Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
6 January 2006 to 18 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid and/or internationally accepted testing guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dimyrcetol
- Physical state: clear, colorless liquid
- Analytical purity: 99% (Certificate of Purity)
- Impurities: None identified
- Purity test date: 15 December 2005
- Lot/batch No.: SM/4103311 and SM/5073201
- Expiration date of the lot/batch: December 2006
- Stability under test conditions: Solutions in corn oil determined to be stable for at least 14 days
- Storage condition of test material: room temperature in the dark until 6 January 2006, thereafter approximately 4 deg C in the dark
- Other: The integrity of the supplied data relating to the identity, purity and stability of the test material was the responsibility of the Sponsor.

Statement from the Test Article Supplier (International Flavors and Fragrances, Lot SM 410331)
Dimyrcetol is a fragrance ingredient manufactured by IFF. It is not a single component but a mixture of two chemical entities, 2,6-dimethyl-7-octen-2-ol formate and 2,6-dimethyl-7-octen-2-ol. The percentage of each component from the certificate of analysis dated December 15, 2005, is 54.8% and 44.2%, respectively. These two components derive from the chemistry used to manufacture dimyrcetol. It is an equilibrium mixture of tertiary formate and tertiary alcohol.

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Cr1:CD IGS (SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Males: 228 to 286 g; Females: 174 to 235 g
- Fasting period before study: None
- Housing: In groups of 3 or 4 by sex in polypropylene grid-floor suspended cages
- Diet: Pelleted diet, Rodent 5LF2 (Certified), ad libitum
- Water: Mains tap, ad libitum
- Acclimation period: 20 days after receipt
- Environmental enrichment: wooden chew blocks (B&K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd., Chesire, UK)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 15 +/- 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 6 January 2006 To: 18 September 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in corn oil (see Table 1). The stability and homogeneity of the test material formulations were determined by SafePharm Analytical Laboratory. The material was sufficiently stable to allow weekly preparation. These were stored at 4 deg C in the dark.

VEHICLE
- Justification for use and choice of vehicle: The test material was sufficiently soluble and stable in corn oil
- Concentration in vehicle: See Table 1
- Amount of vehicle (if gavage): constant 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material was determined by gas chromatography (GC) using and external standard technique. Test material formulations were diluted with acetone to give final, theoretical concentrations of approximately 0.1 mg/ml. Standard solutions of the test material was prepared in acetone at nominal concentrations of 0.1 mg/ml.

Homogeneity Determinations
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.

Stability Determinations
The test material formulations were sampled and analyzed initially and then after storage at approximately +4 deg C in the dark for 14 days.

Verification of Test Material Formulations
The test material formulations were sampled and analyzed within 3 days of preparation. The concentration found of test material in formulations was from 91 to 110 % of the nominal.

The mean concentrations of test material (and % of nominal) in the formulations over the dosing period was as follows: 2.41 mg/ml (97%); 11.9 (95%); 121 (97%); and 242 (97%).
Duration of treatment / exposure:
Ninety consecutive days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 50, 500 and 1000 mg/kg/day
Basis:
other: nominal in corn oil
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The test material was administered by gavage for 90 consecutive days using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated identically with 4 ml/kg/day of corn oil. The volume of test material and control (vehicle) administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

The dose levels were chosen based on the results of a range-finder study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before and after dosing and one to five hours after dosing during the work week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12, functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, and tail elevation.

BODY WEIGHT: Yes
Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.
FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
Weekly food efficiency values were calculated

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Daily visual inspection of water bottles during Week 1 revealed possible intergroup differences. Water consumption was therefore measured and recorded for each group from Day 15 onwards.

OPHTHALMOSCOPIC EXAMINATION:
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observations of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% "Tropicamide" solution (Alcon Laboratories (UK) Ltd., Imperial Way, Watford, Hertfordshire),

HAEMATOLOGY:
Haematological investigations were performed on all animals from each test and control group during Week 7 and at the end of the study (Day 90). Blood samples were obtained from a lateral tail vein. Where necessary, repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling. The following parameters were measured: hemoglobin, erythrocyte count, hematocrit, erythrocyte indices (mean corpuscular hemoglobin, volume, hemoglobin concentration), total leucocyte count, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), and platelet count. Cresyl blue stained slides were prepared for reticulocyte counts but were not assessed. Prothrombin time was assessed by 'Thrombomax HS with calcium' and Activated partial thromboplastin time by 'Actin FS' using samples collected in sodium citrate (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
Blood chemical analyses were performed on all animals from each test group during Week 7 and at the end of the study (Day 90). The following were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, and total bilirubin.

