Registration Dossier

Administrative data

Description of key information

Subchronic NOAEL (oral, rat, male/female): 80 mg/kg bw/day (OECD 408/GLP)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 August 2019 - 15 June 2020
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:
According to ECHA Decision No CCH-D-2114453248-46-01/F "modified to include urinalysis and a full histopathological examination which is to include immunohistochemical investigation of renal pathology to determine if the pathology is mediated by alpha-2u globulin nephropathy."
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hangzhou Grascent Co., Ltd.; 0100179438
- Expiration date of the lot/batch: 28 February 2021
- Purity: ≥ 75 % (GLC); ≥ 77 % (ketone-test) impurities were not taken into account for calculation of final formulation


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

- Other information: [3R-(3α,3aβ,7β,8aα)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one = Methyl Cedryl Ketone
Species:
rat
Strain:
other: Wistar Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks old
- Weight at study initiation: males: 181 – 206 g (mean: 193.2 g, ± 20 % = 154.5 – 231.8 g); females: 140 – 170 g (mean: 152.5 g, ± 20 % = 122.0 – 183.0 g)
- Housing: The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food and water are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22  3 °C
- Humidity (%): 55  10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark
Route of administration:
oral: gavage
Details on route of administration:
The test item formulation or vehicle were administered at a single dose to the animals by oral gavage.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in corn oil. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved.
Based on the results of stability testing (Eurofins Munich Study No. 191873), the test item formulations were prepared at least every 10 days as given by Eurofins Munich Study No. 191873. The prepared formulation was stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration.
The application volume for all groups was 4 mL/kg body weight (Table 2). For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was soluble in corn oil.
- Lot/batch no. (if required): MKCH1635 (Sigma Aldrich)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 191873).

Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 191873 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1, 5, 9 and in the last week of treatment (16 samples in total).

Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 191874) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at below -15 °C at BSL Munich (test facility) and will be discarded after completion of the final study report.

For a detailed description of the individual findings see Annex 4.

Concentration analysis of formulation samples was determined at three concentrations, 6.25 mg/mL, 20 mg/mL and 62.5 mg/mL in study weeks 1, 5, 9 and in the last week of the study. The mean recoveries observed for the LD dose group was between 94.5 % and 102.2 % of the nominal value, between 84.9% and 100.0 % for the MD dose group and between 91.0 % and 101.4 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 99.3 %, 95.7 %, and 96.3 % of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %. However, one sample (no. 15, MD week last) did not meet this criterion with a recovery of 84.9 %. The precision of the measurement was high and the validity of the run was confirmed by the quality control samples. Hence the difference appears to be a result of sample preparation, not sample work up.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle (corn oil); Control (C)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Low dose (LD)
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Mid dose (MD)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
High dose (HD)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Refer to Supporting, RL1, rat (DRF)/Hangzhou, 2019/Repeated dose toxicity: oral.002
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes; spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes; All animals were observed for clinical signs during the entire treatment period of 90 days. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes; The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was measured weekly during the treatment period.

OPHTHALMOSCOPIC EXAMINATION: Yes; Ophthalmological examination using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period.

HAEMATOLOGY: Yes; Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined (Table 3). Coagulation parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined (Table 4).

CLINICAL CHEMISTRY: Yes; Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals. After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined (Table 5). For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals were retained at the end of treatment (80 animals) and stored at <-15 °C. T3, T4 and TSH serum levels were determined of main study animals (80 animals; Table 6).

URINALYSIS: Yes; A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. Additionally, urine colour / appearance were recorded. The following parameters (Table 7) were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).

FUNCTIONAL OBSERVATIONS: Yes; Once before the first exposure and towards the end of the exposure period but not earlier than in week 11 multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests. These tests were conducted in all animals.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes. One day after the last administration (study day 91) all surviving animals of the treatment period were sacrificed using anaesthesia (ketamine/xylazin) and were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.

