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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The reliability is based on read across for a structural analogue compound, myrcene (CAS 123-35-3). The myrcene study report was conclusive and done to valid OECD and EU guidelines and the study was conducted under GLP conditions. The study for myrcene was conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
Myrcene and Farnesene have similar chemical structures and therefore are expected to have similar physical/chemical characteristics. Thus, read across of myrcene data is a reasonable approach.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The reliability is based on read across for a structural analogue compound, myrcene (CAS 123-35-3). The myrcene study report was conclusive and done to valid OECD and EU guidelines and the study was conducted under GLP conditions. The study for myrcene was conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
Myrcene and Farnesene have similar chemical structures and therefore are expected to have similar physical/chemical characteristics. Thus, read across of myrcene data is a reasonable approach.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, USA)
- Age at study initiation: 5 weeks
- Housing: 5 animals/cage; polycarbonate cages; changed at least twice weekly
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardners, USA), ad libitum; changed at least weekly
- Water (e.g. ad libitum): Tap water (City of Columbus municipal supply) via automatic watering system, ad libitum
- Acclimation period: 11 (males) or 12 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3 °F
- Humidity (%): 50 ± 15 %
- Air changes (per h): 10/h
- Photoperiod (h dark / h light): 12 h dark / 12 h fluorescent light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- USP-grade corn oil was obtained in multiple lots from Spectrum Chemicals and Laboratory Products (Gardena,
CA) and was used as the vehicle. Periodic analyses of the corn oil vehicle
performed by the study laboratory using potentiometric titration demonstrated peroxide concentrations below the
acceptable limit of 3 mEq/kg.

The appropriate volumes of beta-myrcene and corn oil were
combined in a calibrated glass mixing container and mixed
on a paint shaker for 5 minutes. The dose
formulations were prepared approximately monthly.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies of a 50 mg/mL beta-myrcene dose formulation were performed by the analytical chemistry laboratory
on lot 09116TQ obtained from Aldrich Chemical Company (Milwaukee, WI). Analyses were performed with a
GC-FID Hewlett-Packard (Palo Alto, CA) system that included a Stabilwax® Crossbond® 30 m × 0.25 mm, 0.25-
μm film thickness column (Restek), helium carrier gas at a flow rate of 1.5 mL/minute, and an oven temperature
program starting at 40° C for 3 minutes and then increased to 220° C at 15° C/minute. Stability was confirmed for
at least 37 days for dose formulations stored in amber glass bottles sealed with Teflon®-lined lids at temperatures
up to room temperature, and for up to 3 hours under simulated animal room conditions.
Periodic analyses of the dose formulations of beta-myrcene were conducted by the study laboratory using GC-FID by
the system used for the purity analyses. During the 3-month studies, the dose formulations were analyzed at the
beginning, midpoint, and end of the studies; all 13 dose formulations analyzed for rats and all 15 for mice were
within 10% of the target concentrations. Animal room samples of these dose formulations were also
analyzed; all animal room samples for were within 10% of the target concentrations.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 times/week
Remarks:
Doses / Concentrations:
0.25g/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.5 g/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1 g/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2 g/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
4 g/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 males
10 females
+ 10 males/females administered same dose for 23 days.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: published data
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Additional groups of 10 males and 10 female special study rats were administered the same doses for 23 days.
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Initially, weekly and at the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, weekly and at the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 23 and at the end of the study
- Anaesthetic used for blood collection: Yes (CO2/O2 mixture)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined: Hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 23 and at the end of the study
- Anaesthetic used for blood collection: Yes (CO2/O2 mixture)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined: Urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbital dehydrogenase, and total bile acids

REPRODUCTIVE PARAMETERS: Yes
- Time schedule for examinations: Sperm samples were collected at the end of the study; vaginal samples were collected for up to 12 consecutive days prior to the end of the studies
- How many animals: Sperm samples were collected from core study male animals in the 0, 250, 500, and 1000 mg/kg bw/day groups for sperm motility evaluations; vaginal samples were collected from core study females dosed with 0, 250, 500, and 1000 mg/kg bw/day for vaginal cytology evaluations
- Parameters examined: Sperm samples: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration; Vaginal samples: Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage; % of time spent in various estrous cycle stages and estrous cycle length were evaluated
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsies were performed on all core study animals and organs weighed were heart, right kidney, liver, lung, right testis, thymus; left cauda, left epididymis and left testis

HISTOPATHOLOGY: Yes
- Complete histopathologic examinations were performed on all core study rats in 0, 2000 and 4000 mg/kg bw/day groups and all animals that died early; tissues were fixed and preserved in 10% neutral buffered formalin and stained in hematoxylin/eosin
- In addition to gross lesions and tissue masses, the following tissues were examined to the no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), right testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus and right kidney
Other examinations:
None
Statistics:
- Survival: Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Organ and body weight data: Analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, spermatid and epididymal spermatozoal data: Analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964) or Jonckheere’s test (Jonckheere, 1954)
- Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across doses.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979) or chi-square statistics.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
- No dose-related clinical findings were noted in animals surviving to the end of study.
- Dose-related clinical findings that occurred in animals that died early (some by moribund sacrifice) included thinness, lethargy, abnormal breathing, and ruffled fur. The cause of death of animals that died early was not determined. No clinical findings were seen in either sex receiving 1000 mg/kg bw/day or less.