URINALYSIS: Yes
Urine analyses were performed on all animals from each test group and control during Week 7 and at the end of the study (Day 90). Urine was measure for volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, and blood.

NEUROBEHAVIOURAL EXAMINATION: Yes
Prior to the start of testing and during Week 12, functional/behavioral toxicity was monitored for all animals.
Motor Activity: Twenty purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the monitors. Tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation time was 30 minutes for each animal. The time each animal was active and mobile was recorded for the overall 30 minute period and also during the last 20% of the period (considered to be asymptotic).

Forelimb/Hindlimb Grip Strength: An automated grip-strength monitor was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was recorded. Three consecutive trials were performed for each animal.

Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. These included: grasp response, vocalization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex, and blink reflex.

OTHER:
Oestrous cycles were analyzed during the study. During weeks 6 and 7 and weeks 12 and 13, vaginal smears were taken daily and a sample placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrous was recorded.
Sacrifice and pathology:
Sacrifice
On completion of dosing, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.

Gross Pathology
All animals were subjected to full external and internal examinations, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, and uterus.

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta, bone with marrow (femur including stifle join), bone and bone marrow (sternum), brain (including cerebrum, cerebellum and pons) caecum, colon, duodenum, right epidiymides, eyes, gross lesions, heart, ileum (including Peyer's patches), jejunum, kidneys, liver, lungs with bronchi, lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, midthoracic and lumbar), spleen, stomach, right testis, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, and uterus. Testes preserved in Bouins fluid and then 70% IMS after forty-eight hours. Eyes fixed in Davidson's fluid. Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative. All tissues from control and 1000 mg/kg/day dose group animals were prepared as parafin blocks, sectioned at 5 micrometers, and stained with hematoxylin and eosin for subsequent microscopic analysis. Since there were indications of treatment-related hepatic, renal and bone marrow changes, examinations of these tissues were prepared for all other treatment groups.
Other examinations:
Semen Assessment
At necropsy, the following evaluations were performed on all males:
i) The left testes and epididymis were removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at aprox. -20 deg C. At an appropriate later date the tissue were thawed, re-weighed and homogenized in a suitable saline/detergent mixture. Samples of the homogenate were stained with a DNA-specific fluorescent stain and a sub-sample was analyzed for numbers of homogenization-resistant spermatids.
iii) For the epididymis, the distal region was incised and a sample of the luminal fluid collected and transferred to a buffered solution for analysis of sperm motility and sperm morphology. A minimum of 200 individual sperm were assessed using an automated semen analyzer, to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using a computer assisted sperm analyzer.
iv) A sample of semen was preserved in formalin and then stained with eosin. A sub-sample was placed on a glass slide with a coverslop and morphometric analysis of sample semen performed manually.
v) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then frozen at approx. -20 deg C. At an appropriate later date the tissue was thawed, reweighed and homogenized in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a subsample was analyzed for homogenization resistant spermatids.
vi) For both ii) and iv) above, samples from control and high dose groups were evaluated only as no significant effects were seen.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Where appropriate, quantitative data were analyzed by the Provantis Tables(TM) and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analyzed to find the lowest treatment level showing a significant effect, using William's test for parametric data or Shirley's test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analyzed by a stepwise Dunnett (parametric) or Steel (non-parametric). Sperm analysis was assessed using Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.

Histopathology data were analyzed using the following methods to determine significant differences between control and treatment groups for the individual sexes: Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater; Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinically observable signs of toxicity were detected in test or control animals throughout the study period.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights in high dose male rats were decreased versus controls at week 13 but the reduction was not statistically significant. Statistically significant reductions in bodyweight gain were evident in animals of both sexes treated at 500 or 1000 mg/kg/day (Table 2). These reductions were generally sporadic in nature, were present generally only at the highest dose level and were not statistically significant at week 13. No such effects were observed at the 50 or 10 mg/kg/day dose levels.

FOOD CONSUMPTION/FOOD EFFICIENCY
Animals of both sexes treated with either 500 or 1000 mg/kg/day showed reductions in mean food efficiency (data not shown). No statistical significance was assigned to these results. No such effects were seen at the lower dose levels.

WATER CONSUMPTION
Animals of both sexes treated with either 500 or 1000 mg/kg/day showed increases in water consumption from day 15 onwards (data not shown). In males, overall water consumption was increased (>10%) in the three highest dose groups at week 13 and in female rats in the two highest dose groups (Table 3). No such effects were detected at lower dose levels.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related ocular effects were observed.