Organ Weights
The wet weight of the organs (Table 8) of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation. Organ weights of the animal euthanised for animal welfare reasons were not recorded.

HISTOPATHOLOGY: Yes. The following tissues (Table 9) from all animals were preserved in 4 % neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.

The animal intercurrently euthanised for animal welfare reasons (female animal No. 72 (HD group)) was also subjected to a gross necropsy and the organs preserved for a histopathological examination.
The afore-listed organs (Table 9) were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (HE) staining for the animals of the groups 1 and 4 (control and HD) sacrificed at the end of the treatment period and the animal euthanised before the planned day of sacrifice.

For organs and tissues showing treatment-related changes in the high dose group, these examinations were extended to animals of all other dosage groups.

Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services GmbH, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath Services GmbH, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology).

Diagnostic Criteria
Histological changes were described, wherever possible, according to distribution, severity and morphologic character. Severity scores were assigned Grade 1 to 5.


Grade 1, Minimal
This corresponds to a histopathologic change ranging from inconspicuous to barely noticeable but so minor, small, or infrequent as to warrant no more than the least assignable grade. For multifocal or diffusely distributed lesions, this grade was used for processes where less than approximately10% of the tissue in an average high-power field was involved.
Grade 2, Slight
This corresponds to a histopathologic change that is a noticeable but not a prominent feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where between approximately 10% and 25% of the tissue in an average high-power field was involved.
Grade 3, Moderate
This corresponds to a histopathologic change that is a prominent but not a dominant feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where between approximately 25% and 50% of the tissue in an average high-power field was involved.
Grade 4, Marked
This corresponds to a histopathologic change that is a dominant but not an overwhelming feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where between approximately 50% and 95% of the tissue in an average high-power field was involved.
Grade 5, Severe
This corresponds to a histopathologic change that is an overwhelming feature of the tissue. For multifocal or diffusely distributed lesions, this grade was used for processes where greater than approximately 95% of the tissue in an average high-power field was involved.





Other examinations:
Analysis of alpha2u-globulin in Kidneys
The processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath Services GmbH, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Selected samples were embedded in paraffin, cut at a thickness of 4 µm and stained with HE.

An indirect immunohistochemistry optimised method using the anti-alpha2u-globulin (primary antibody) and BOND Polymer Refine Detection system (containing secondary Rabbit anti mouse IgG, HRP conjugated anti-rabbit Polymer-IgG and the substrate chromogen, 3,3’-Diaminobenzidine tetrahydrochloride hydrate (DAB)) was performed on the kidneys from all the male rats and from females of the control and high dose group using Leica Bond-III system, automated immunohistochemistry stainer.

IHC slides were evaluated using light microscopy and a semi-quantitative method for the analysis of immunostaining. The number and intensity of specifically stained cells was assessed accordingly to their severity and distribution and entered into PathData system: negative (0, no stained cells), minimal (1, rare cells with low staining intensity), slight (2, several cells with low/medium staining intensity), moderate (3, many cells with medium/high staining intensity), marked (4, significant numbers of cells with high staining intensity), diffuse (5, mostly all the cellular structures are immunolabeled).

Histomorphometry by Image Analysis
One immunohistochemistry kidney section from all males in the study and females only from the Control and High Dose groups (plus the positive and negative control blocks), were imaged scanned by a Märzhäuser motorized microscope stage and an Olympus XC30 camera mounted on an Olympus BX43 microscope system at 4x magnification. Quantitative evaluation was made in Olympus imaging and image analysis software cellSens v1.18. The cortex of the kidney was defined as a region of interest. The area of positive anti-alpha2u-globulin immunohistochemistry staining was measured in the region of interest using pixel thresholds by HUE, saturation, and intensity. Illustrative images with measurements are displayed in Figure 1 and Figure 2 (Annex 3).