MORTALITY
- Mortality in core study rats was 0/10 male and 0/10 female at 0 and 250 mg/kg bw/day; 1/10 males and 0/10 females at 500 mg/kg bw/day; 1/10 males and 1/10 females at 1000 mg/kg bw/day; 2/10 males and 4/10 females at 2000 mg/kg bw/day and 10/10 males and 10/10 females at 4000 mg/kg bw/day.
- Special study rats in the 4000 mg/kg bw/day groups died by the end of the first week. Survival of special study rats in the 250, 500 and 1000 mg/kg bw/day groups of both sexes was similar to that of the vehicle controls.


BODY WEIGHT AND WEIGHT GAIN
- Mean body weights were significantly decreased in the 500, 1000 and 2000 mg/kg bw/day groups.


HAEMATOLOGY & CLINICAL CHEMISTRY:
- In rats on Day 23, a decrease (approximately 25 %) in leukocyte counts, characterized by a decrease in lymphocytes, occurred in 2000 mg/kg bw/day males and females and was consistent with a transient, physiological (corticosteroid-induced-type) response. Such a response is consistent with the decreases in final body weights in the 2000 mg/kg bw/day males and females.
- In core study rats at week 14, dose-related decreases (up to 30 %) in creatinine concentration occurred in males and females and would be consistent with the decreased body weights. The leukon and creatinine effects were likely secondary biological effects.
- Other changes in hematology and clinical chemistry parameters seem inconsistent between sexes and in comparison to similar or complimentary markers.


ORGAN WEIGHTS
- Absolute and relative right kidney and liver weights of both sexes in all dosed groups were significantly greater than those of the vehicle controls with the exception of the absolute liver weight of the 2000 mg/kg bw/day males.
- Decreased absolute and relative thymus weights occurred in the 2000 mg/kg bw/day males.


REPRODUCTIVE PARAMETERS:
- No significant changes seen in the weights neither of the reproductive organs nor in the sperm parameters or estrous cyclicity of the male or female rats at any dose level


HISTOPATHOLOGY: NON-NEOPLASTIC
- Kidney: In rats evaluated on Day 23, the incidences and severities of chronic progressive nephropathy (CPN) and renal tubule necrosis were increased in 2000 mg/kg bw/day males. At the end of the 3 month study, the incidences of renal tubule necrosis were significantly increased in all dosed groups of males and females. Treatment-related increases in the incidences and severities of hyaline droplet accumulation were found in 250, 500 and 1000 mg/kg bw/day males; hyaline droplet accumulation was not observed in the 2000 mg/kg bw/day males and in any of the females
- Nose: At 3 months, the incidences and severities of olfactory epithelium degeneration in 2000 mg/kg bw/day males and females were significantly increased, and the severities were increased. The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased.
- Spleen: All 2000 mg/kg bw/day males and females had splenic atrophy.
- Mesenteric lymph node: Significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females.
- Stomach: Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females.
- Eye: Incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females
Critical effects observed:
not specified

Table 1: Selected clinical pathology data for rats in the 3‑month gavage study of β-myrcenea

 

Vehicle Control

0.25 g/kg

0.5 g/kg

1 g/kg

2 g/kg

Male

Hematology

 

 

 

 

 

n

 

 

 

 

 

Day 23

10

10

10

9

5

Week 14

9

10

9

9

8

Leukocytes (103/μL)

 

 

 

 

 

Day 23

10.54 ± 0.55

10.24 ± 0.40

9.65 ± 0.37

10.09 ± 0.64

7.70 ± 0.46**

Week 14

7.32 ± 0.28

8.49 ± 0.50

7.86 ± 0.55

7.52 ± 0.61

7.33 ± 0.39

Lymphocytes (103/μL)

 

 

 

 

Day 23

9.22 ± 0.53

8.78 ± 0.42

8.43 ± 0.33

8.65 ± 0.54

6.00 ± 0.41**

Week 14

6.15 ± 0.29

7.31 ± 0.50

6.70 ± 0.56

6.45 ± 0.59

5.66 ± 0.19

Clinical Chemistry

 

 

 

 

 

n

 

 

 

 

 

Day 23

10

10

10

9

6

Week 14

10

10

9

9

8

Creatinine (mg/dL)

 

 

 

 

 