HAEMATOLOGY
During Week 7, males treated with 1000 and 500 mg/kg/day showed statisitically significant reductions in platelet counts. Animals of either sex treated with 1000 mg/kg/day and females treated with 500 mg/kg/day showed statistically significant reductions in activated partial thromboplastim time (APPT, Table 4). Week 13 assessments revealed reductions in hemoglobin, hematocrit, and erythrocyte counts for females treated with 1000 mg/kg/day, with the effect on erythrocyte count also extending into the female 500 mg/kg/day dose group. The effect on platelet count continued into Week 13 assessments and extended into the male 50 mg/kg/day dose group. No similar effects were noted in females treated with 50 mg/kg/day or animals of either sex treated with 10 mg/kg/day. See Table 4 (selected values).

Based on an independent and expert review of the hematological data from this study, it was concluded that dimycetol did not produce any treatment-related hematological effects (report attached).

CLINICAL CHEMISTRY
During Week 7, a statistically significant increase in cholesterol was evident for animals of either sex treated with 1000 and 500 mg/kg/day (Table 5). Males treated with 1000 mg/kg/day also showed statistically significant increases in aspartate aminotransferase and alanine aminotransferase (Table 5). Females from this treatment group also showed statistically significant increases in phosphorous and chloride (data not shown) together with statistically significant reductions in albumin/globulin ratios (Table 5).

Elevated cholesterol levels were still evident during Week 13 for animals of either sex treated at 500 and 1000 mg/kg/day (Table 5). Females treated with 1000 mg/kg/day also continued to show a statistically significant reduction in albumin/globulin ratio (Table 5). Elevations in plasma urea and creatinine levels were evident in males treated with 1000, 500 and 50 mg/kg/day (Table 5). An association between the slight elevations and the renal abnormalities detected cannot be entirely excluded. The majority of values, however, were within the range of historical control data and in the absence of a true dose related response the possibility of kidney dysfunction is of minimal biological importance. No such effects were reported in females treated with 50 mg/kg/day or animals of either sex at 10 mg/kg/day.

OESTRUS CYCLE ASSESSMENTS
There were no treatment-related effects on female oestrous cycles or on the type or proportion of females with anomalous oestrous cycles.

SPERM ANALYSIS
Homogenization resistant spermatid counts in the testes of the high dose males were statistically significantly reduced versus the control (p < 0.01). A statistically significant increase in sperm counts in the cauda epididymis was recorded (p < 0.01).
In the testes:
- control counts (millions/g), 174 +/- 15.0; at 1000 mg/kg bwt/d, 136 +/- 22.5
In the cauda epididymis:
- control counts, 188.2 +/- 107.0; at 1000 mg/kg bwt/d, 372 +/- 82.6

URINALYSIS
Animals of either sex treated with 1000 mg/kg/day showed increased urine volumes during Week 7 and 13. Specific gravities were also reduced in these animals. No such effects were detected at animals treated with lower doses.

NEUROBEHAVIOUR
There were no toxicologically significant changes in the measured behavioural parameters, functional performance parameters, or sensory reactivity.

ORGAN WEIGHTS
Selected organ weight data is shown in Table 6. Animals of both sexes treated with 500 and 1000 mg/kg/day showed statistically significant increases in liver and kidney weights both absolute and relative to terminal bodyweight. Liver weight effects extended to males treated with 50 mg/kg/day. No such effects were detected in females treated at 50 mg/kg/day or in animals of either sex at 10 mg/kg/day. Adrenal weights were increased in all dose male groups but only at the highest dose in females. Similarly, thymus weights were increased in all dosed male groups with no similar effects noted in females.

GROSS PATHOLOGY
No macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
LIver: Centrilobular hepatocyte enlargement, occasionally associated with lipid-type vacuolation of hepatocytes, was observed in relation to treatment for males (p < 0.001) and for females (p < 0.001) at the 1000 mg/kg/day dose level. Two animals of either sex were similarly affected in the 500 mg/kg/day dose group and, although occasionally observed in control animals, cannot be reliably excluded as a treatment-related effect. Hepatocyte enlargement is commonly seen in rodent liver and, in the absence of inflammatory or degenerative changes, is generally considered adaptive in nature.