Arithmetic mean values of the percentage positive area of the cortex were used for further descriptive statistics. Statistical tests were performed using GraphPad Prism 8. The Shapiro-Wilk test for normality was performed. When the data was following normal distribution, the comparisons would be performed with the unpaired t-test. When data did not follow normal distribution, the Mann-Whitney test was used (p-values <0.05 were considered significant).

Statistics:
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and are presented as percentage.

All results are reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).

Toxicology and pathology data was captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.3.4, Pathology Data Systems Ltd.).

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Slight to severe salivation was noted in all males of the HD group and in 1/10 of the MD group. Slight to severe salivation was noted in all females of the HD group, in 2/10 of the MD group and in 1/10 in the LD group. Furthermore, moving the bedding observed in all males and all females of the HD group. Clinical symptoms like hairless area and scratch/cut were observed mostly transiently and within the normal background frequency. Female animal no. 72 (HD group) was euthanised in a moribund condition showing severe reduces spontaneous activity, abnormal breathing and nasal discharge. No treatment related clinical signs were recorded in the control group. (Annex 1).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female animal No. 72 (HD group) was euthanised in a moribund condition on study day 14. The animal showed severe reduces spontaneous activity, abnormal breathing and nasal discharge. The remaining animals survived the scheduled period (Annex 1).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of male and female treated groups and the control group increased during the treatment period (day 1 compared to day 90), however there was statistically significant decrease in the male HD group starting from day 8 to day 90 compared to the control group. Mean body weight in males was decreased between ~6 % (p < 0.05) and ~21 % (p < 0.001) (day 8-90) in HD group when compared to control. In females, the mean body weight was statistically significantly decreased on day 29 and 36 only (~8 % and ~6 % respectively, below control (p < 0.05)). The mean body weights of male and female HD animals were within the range of the historical control data (Annex 5).

Overall, the body weight change (day 1-90) of the treated group of males showed a decrease in body weight with dose dependency and statistical significance only in the HD group. The change was a statistically significant decrease in the male treated groups (LD -~8 %, MD -~11 % and HD -~48 % (p< 0.001) respectively, below control). In females, the overall body weight change (day 1-90) was decreased in HD group (~16%) without statistical significance. The weekly body weight change during the treatment period showed statistical significance decrease in the male HD group in week 1, 3, 4, 7-9 (~48 %, ~53 %, ~59 %, ~154 %, ~64 %, ~105 % respectively, below control) and an increased change without statistical significance was found in the male HD group in week 11 (~431 % above control). In females, a decreased body weight change with statistical significance was found in the HD group on week 4 (~120 % above control). The statistical significances in the female HD groups were not considered to be an effect of the test item and there were no dose dependent changes noted between the treated groups of females (Annex 1).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In males of the HD group, decrease in mean food consumption (n=2 cages) was observed on week 5, 9, 11 (~11 %, ~34 % and ~11 % respectively, below control) and in LD group lower mean food consumption was observed on week 5 (~15 % below control) and higher consumption on week 7 (~55 % above control) when compared to control. In MD male, higher mean food consumption was observed on week 3 (~16 % above control) when compared to control. In the female HD group, decrease in mean food consumption was noted on week 1, 4-5, 12-13 (~15 %, ~16-~17 % and ~10-~13 % respectively, below control) and higher mean food consumption on week 9-10 (~54-~57 % above control) when compared to control. Single incidence of lower mean food consumption was observed in LD group on week 1, 8 and 12 (~10 %, ~15 % and ~10 %, below control) and in MD group on week 7 (~12 % below control) when compared to control.

Overall, the mean total food consumption (day 1-90) was found to be comparable in all male and female dose groups when compared to the control group. (Annex 1)..
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmologic findings were observed in any of the animals of this study.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In haematology a statistically significant decrease in male and female HD animals in RBC (~10 % (p < 0.001) and ~7 % (p < 0.05) below controls), in male and female HD animals in HGB (~7 % (p < 0.01) and ~6 % (p < 0.01) below controls), in male HD and in female MD and HD animals in HCT (~11 % (p < 0.001), ~6 % (p < 0.05) and ~9 % (p < 0.001), respectively, below controls) and in male MD in MCV (~3 % (p < 0.05) below control) were found.