Day 23

0.44 ± 0.02

0.49 ± 0.01

0.47 ± 0.02

0.49 ± 0.01*

0.52 ± 0.02**

Week 14

0.56 ± 0.02

0.58 ± 0.01

0.50 ± 0.00*

0.47 ± 0.02**

0.40 ± 0.00**

Female

Hematology

 

 

 

 

 

n

 

 

 

 

 

Day 23

9

7

8

8

5

Week 14

10

9

10

9

6

Leukocytes (103/μL)

 

 

 

 

 

Day 23

11.08 ± 0.37

12.14 ± 0.67

10.45 ± 0.36

9.60 ± 0.58

8.36 ± 0.48**

Week 14

7.83 ± 0.66

7.53 ± 0.29

8.45 ± 0.62

8.92 ± 0.66

7.72 ± 0.52

Lymphocytes (103/μL)

 

 

 

 

Day 23

9.74 ± 0.34

10.76 ± 0.63

9.26 ± 0.34

8.46 ± 0.51

7.27 ± 0.63**

Week 14

6.76 ± 0.52

 6.23 ± 0.24

7.14 ± 0.47

7.83 ± 0.58

6.42 ± 0.43

Clinical Chemistry

 

 

 

 

 

n

10

10

10

9

6

Creatinine (mg/dL)

 

 

 

 

 

Day 23

0.49 ± 0.01

0.48 ± 0.01

0.48 ± 0.01

0.48 ± 0.02

0.48 ± 0.02

Week 14

0.57 ± 0.02

0.50 ± 0.02**

0.48 ± 0.01**

0.49 ± 0.01**

0.43 ± 0.02**

* Significantly different (P≤0.05) from the vehicle control group by Dunn’s or Shirley’s test

** P≤0.01

aMean ± standard error. Statistical tests were performed on unrounded data. No data available for 4 g/kg males or females due to 100% mortality.

Table 2:Selected organ weights and organ-weight-to-body-weight ratios for rats in the 3‑month gavage study of β-myrcenea

 

Vehicle Control

0.25 g/kg

0.5 g/kg

1 g/kg

2 g/kg

Male

 

n

10

10

9

9

8

Necropsy body wt

341 ± 7

335 ± 7

318 ± 5*

300 ± 8**

255 ± 8**

R. Kidney

 

 

 

 

 

Absolute

0.964 ± 0.020

1.186 ± 0.021**

1.306 ± 0.028**

1.524 ± 0.033**

1.792 ± 0.064**

Relative

2.826 ± 0.049

3.545 ± 0.033**

4.109 ± 0.045**

5.099 ± 0.092**

7.014 ± 0.121**

Liver

 

 

 

 

 

Absolute

11.47 ± 0.21

12.76 ± 0.35*

12.78 ± 0.29*

 13.44 ± 0.32**

12.55 ± 0.43

Relative

33.688 ± 0.742

38.084 ± 0.398**

40.205 ± 0.563**

44.930 ± 0.653**

49.121 ± 0.647**

Thymus

 

 

 

 

 

Absolute

0.350 ± 0.016

0.340 ± 0.018

0.285 ± 0.009**

0.272 ± 0.015**

0.205 ± 0.017**

Relative

1.024 ± 0.040

1.013 ± 0.048

0.899 ± 0.038

0.913 ± 0.052

0.795 ± 0.052**

Female

n

10

10

10

9

6

Necropsy body wt

196 ± 3

196 ± 3

187 ± 3

 188 ± 3

185 ± 5

R. Kidney

 

 

 

 

 

Absolute

0.633 ± 0.012

 0.799 ± 0.012**

0.828 ± 0.019**

0.953 ± 0.027**

1.197 ± 0.043**

Relative

3.229 ± 0.056

4.091 ± 0.062**

4.418 ± 0.056**

5.055 ± 0.085**

6.483 ± 0.143**

Liver

 

 

 

 

 

Absolute

5.990 ± 0.162

6.717 ± 0.109**

7.022 ± 0.164**

 7.819 ± 0.219**

9.421 ± 0.326**

Relative

30.533 ± 0.641

34.407 ± 0.622**

37.463 ± 0.463**

41.499 ± 0.831**

51.003 ± 0.867**

Thymus

 

 

 

 

 

Absolute

0.265 ± 0.009

0.256 ± 0.009

0.248 ± 0.012

0.266 ± 0.009

0.224 ± 0.008*

Relative

1.353 ± 0.046

1.313 ± 0.050

1.321 ± 0.057

1.410 ± 0.036

1.213 ± 0.042

* Significantly different (P≤0.05) from the vehicle control group by Williams’ or Dunnett’s test

** P ≤ 0.01

aOrgan weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error). No data available for 4 g/kg males or females due to 100% mortality.