Kidney: A greater incidence and/or severity of groups of basophilic tubules and/or globular accumulations of eosinophilic material, were observed as a treatment-related effect in males only at 1000 mg/kg/day (p < 0.01 - 0.001), 500 mg/kg/day (p < 0.01 - 0.001), 50 mg/kg/day (p < 0.01 - 0.001), and 10 mg/kg/day (p < 0.05). These findings are consisten with hydrocarbon nephropathy, which results from an excessive accumulation of alpha-2-microglobulin, indicated by positive staining with Mallory's Heidenhain stain, in renal proximal tubular epithelial cells, frequently associated with tubular degenerative changes. Tubular necrosis was also observed for males treated with 1000 mg/kg/day (p < 0.001) or at 500 mg/kg/day (p < 0.01).

Bone Marrow: The incidence of higher grades of severity of adipose infiltration of the bone marrow was greater in treated males at the 1000 mg/kg/day dose level (p < 0.05). However, group incidences of severity varied inconsistently without evidence of a dose response. A similar effect was not observed in females. Such group differences can occur spontaneously, however, with hematological changes noted in this study in high dose animals, a marginal treatment-related effect cannot be ruled out.

OTHER FINDINGS
All remaining morphological changes were those commonly observed in laboratory rats of this age and strain and were not considered of toxicological significance.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: Bodyweight, food utilization, liver, kidney, and hematological effects.
Dose descriptor:
NOAEL
Sex:
male
Basis for effect level:
other: A greater incidence and/or severity of groups of basophilic tubules and/or globular accumulations of eosinophilic material were observed in relation to treatment for males from all treatment groups.
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: Hematological effects
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Apparent diuresis effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2: Group Mean Weekly Bodyweight Gains for Rats in 90-Day Oral Gavage Study (1)

Weekly Bodyweight Gains (SD) Relative to Start Date

Dose Group (mg/kg/day)

Base Weight Week 1- Mean (SD)

1

2

3

4

5

6

7

8

9

10

11

12

13

Abs. Gain

% Gain

Males

0 (Control)

252.8 (14.7)

55.8 (8.2)

39.6 (5.9)

37.4 (5.8)

23.6 (9.7)

22.8 (9.7)

23.1 (7.9)

11.0 (5.9)

15.9 (8.4)

18.9 (7.7)

12.6 (6.4)

12.9 (6.3)

-8.7 (3.3)

15.5 (6.0)

280.4 (58.9)

110.5 (19.5)

500

252.9 (15.9)

58.9 (9.2)

40.5 (9.0)

35.0 (5.2)

26.6 (5.2)

24.6 (6.6)

17.8* (4.8)

10.9 (5.5)

20.4 (6.2)

17.7 (8.0)

13.7 (6.0)

16.1 (6.2)

-7.7 (2.9)

21.0 (4.6)

295.5 (47.4)

116.7 (15.7)

1000

254.7 (20.0)

53.0 (10.7

36.7 (7.3)

31.6 (6.7)

24.7 (6.4)

20.2 (6.3)

17.0* (6.7)

4.0* (7.6)

17.9 (4.8)

13.6 (3.9)

8.2 (4.7)

4.6** (4.7)

-10.8 (4.9)

15.8 (6.4)

236.5 (38.1)

93.2 (15.6)

Females

0 (Control)

190.3 (9.8)

24.0 (6.7)

15.5 (6.7)

12.0 (8.0)

13.6 (4.4)

8.0 (5.7)

3.1 (7.8)

5.3 (5.9)

6.7 (10.0)

8.7 (7.2)

3.0 (4.3)

6.2 (7.1)

-10.7 (5.7)

10.1 (5.3)

105.5 (14.8)

55.5 (7.7)

500

193.0 (9.1)

19.8 (7.1)

23.9 (8.4)

16.7 (5.9)

8.3 (4.6)

12.5 (8.5)

3.4 (4.0)

5.1 (4.7)

7.7 (6.7)

3.2* (6.1)

4.3 (6.8)

8.2 (4.3)

-9.2 (4.0)

8.4 (6.0)

112.3 (22.0)

58.1 (11.0)

1000

197.5 (16.2)

26.5 (7.6)

18.9 (6.3)

10.6 (7.0)

14.2 (11.1)

14.3* (4.2)

0.4 (6.9)

0.4 (6.7)

17.0** (7.1)

-1.4** (7.2)

-2.0* (2.9)

5.8 (6.9)

-10.7 (6.7)

7.1 (10.4)

101.1 (15.3)

51.6 (9.1)

* p 0.05 ** p 0.01 (1) Data for 10 and 50 mg/kg/day dose levels not shown. Values in parentheses are SD.

