these studies are only of 28 days duration.