Table 3:Incidences of selected nonneoplastic lesions in rats in the 3‑month gavage study of β-myrcenea

 

Vehicle Control

0.25 g/kg

0.5 g/kg

1 g/kg

2 g/kg

Male

Kidneyb

7

10

9

10

10

Renal Tubule, Necrosisc

0

10** (1.0)d

9** (1.1)

10** (1.8)

10** (2.9)

Nephropathy

7 (1.0)

10 (1.0)

9 (1.3)

8 (1.0)

 9 (1.9)

Nephrosis

0

0

1 (1.0)

10** (1.0)

9** (2.7)

Renal Tubule, Accumulation, Hyaline Droplet

0

10** (2.0)

9** (2.4)

 10** (2.1)

0

Nose

10

10

10

10

10

Olfactory Epithelium, Degeneration

0

0

0

2 (1.0)

8** (2.6)

Inflammation, Suppurative

0

0

0

1 (1.0)

 3 (1.0)

Inflammation, Chronic

0

0

1 (1.0)

6** (1.0)

8** (1.1)

Spleen

10

10

10

10

10

Atrophy

0

0

1 (2.0)

0

10** (1.8)

Mesenteric Lymph Node

10

0

1

1

10

Atrophy

0

 

0

1 (1.0)

6** (1.7)

Harderian Gland

10

10

10

10

9

Pigmentation, Porphyrin

1 (1.0)

3 (1.0)

7** (1.0)

8** (1.1)

9** (1.3)

Female

 

Kidney

10

10

10

10

10

Renal Tubule, Necrosis

0

10** (1.0)

10** (1.0)

9** (2.2)

9** (2.4)

Nephropathy

1 (1.0)

 2 (1.0)

3 (1.0)

4 (1.0)

1 (1.0)

Nephrosis

0

0

0

10** (1.0)

7** (1.1)

Nose

10

0

10

10

10

Olfactory Epithelium, Degeneration

0

 

0

2 (1.5)

 7** (2.9)

Inflammation, Suppurative

0

 

0

1 (1.0)

6** (1.7)

Inflammation, Chronic

0

 

0

9** (1.0)

 4* (1.5)

Spleen

10

 

10

10

10

Atrophy

0

 

0

1 (1.0)

10** (1.8)

Mesenteric Lymph Node

10

 

0

3

10

Atrophy

0

 

 

2** (1.0)

4* (1.8)

Forestomach

10

 

0

10

10

Inflammation, Acute

0

 

 

0

4* (1.8)

* Significantly different (P≤0.05) from the vehicle control group by the Fisher exact test

** P ≤ 0.01

aNo data presented for 4 g/kg males or females due to early mortality.

bNumber of animals with tissue examined microscopically

cNumber of animals with lesion

dAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked

Conclusions:
No repeat dose tocicity studies are available for farnsene. In a read-across study the structrally related compound beta-myrcene was investigated in rats following oral administration for 3 months, at levels bewteen 250 and 4000 mg/kg/day. See read across justification section 13.
A significant increase in liver and kidney weights in males and females, associated with minimal renal tubule necrosis and only in males, hyaline droplet accumulation, were observed at 250 mg/kg bw/day, the lowest dose tested. Therefore, no NOAEL was identified and a LOAEL was set at 250 mg/kg bw/day.
Executive summary:

No repeat dose toxicity data are available for farnesene.

A read-across study to evaluate the cumulative toxic effects of repeated exposure to β‑myrcene was conducted according to the method equivalent or similar to OECD Guideline 408 in compliance with Good Laboratory Practices.

 

Groups of 20 F334/N rats (10/sex/dose) were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day β-myrcene in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses for 22 days. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.

 

All rats exposed to 4000 mg/kg bw/day died and 2/10 males and 4/10 females died in the 2000 mg/kg bw/day group. Dose-related clinical findings in animals that died early included thinness, lethargy, abnormal breathing and ruffled fur. Mean body weights were significantly decreased in the 500, 1000 and 2000 mg/kg bw/day groups. A significant increase in liver and kidney weights was observed in all dosed animals as well as renal tubule necrosis. A significant decrease in creatinine was also detected in all dosed females and in males at 500 mg/kg bw/day and higher doses. The incidences and severities of olfactory epithelium degeneration were increased in males and females of the 2000 mg/kg bw/day group.The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased. All 2000 mg/kg bw/day males and females had splenic atrophy. In the mesenteric lymph node, significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females. Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females. The incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.