Table 3: Mean Water Consumption Overall (Days 15 to 91, g/animal/day)

Males

Females

0

Control

50

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

0

Control

50

mg/kg/day

500

mg/kg/day

1000

mg/kg/day

1761

2063

2384

2658

1615

1593

1945

2839

Table 4: Selected Hematological Parameters (SD) from Rats Dosed Orally for 90 Days

7 Weeks

13 Weeks

Group mg/kg/day

Hb

g/dl

RBC

10^12/l

Hct

%

PLT

10^9/l

APPT

Seconds

Hb

g/dl

RBC

10^12/l

Hct

%

PLT

10^9/l

APPT

Seconds

Male

0 (Control

15.18 (0.68)

8.046 (0.302)

44.82 (2.01)

911.7 (87.1)

15.81 (1.98)

15.57 (0.28)

8.599 (0.250)

45.53 (1.08)

990.4 (71.9)

15.21 (2.41)

10

15.27 (0.63)

7.984 (0.193)

45.06 (1.82)

870.2 (83.0)

16.18 (0.95)

15.70 (0.61)

8.530 (0.342)

45.98 (2.19)

1006.6 (96.7)

15.63 (1.07)

50

15.33 (0.61)

8.064 (0.370)

45.34 (1.73)

890.1 (85.3)

15.82 (1.28)

15.50 (0.67)

8.524 (0.343)

45.55 (2.28)

913.9* (111.3)

14.56 (2.66)

500

15.30 (0.51)

8.058 (0.263)

45.00 (1.80)

825.6** (80.8)

15.81 (1.22)

15.52 (0.56)

8.538 (0.345)

45.49 (1.81)

895.5* (106.6)

13.68 (0.98)

1000

15.09 (0.71)

7.851 (0.429)

44.35 (2.28)

814.5** (71.2)

14.16* (2.15)

15.43 (0.49)

8.351 (0.298)

45.21 (1.54)

884.6* (93.7)

14.13 (2.32)

Female

0 (Control

15.14 (0.77)

8.063 (0.307)

43.93 (2.10)

882.2 (116.1)

14.60 (1.42)

15.18 (0.70)

7.936 (0.284)

43.78 (1.90)

1004.5 (75.2)

14.09 (1.04)

10

14.69 (0.49)

7.772 (0.438)

42.49 (1.47)

815.3 (148.1)

13.97 (1.70)

14.79 (0.58)

7.645 (0.445)

42.30 (1.93)

919.7 (87.4)

13.57 (1.05)

50

15.22 (0.45)

8.112 (0.322)

44.19 (1.27)

814.2 (147.8)

14.08 (1.74)

15.16 (0.34)

7.928 (0.236)

43.72 (0.75)

944.2 (109.5)

13.76 (2.02)

500

14.83 (0.70)

7.641 (0.471)

42.70 (2.31)

803.4 (192.1)

13.20* (1.37)

14.99 (0.48)

7.577* (0.365)

43.06 (1.49)

917.6 (93.9)

13.85 (1.54)

1000

14.91 (0.36)

7.879 (0.295)

43.21 (1.20)

838.9 (126.3)

12.92* (1.25)

14.65* (0.36)

7.647* (0.224)

42.19* (1.07)

954.1 (80.4)

13.54 (1.39)

* p 0.05 ** p 0.01. Values in parentheses are SD.

Table 5: Group Mean (SD) Blood Clinical Values

Males

Females

Dose

Level

Urea

mg/dl

A/G

Ratio

Creatinine

mg/dl

Cholesterol

mg/dl

Urea

mg/dl

A/G

Ratio

Creatinine

mg/dl

Cholesterol

mg/dl

Week 7

0

Control

22.8

(5.1)

1.204

(0.176)

0.788

(0.064)

80.4

(14.3)

37.7

(4.9)

1.191

(0.073)

0.803

(0.055)

65.0

(9.6)

10

mg/kg/d

24.2

(2.9)

1.104

(0.127)

0.793

(0.040)

78.0

(8.2)

33.1

(4.8)

1.175

(0.110)

0.762

(0.038)

66.8

(12.0)

50

mg/kg/d

25.9*

(4.8)

1.196

(0.195)

0.786

(0.044)

82.9

(11.1)

33.4

(4.3)

1.183

(0.100)

0.780

(0.042)

70.9

(9.6)

500

mg/kg/d

28.7**

(2.8)

