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Administrative data

Description of key information

In a GLP compliant subchronic oral toxicity study with the analogue substance dimyrcetol according to OECD guideline 408, the NOAEL was found to be 500 mg/kg bw/day in male and female rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 January 2006 to 24 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Adopted 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Cr1:CD IGS (SD) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 228 to 286 g (males); 174 to 235 g (females)
- 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
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 in the testing laboratory. The material was sufficiently stable to allow weekly preparation. These were stored at 4 °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: 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 °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
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose
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 where 1000 mg/kg bw only induced a slight decrease in body weights of the animals.
Positive control:
Not included
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:
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:
GROSS PATHOLOGY: Yes. On completion of dosing, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to full external and internal examinations, and any macroscopic abnormalities were recorded. 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: Yes. 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 °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.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinically observable signs of toxicity were detected in test or control animals throughout the study period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals of both sexes treated with either 500 or 1000 mg/kg/day showed reductions in mean food consumption (data not shown). No statistical significance was assigned to these results. No such effects were seen at the lower dose levels.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
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 and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
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.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ocular effects were observed.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
During Week 7, males treated with 1000 and 500 mg/kg/day showed statistically significant reductions in platelet counts. Animals of each sex treated with 1000 mg/kg/day and females treated with 500 mg/kg/day showed statistically significant reductions in activated partial thromboplastin 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 toxicologically relevant, treatment-related hematological effects (report attached).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
During Week 7, a statistically significant increase in cholesterol was evident for animals of each 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.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the measured behavioural parameters, functional performance parameters, or sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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 pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic abnormalities were detected.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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 consistent 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
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
Dose descriptor:
NOEL
Effect level:
50 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
food efficiency
haematology
histopathology: non-neoplastic
Remarks on result:
other: The NOEL for female rats was 50 mg/kg bw according to the authors of this study. This NOEL was not used for risk assessment as an expert statement did not consider these effects to be toxicologically relevant.
Dose descriptor:
NOEL
Effect level:
< 10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
not determinable
Remarks:
The NOEL was below 10 mg/kg bw according to the authors of this study. This value was not used for risk assessment as an expert statement did not consider these effects to be toxicologically relevant.
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
Remarks on result:
other: The NOAEL was 10 mg/kg bw in male animals according to the authors of this study. This NOAEL was not used for risk assessment as an expert statement did not consider these effects to be toxicologically relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
water consumption and compound intake
Key result
Critical effects observed:
not specified

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.


 


Table 6: Selected Group Mean Organ Weights (SD) from Rats Dosed Orally for 90 Days (g absolute and % of terminal bw)


 














































































































































































































 



Males



Females



Organ



0


Control



10


mg/kg/day



50


mg/kg/day



500


mg/kg/day



1000


mg/kg/day



0


Control



10


mg/kg/day



50


mg/kg/day



500


mg/kg/day



1000


mg/kg/day



 



 



 



 



 



 



 



 



 



 



 



Terminal Bodyweights



533.2 (69.4)



540.9 (54.4)



567.5 (58.9)



548.4 (57.4)



491.2 (46.9)



295.8 (18.7)



315.0 (25.4)



302.6 (17.3)



305.3 (26.1)



298.6 (18.2)



 



 



 



 



 



 



 



 



 



 



 



Adrenals (g)



0.04126 (0.00608)



0.04852* (0.01086)



0.05621* (0.00984)



0.05270* (0.01294)



0.04808* (0.00486)



0.06427 (0.01342)



0.06422 (0.01424)



0.05884 (0.00983)



0.06839 (0.00914)



0.07832* (0.01537)



Adrenals (%)



0.008 (0.001)



0.009* (0.002)



0.010* (0.002)



0.010* (0.002)



0.010* (0.001)



0.022 (0.004)



0.020 (0.005)



0.019 (0.003)



0.023 (0.004)



0.026* (0.006)



 



 



 



 



 



 



 



 



 



 



 



Kidneys (g)



3.33635 (0.48475)



3.54299 (0.40387)



3.62826 (0.39775)



3.85347** (0.46511)



3.66888** (0.42372)



1.99385 (0.14621)



2.13026 (0.14764)



2.10059 (0.13005)



2.14211* (0.14416)



2.21382** (0.19731)



Kidneys (%)



0.626 (0.038)



0.655 (0.024)



0.642 (0.062)



0.705** (0.079)



0.748** (0.057)



0.675 (0.051)



0.679 (0.051)



0.0695 (0.047)



0.705* (0.054)



0.743** (0.075)



 



 



 



 



 



 



 



 



 



 



 



Liver (g)



16.3870 (2.80357



16.9835 (2.05324)



18.8533* (2.07852)



20.4133** (2.93092)



20.6495** (2.75768)



9.10466 (1.01145)



10.2428 (1.25567)



9.49078 (1.02122)



11.0799** (1.05770)



13.2904** (0.57097



Liver (%)



3.065 (0.228)



3.137 (0.150)



3.328* (0.250)



3.717** (0.325)



4.193** (0.260)



3.076 (0.246)



3.254 (0.338)



3.136 (0.284)



3.629** (0.161)



4.461** (0.251)



 



 



 



 



 



 



 



 



 



 



 



Thymus (g)



0.40283 (0.07284)



0.47612* (0.08081)



0.54487* (0.08640)



0.51130* (0.13133)



0.41289* (0.04930)



0.35774 (0.08586)



0.35725 (0.07546)



0.36560 (0.09293)



0.37254 (0.07377)



0.32936 (0.06752)



Thymus (%)



0.076 (0.014)



0.088* (0.012)



0.097* (0.020)



0.094* (0.024)



0.085* (0.012)



0.121 (0.031)



0.114 (0.023)



0.121 (0.030)



0.122 (0.019)