 

As a significant increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females, as well as hyaline droplet accumulation only in males, were observed at 250 mg/kg bw/day, no NOAEL could be identified in this study. The LOAEL was set at 250 mg/kg bw/day.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): beta-myrcene
- Molecular formula (if other than submission substance): C10H16
- Molecular weight (if other than submission substance): 136.24
- Smiles notation (if other than submission substance): See attached
- Structural formula attached as image file (if other than submission substance): see attached
Name of test material (as cited in study report): β-Myrcene
- Source: Millennium Specialty Chemicals (Jacksonville, USA)
- Analytical purity: > 90 %
- Impurities (identity and concentrations): Total impurities (9.55 %): psi-limonene (4.4 %), dl-limonene (1.4 %), water (< 0.016 %)
- Lot/batch No.: 0LB410
- Physical state: Clear to slightly yellow liquid
- Stability under test conditions: No degradation was detected during storage period
- Storage condition of test material: Stored in amber glass bottles sealed with Teflon®-lined lids ≤ –20 °C

Test animals

Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, USA)
- Age at study initiation: 5 weeks
- Housing: 5 animals/cage; polycarbonate cages; changed at least twice weekly
- Diet (e.g. ad libitum): Irradiated NTP-2000 wafer feed (Zeigler Brothers, Inc., Gardners, USA), ad libitum; changed at least weekly
- Water (e.g. ad libitum): Tap water (City of Columbus municipal supply) via automatic watering system, ad libitum
- Acclimation period: 11 (males) or 12 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 3 °F
- Humidity (%): 50 ± 15 %
- Air changes (per h): 10/h
- Photoperiod (h dark / h light): 12 h dark / 12 h fluorescent light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- USP-grade corn oil was obtained in multiple lots from Spectrum Chemicals and Laboratory Products (Gardena,
CA) and was used as the vehicle. Periodic analyses of the corn oil vehicle
performed by the study laboratory using potentiometric titration demonstrated peroxide concentrations below the
acceptable limit of 3 mEq/kg.

The appropriate volumes of beta-myrcene and corn oil were
combined in a calibrated glass mixing container and mixed
on a paint shaker for 5 minutes. The dose
formulations were prepared approximately monthly.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies of a 50 mg/mL beta-myrcene dose formulation were performed by the analytical chemistry laboratory
on lot 09116TQ obtained from Aldrich Chemical Company (Milwaukee, WI). Analyses were performed with a
GC-FID Hewlett-Packard (Palo Alto, CA) system that included a Stabilwax® Crossbond® 30 m × 0.25 mm, 0.25-
μm film thickness column (Restek), helium carrier gas at a flow rate of 1.5 mL/minute, and an oven temperature
program starting at 40° C for 3 minutes and then increased to 220° C at 15° C/minute. Stability was confirmed for
at least 37 days for dose formulations stored in amber glass bottles sealed with Teflon®-lined lids at temperatures
up to room temperature, and for up to 3 hours under simulated animal room conditions.
Periodic analyses of the dose formulations of beta-myrcene were conducted by the study laboratory using GC-FID by
the system used for the purity analyses. During the 3-month studies, the dose formulations were analyzed at the
beginning, midpoint, and end of the studies; all 13 dose formulations analyzed for rats and all 15 for mice were
within 10% of the target concentrations. Animal room samples of these dose formulations were also
analyzed; all animal room samples for were within 10% of the target concentrations.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 times/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.25g/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0.5 g/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1 g/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2 g/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
4 g/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 males
10 females
+ 10 males/females administered same dose for 23 days.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: published data
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Additional groups of 10 males and 10 female special study rats were administered the same doses for 23 days.
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Initially, weekly and at the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, weekly and at the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 23 and at the end of the study
- Anaesthetic used for blood collection: Yes (CO2/O2 mixture)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined: Hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 23 and at the end of the study
- Anaesthetic used for blood collection: Yes (CO2/O2 mixture)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined: Urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbital dehydrogenase, and total bile acids

REPRODUCTIVE PARAMETERS: Yes
- Time schedule for examinations: Sperm samples were collected at the end of the study; vaginal samples were collected for up to 12 consecutive days prior to the end of the studies
- How many animals: Sperm samples were collected from core study male animals in the 0, 250, 500, and 1000 mg/kg bw/day groups for sperm motility evaluations; vaginal samples were collected from core study females dosed with 0, 250, 500, and 1000 mg/kg bw/day for vaginal cytology evaluations
- Parameters examined: Sperm samples: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration; Vaginal samples: Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage; % of time spent in various estrous cycle stages and estrous cycle length were evaluated
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsies were performed on all core study animals and organs weighed were heart, right kidney, liver, lung, right testis, thymus; left cauda, left epididymis and left testis

HISTOPATHOLOGY: Yes
- Complete histopathologic examinations were performed on all core study rats in 0, 2000 and 4000 mg/kg bw/day groups and all animals that died early; tissues were fixed and preserved in 10% neutral buffered formalin and stained in hematoxylin/eosin
- In addition to gross lesions and tissue masses, the following tissues were examined to the no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), right testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus and right kidney
Other examinations:
None
Statistics:
- Survival: Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Organ and body weight data: Analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, spermatid and epididymal spermatozoal data: Analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964) or Jonckheere’s test (Jonckheere, 1954)
- Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across doses.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979) or chi-square statistics.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
- No dose-related clinical findings were noted in animals surviving to the end of study.
- Dose-related clinical findings that occurred in animals that died early (some by moribund sacrifice) included thinness, lethargy, abnormal breathing, and ruffled fur. The cause of death of animals that died early was not determined. No clinical findings were seen in either sex receiving 1000 mg/kg bw/day or less.