0.965*

(0.155)

0.838

(0.049)

92.6*

(8.5)

31.4*

(5.9)

1.129

(0.083)

0.752

(0.065)

78.6**

(10.5)

1000

mg/kg/d

28.5**

(2.3)

1.070*

(0.174)

0.817

(0.019)

99.9**

(19.0)

32.1*

(9.7)

1.036**

(0.090)

0.777

(0.132)

96.7**

(10.2)

Week 13

0

Control

24.6

(2.4)

1.014

(0.100)

0.807

(0.039)

68.1

(14.7)

44.4

(14.8)

1.219

(0.092)

1.000

(0.394)

72.0

(12.3)

10

mg/kg/d

25.7

(2.1)

1.060

(0.080)

0.822

(0.054)

66.3

(8.6)

33.9**

(4.6)

1.287

(0.172)

0.896

(0.041)

80.8

(16.6)

50

mg/kg/d

27.2*

(2.2)

1.006

(0.107)

0.854*

(0.036)

75.2

(13.8)

33.5**

(6.1)

1.175

(0.308)

0.899

(0.050)

77.5

(9.4)

500

mg/kg/d

27.8*

(3.2)

0.943

(0.090)

0.869*

(0.034)

80.8*

(9.9)

30.0**

(6.9)

1.197

(0.082)

0.881

(0.064)

89.1**

(10.0)

1000

mg/kg/d

27.0*

(2.3)

0.975

(0.066)

0.820*

(0.033)

87.2**

(18.6)

30.8**

(4.8)

1.099*

(0.106)

0.904

(0.055)

110.8**

(16.6)

* p 0.05 ** p 0.01. Values in parenheses are SD.

see Overall Remarks Section for Table 6

Applicant's summary and conclusion

Executive summary:

In the conclusions from the authors of this study, the oral administration of dimyrcetol to rats for a period of 90 consecutive days at dose levels of 10, 50, 500 and 1000 mg/kg bwt/day resulted in treatment-related effects in male rats at all dose levels and in female rats at 500 and 1000 mg/kg bwt/day. For males, a NOEL value could not be established. For females, a NOEL value of 50 mg/kg bwt/day has been demonstrated. Histopathological changes in the kidneys were consistent with well-documented changes in the male rat in response to treatment with some hydrocarbons. This effect was not considered indicative of a hazard to human health, and, for the purposes of hazard evaluation, the "no-observed-adverse-effect-level" for male was assigned as 10 mg/kg bwt/day (hematological effects).

There were no treatment-related oestrus cycle effects in female rats. Measured behavioral parameters, functional performance or sensory reactivity were not affected by test chemical administration. Gross pathology revealed no macroscopic abnormalities.

Reduced spermatid counts at the highest dose level were noted in the testes of male rats, with no similar reductions in the cauda epididymis. No histopathological changes were associated with the reduced spermatid counts. It was the conclusions of the authors of this study that reduced spermatid counts were not related to dimyrcetol administration. However, different staining procedures were used to enumerate spermatids in the testes versus the cauda epididymides, thus making direct comparisons difficult. Also, histopathological analysis would not necessarily detect minimal to moderate changes in spermatid count. Due to these methodological and interpretational inconsistencies, and because only the highest dose level was assayed for spermatid counts in this study, no further conclusions can be drawn concerning the significance of these findings.

Absolute and relative liver weights were increased at the two highest dose levels. These effects extended to the 50 mg/kg/d dose level in male rats. Histopathological analysis indicated these effects were adaptive in nature and not toxicologically significant. Absolute and relative kidney weights were also significantly increased at the two highest dose levels. Adipose infiltration of bone marrow was significantly greater in males at the highest dose level. A treatment-related effect on bone marrow cannot be ruled out.

In an independent and expert review of the current study results (attached), it was concluded that dimyrcetol did not produce any treatment-related effects based on hematological data. These conclusion were based principally on the minimal nature of the changes and on comparisons with historical control data from the testing laboratory.

For the purpose of human risk assessment, the highest dose level (1000 mg/kg/d) in the current study was assigned as the LOAEL, based on apparent diuresis effects caused by the test chemical. The 500 mg/kg/d dose level was considered the NOAEL. These effects were manifested as increased water consumption and increased urine volumes and decreased A/G ratios. Although renal effects cannot be completely ruled out in the current study, the changes noted in certain measured values including blood urea and creatinine were generally within the range of normal control data and, in the absence of a true dose-related response, the effects were considered of minimal significance.