0.110 (0.023)



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

Conclusions:
In this GLP compliant subchronic oral toxicity study according to OECD guideline 408, the NOAEL was found to be 500 mg/kg bw/day in male and female rats.
Executive summary:

In a GLP compliant 90-day repeated dose oral toxicity study according to OECD guideline 408 with dimyrcetol, the authors of this study concluded that 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Reliable without restrictions

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

WoE, Read-across, subchronic oral toxicity, RL1


The repeated dose toxicity of the analogue chemical, dimyrcetol, has been determined in a GLP compliant subchronic oral gavage study according to OECD guideline 408 in rats. Rats were treated with dimyrcetol at dose levels of 0, 10, 50, 500 or 1000 mg/kg bwt/day. Body weights in males were decreased at the highest dose level but not significantly. Significant reductions in body weight gains, most evident at the highest dose level but not at all time points measured, occurred in both males and females at levels of 500 and 1000 mg/kg bw/day. Diuresis was evident as increases in urine volumes (statistically significant only for females at the highest dose level) and decreased albumin/globulin ratios. In male rats, nephropathy (consistent with alpha-2u-globulin accumulation) was present at all dose levels. Evidence for this consisted of observations of increased incidence or severity of basophilic tubules and/or granular accumulations of eosinophilic material indicative of alpha-2u-globulin accumulation following staining with Mallory’s Heidenhain stain. 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). 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. Adrenal and thymus weights were significantly increased for all groups of dosed males. Although statistically significant, increased weights were not associated with any reported histopathological changes. The histopathological changes associated with alpha-2u-globulin deposition were not considered relevant to human hazard and were not used in the hazard evaluation process. A NOAEL value of 10 mg/kg bwt/day was assigned to this study by the authors and was based on the hematological effects seen in male rats. An expert review of the hematological findings from this study concluded that based on the minimal nature of the effects observed, with most values falling within historical control ranges, diymyrcetol did not produce any treatment-related hematological effects. In addition, the expert review also concluded that the suggested possibility of bone-marrow hypoplasia in male rats in this study was a misinterpretation, and that when all data were considered, and lacking a similar effect in female rats, the bone marrow was not considered to be a target tissue in this study. Of additional concern in this study were reduced spermatid counts at the highest dose level 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. Due to questions concerning the methodology used to determine spermatid counts, and because only the highest dose level was assayed in this study, no further conclusions can be drawn concerning the significance of these findings. Body weight changes, most pronounced at the highest dose level tested, are suggestive of a general systemic effect for dimyrcetol. In the absence of a well-defined mode-of action for dimyrcetol; the lack of chronic toxicity information; and the possibility that long-term diuresis may represent an adverse effect: a NOAEL value of 500 mg/kg bwt/day value was assigned to this study and was used for the derivation of DNEL values. It is not possible to state if any of the effects observed in studies with dimyrcetol were due to the presence of the formate ester.


 


WoE, Read-across, 14-day oral range finding study, RL2


The repeated dose toxicity of the analogue chemical, dimyrcetol, has been determined in a subacute oral gavage study in rats. In the 14-day range-finding oral gavage study conducted prior to a 90-day sub-chronic study, rats (3 males and 3 females per treatment group) were treated at 0 (control) and 1000 mg/kg bw/day. All animals displayed increased salivation 10 minutes following dosing. Body weights of male rats were slightly reduced on day 14. At necropsy, males displayed pale kidneys. No such effects were noted in female rats. Based on slightly decreased body weights in male rats, 1000 mg/kg bw/day was assigned as the LOAEL in this study. 


 


WoE, 21-day oral range finding study, RL2


The study was conducted similar to OECD guideline 407 under GLP conditions. The purpose of this study was to assess the palatability and systemic toxic potential of the test item, when administered in the diet to Sprague Dawley rats for 21 days. Three groups, each comprising four male and four female Crl:CD(SD) rats, received the test item at dietary concentrations of 3750, 7500 or 15000 ppm. A similarly constituted control group received untreated basal diet. During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken. The overall mean achieved doses in animals receiving 3750, 7500 or 15000 ppm were 225, 456 and 886 mg/kg/day in males and 235, 485 and 842 mg/kg/day in females, respectively. There were no premature decedents or treatment-related changes in clinical condition at all dose levels. Some short periods of reduced body weight gain or minor weight loss were apparent for males and females at all dose levels. These reduced gains or losses predominately occurred during the initial period of treatment. Subsequently, overall body weight gain for the treatment period was slightly lower for males at 3750 ppm, moderately lower for males receiving 7500 or 15000 ppm and for females receiving 3750 or 7500 ppm and considerably lower for females receiving 15000 ppm compared to controls. Overall food consumption was slightly lower for males and females receiving 15000 ppm when compared with controls. Overall mean food intake was similar to controls for male and females receiving 3750 or 7500 ppm after an initial reduction observed on Day 1 of the study. Visual water consumption at all dose levels was unaffected by treatment. There was an indication that absolute and body weight relative liver weights were slightly higher in females that received 15000 ppm when compared with controls, but no changes were apparent in males. There were no test item-related macroscopic changes observed at necropsy. In conclusion, it was considered that a high dose level of 15000 ppm, equivalent to 886 mg/kg/day in males and 842 mg/kg/day in females, and that the dose levels used in this palatability study could be selected for the preliminary study used to determine the dose levels for the main OECD 443 guideline study.


 


Please refer to Section 13 of this IUCLID for read-across documentation and rationale for the selection of analogue chemicals to dihydromyrcenol.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The test substance is assessed on the basis of the analogue substance dimyrcetol. The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result, the test substance is not classified for special target organ toxicity, repeated dose according to Regulation (EC) No 1272/2008 (CLP), as amended for sixteenth time in Regulation (EU) No 2021/743.