MORTALITY
- Mortality in core study rats was 0/10 male and 0/10 female at 0 and 250 mg/kg bw/day; 1/10 males and 0/10 females at 500 mg/kg bw/day; 1/10 males and 1/10 females at 1000 mg/kg bw/day; 2/10 males and 4/10 females at 2000 mg/kg bw/day and 10/10 males and 10/10 females at 4000 mg/kg bw/day.
- Special study rats in the 4000 mg/kg bw/day groups died by the end of the first week. Survival of special study rats in the 250, 500 and 1000 mg/kg bw/day groups of both sexes was similar to that of the vehicle controls.


BODY WEIGHT AND WEIGHT GAIN
- Mean body weights were significantly decreased in the 500, 1000 and 2000 mg/kg bw/day groups.


HAEMATOLOGY & CLINICAL CHEMISTRY:
- In rats on Day 23, a decrease (approximately 25 %) in leukocyte counts, characterized by a decrease in lymphocytes, occurred in 2000 mg/kg bw/day males and females and was consistent with a transient, physiological (corticosteroid-induced-type) response. Such a response is consistent with the decreases in final body weights in the 2000 mg/kg bw/day males and females.
- In core study rats at week 14, dose-related decreases (up to 30 %) in creatinine concentration occurred in males and females and would be consistent with the decreased body weights. The leukon and creatinine effects were likely secondary biological effects.
- Other changes in hematology and clinical chemistry parameters seem inconsistent between sexes and in comparison to similar or complimentary markers.


ORGAN WEIGHTS
- Absolute and relative right kidney and liver weights of both sexes in all dosed groups were significantly greater than those of the vehicle controls with the exception of the absolute liver weight of the 2000 mg/kg bw/day males.
- Decreased absolute and relative thymus weights occurred in the 2000 mg/kg bw/day males.


REPRODUCTIVE PARAMETERS:
- No significant changes seen in the weights neither of the reproductive organs nor in the sperm parameters or estrous cyclicity of the male or female rats at any dose level


HISTOPATHOLOGY: NON-NEOPLASTIC
- Kidney: In rats evaluated on Day 23, the incidences and severities of chronic progressive nephropathy (CPN) and renal tubule necrosis were increased in 2000 mg/kg bw/day males. At the end of the 3 month study, the incidences of renal tubule necrosis were significantly increased in all dosed groups of males and females. Treatment-related increases in the incidences and severities of hyaline droplet accumulation were found in 250, 500 and 1000 mg/kg bw/day males; hyaline droplet accumulation was not observed in the 2000 mg/kg bw/day males and in any of the females
- Nose: At 3 months, the incidences and severities of olfactory epithelium degeneration in 2000 mg/kg bw/day males and females were significantly increased, and the severities were increased. The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased.
- Spleen: All 2000 mg/kg bw/day males and females had splenic atrophy.
- Mesenteric lymph node: Significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females.
- Stomach: Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females.
- Eye: Incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.

Effect levels

Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Selected clinical pathology data for rats in the 3‑month gavage study of β-myrcenea

 

Vehicle Control

0.25 g/kg

0.5 g/kg

1 g/kg

2 g/kg

Male

Hematology

 

 

 

 

 

n

 

 

 

 

 

Day 23

10

10

10

9

5

Week 14

9

10

9

9

8

Leukocytes (103/μL)

 

 

 

 

 

Day 23

10.54 ± 0.55

10.24 ± 0.40

9.65 ± 0.37

10.09 ± 0.64

7.70 ± 0.46**

Week 14

7.32 ± 0.28

8.49 ± 0.50

7.86 ± 0.55

7.52 ± 0.61

7.33 ± 0.39

Lymphocytes (103/μL)

 

 

 

 

Day 23

9.22 ± 0.53

8.78 ± 0.42

8.43 ± 0.33

8.65 ± 0.54

6.00 ± 0.41**

Week 14

6.15 ± 0.29

7.31 ± 0.50

6.70 ± 0.56

6.45 ± 0.59

5.66 ± 0.19

Clinical Chemistry

 

 

 

 

 

n

 

 

 

 

 

Day 23

10

10

10

9

6

Week 14

10

10

9

9

8

Creatinine (mg/dL)

 

 

 

 

 

Day 23

0.44 ± 0.02

0.49 ± 0.01

0.47 ± 0.02

0.49 ± 0.01*

0.52 ± 0.02**

Week 14

0.56 ± 0.02

0.58 ± 0.01

0.50 ± 0.00*

0.47 ± 0.02**

0.40 ± 0.00**

Female

Hematology

 

 

 

 

 

n

 

 

 

 

 

Day 23

9

7

8

8

5

Week 14

10

9

10

9

6

Leukocytes (103/μL)

 

 

 

 

 

Day 23

11.08 ± 0.37

12.14 ± 0.67

10.45 ± 0.36

9.60 ± 0.58

8.36 ± 0.48**

Week 14

7.83 ± 0.66

7.53 ± 0.29

8.45 ± 0.62

8.92 ± 0.66

7.72 ± 0.52

Lymphocytes (103/μL)

 

 

 

 

Day 23

9.74 ± 0.34

10.76 ± 0.63

9.26 ± 0.34

8.46 ± 0.51

7.27 ± 0.63**

Week 14

6.76 ± 0.52

 6.23 ± 0.24

7.14 ± 0.47

7.83 ± 0.58

6.42 ± 0.43

Clinical Chemistry

 

 

 

 

 

n

10

10

10

9

6

Creatinine (mg/dL)

 

 

 

 

 

Day 23

0.49 ± 0.01

0.48 ± 0.01

0.48 ± 0.01

0.48 ± 0.02

0.48 ± 0.02

Week 14

0.57 ± 0.02

0.50 ± 0.02**

0.48 ± 0.01**

0.49 ± 0.01**

0.43 ± 0.02**

* Significantly different (P≤0.05) from the vehicle control group by Dunn’s or Shirley’s test

** P≤0.01

aMean ± standard error. Statistical tests were performed on unrounded data. No data available for 4 g/kg males or females due to 100% mortality.

Table 2:Selected organ weights and organ-weight-to-body-weight ratios for rats in the 3‑month gavage study of β-myrcenea

 

Vehicle Control

0.25 g/kg

0.5 g/kg

1 g/kg

2 g/kg

Male

 

n

10

10

9

9

8

Necropsy body wt

341 ± 7

335 ± 7

318 ± 5*

300 ± 8**

255 ± 8**

R. Kidney

 

 

 

 

 

Absolute

0.964 ± 0.020

1.186 ± 0.021**

1.306 ± 0.028**

1.524 ± 0.033**

1.792 ± 0.064**

Relative

2.826 ± 0.049

3.545 ± 0.033**

4.109 ± 0.045**

5.099 ± 0.092**

7.014 ± 0.121**

Liver

 

 

 

 

 

Absolute

11.47 ± 0.21

12.76 ± 0.35*

12.78 ± 0.29*

 13.44 ± 0.32**

12.55 ± 0.43

Relative

33.688 ± 0.742

38.084 ± 0.398**

40.205 ± 0.563**

44.930 ± 0.653**

49.121 ± 0.647**

Thymus

 

 

 

 

 

Absolute

0.350 ± 0.016

0.340 ± 0.018

0.285 ± 0.009**

0.272 ± 0.015**

0.205 ± 0.017**

Relative

1.024 ± 0.040

1.013 ± 0.048

0.899 ± 0.038

0.913 ± 0.052

0.795 ± 0.052**

Female

n

10

10

10

9

6

Necropsy body wt

196 ± 3

196 ± 3

187 ± 3

 188 ± 3

185 ± 5

R. Kidney

 

 

 

 

 

Absolute

0.633 ± 0.012

 0.799 ± 0.012**

0.828 ± 0.019**

0.953 ± 0.027**

1.197 ± 0.043**

Relative

3.229 ± 0.056

4.091 ± 0.062**

4.418 ± 0.056**

5.055 ± 0.085**

6.483 ± 0.143**

Liver

 

 

 

 

 

Absolute

5.990 ± 0.162

6.717 ± 0.109**

7.022 ± 0.164**

 7.819 ± 0.219**

9.421 ± 0.326**

Relative

30.533 ± 0.641

34.407 ± 0.622**

37.463 ± 0.463**

41.499 ± 0.831**

51.003 ± 0.867**

Thymus

 

 

 

 

 

Absolute

0.265 ± 0.009

0.256 ± 0.009

0.248 ± 0.012

0.266 ± 0.009

0.224 ± 0.008*

Relative

1.353 ± 0.046

1.313 ± 0.050

1.321 ± 0.057

1.410 ± 0.036

1.213 ± 0.042

* Significantly different (P≤0.05) from the vehicle control group by Williams’ or Dunnett’s test

** P ≤ 0.01

aOrgan weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error). No data available for 4 g/kg males or females due to 100% mortality.

Table 3:Incidences of selected nonneoplastic lesions in rats in the 3‑month gavage study of β-myrcenea

 

Vehicle Control

0.25 g/kg

0.5 g/kg

1 g/kg

2 g/kg

Male

Kidneyb

7

10

9

10

10

Renal Tubule, Necrosisc

0

10** (1.0)d

9** (1.1)

10** (1.8)

10** (2.9)

Nephropathy

7 (1.0)

10 (1.0)

9 (1.3)

8 (1.0)

 9 (1.9)

Nephrosis

0

0

1 (1.0)

10** (1.0)

9** (2.7)

Renal Tubule, Accumulation, Hyaline Droplet

0

10** (2.0)

9** (2.4)

 10** (2.1)

0

Nose

10

10

10

10

10

Olfactory Epithelium, Degeneration

0

0

0

2 (1.0)

8** (2.6)

Inflammation, Suppurative

0

0

0

1 (1.0)

 3 (1.0)

Inflammation, Chronic

0

0

1 (1.0)

6** (1.0)

8** (1.1)

Spleen

10

10

10

10

10

Atrophy

0

0

1 (2.0)

0

10** (1.8)

Mesenteric Lymph Node

10

0

1

1

10

Atrophy

0

 

0

1 (1.0)

6** (1.7)

Harderian Gland

10

10

10

10

9

Pigmentation, Porphyrin

1 (1.0)

3 (1.0)

7** (1.0)

8** (1.1)

9** (1.3)

Female

 

Kidney

10

10

10

10

10

Renal Tubule, Necrosis

0

10** (1.0)

10** (1.0)

9** (2.2)

9** (2.4)

Nephropathy

1 (1.0)

 2 (1.0)

3 (1.0)

4 (1.0)

1 (1.0)

Nephrosis

0

0

0

10** (1.0)

7** (1.1)

Nose

10

0

10

10

10

Olfactory Epithelium, Degeneration

0

 

0

2 (1.5)

 7** (2.9)

Inflammation, Suppurative

0

 

0

1 (1.0)

6** (1.7)

Inflammation, Chronic

0

 

0

9** (1.0)

 4* (1.5)

Spleen

10

 

10

10

10

Atrophy

0

 

0

1 (1.0)

10** (1.8)

Mesenteric Lymph Node

10

 

0

3

10

Atrophy

0

 

 

2** (1.0)

4* (1.8)

Forestomach

10

 

0

10

10

Inflammation, Acute

0

 

 

0

4* (1.8)

* Significantly different (P≤0.05) from the vehicle control group by the Fisher exact test

** P ≤ 0.01

aNo data presented for 4 g/kg males or females due to early mortality.

bNumber of animals with tissue examined microscopically

cNumber of animals with lesion

dAverage severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked

Applicant's summary and conclusion

Conclusions:
No repeat dose tocicity studies are available for farnsene. In a read-across study the structrally related compound beta-myrcene was investigated in rats following oral administration for 3 months, at levels bewteen 250 and 4000 mg/kg/day. See read across justification section 13.
A significant increase in liver and kidney weights in males and females, associated with minimal renal tubule necrosis and only in males, hyaline droplet accumulation, were observed at 250 mg/kg bw/day, the lowest dose tested. Therefore, no NOAEL was identified and a LOAEL was set at 250 mg/kg bw/day.
Executive summary:

No repeat dose toxicity data are available for farnesene.

A read-across study to evaluate the cumulative toxic effects of repeated exposure to β‑myrcene was conducted according to the method equivalent or similar to OECD Guideline 408 in compliance with Good Laboratory Practices.

 

Groups of 20 F334/N rats (10/sex/dose) were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day β-myrcene in corn oil by gavage, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female special study rats were administered the same doses for 22 days. Parameters evaluated included survival, clinical observations, body weight, hematological and biochemical estimations in blood, reproductive parameters, necropsy and histopathological examination in all animals.

 

All rats exposed to 4000 mg/kg bw/day died and 2/10 males and 4/10 females died in the 2000 mg/kg bw/day group. Dose-related clinical findings in animals that died early included thinness, lethargy, abnormal breathing and ruffled fur. Mean body weights were significantly decreased in the 500, 1000 and 2000 mg/kg bw/day groups. A significant increase in liver and kidney weights was observed in all dosed animals as well as renal tubule necrosis. A significant decrease in creatinine was also detected in all dosed females and in males at 500 mg/kg bw/day and higher doses. The incidences and severities of olfactory epithelium degeneration were increased in males and females of the 2000 mg/kg bw/day group.The incidences of chronic inflammation in 1000 and 2000 mg/kg bw/day males and females were significantly increased. All 2000 mg/kg bw/day males and females had splenic atrophy. In the mesenteric lymph node, significantly increased incidences of atrophy occurred in 2000 mg/kg bw/day males and 1000 and 2000 mg/kg bw/day females. Acute inflammation of the forestomach occurred in four 2000 mg/kg bw/day females. The incidences of porphyrin pigmentation in the Harderian gland of males administered 500 mg/kg bw/day or greater were significantly increased.

 

As a significant increase in liver and kidney weights associated with minimal renal tubule necrosis in males and females, as well as hyaline droplet accumulation only in males, were observed at 250 mg/kg bw/day, no NOAEL could be identified in this study. The LOAEL was set at 250 mg/kg bw/day.