A statistically significant increase in male (~2 % (p < 0.05) above control) and female MD (~3 % (p < 0.01) above control) animals and male (~4 % (p < 0.001) above control) and female (~3 % (p < 0.001) above control) HD animals in MCHC, in male HD and in female MD and HD animals in RET (~48 % (p < 0.05), ~34 % (p < 0.01) and ~106 % (p < 0.001), respectively, above controls), in male HD and in female MD and HD animals in PLT (~37 % (p < 0.001), ~21 % (p < 0.01) and ~29 % (p < 0.001), respectively, above controls), in male LD in LYM (~11 % (p < 0.01) below control) and NEUT ( ~46 % (p < 0.01) above control) were found. All values were within the range of the historical control data except for the PLT in male HD group which was slightly above (Annex 1).

In blood coagulation a statistically significant increase in male HD animals in PT (~ 11 % (p < 0.001) above control) was detected (Annex 1). This value lies within the historical control data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the observation period a statistically significant decrease in male MD and female MD and HD animals in ALAT (~43 % (p < 0.001), ~44 % (p < 0.001) and ~45 % (p < 0.001), respectively, below controls), in male LD, MD and HD animals in Gluc (~15 % (p < 0.05), ~25 % (p < 0.001) and ~42 % (p < 0.001), respectively, below control), in male and female HD animals in Crea (~22 % (p < 0.05) and ~34 % (p < 0.001) below controls), in male HD animals in TP (~ 8 % (p < 0.001) below control), in male MD and HD animals in ALB (~5 % (p < 0.05) and ~8 % (p < 0.001) below control), in male LD, MD and HD animals in CHOL (~33 % (p < 0.001) and in HD animals ~29% (p < 0.001), respectively, below control), in male HD animals in TBA (~59 % (p < 0.05) above control), in male LD, MD and HD and in female MD animals in TG (~35 % (p < 0.01), ~41 %(p < 0.001), ~34% (p < 0.01) and ~48 % (p < 0.001), respectively, below control) and in male LD, MD, HD animals in HDL and LDL (~27 % (p < 0.001), ~26 % (p < 0.001). ~20 % (p < 0.01), ~43 % (p < 0.001), ~27 % (p < 0.001) and ~47 % (p < 0.001), respectively, below controls) were found. A statistically significant increase in male MD and HD and female LD, MD and HD animals in TBIL (~68 % (p < 0.01), ~331 % (p < 0.001), ~25 % (p < 0.05), ~90 % (p < 0.001) and ~209 % (p < 0.001), respectively, above controls), in male HD animals in Urea (~ 38 % (p < 0.01) above control) and in female HD animals in K (~12 % (p < 0.05) above control) were found (Annex 1).

A statistically significant decrease was found in male HD animals only in T3 (p < 0.01) and T4 (p < 0.001) hormone analysis. No statistically significant difference was found in the TSH hormone in male and female animals when compared to the control (Annex 1)..

All values were within the range of the historical control data except for TBIL in male and female MD and HD groups which were above.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The test item had no toxicologically relevant effect on all urinary parameters analysed, on the day of necropsy (study day 91) with exception of an increase in specific gravity in female MD and HD animals (Annex 1).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item related effects were observed in any parameter of the functional observation battery at the end of the treatment period when compared to observations before treatment except in study week 13 (Annex 1 & 2).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In male animals a statistically significant higher mean brain organ/body weight ratio was seen for the HD group (~26 % (p < 0.001) above control). A statistically significant higher mean spleen organ/body weight ratio was seen for the HD group (~38 % (p < 0.001) above control). A statistically significant lower mean thymus weight and organ/brain weight ratio was seen for the HD group (~25 % (p < 0.01) and ~22 % (p < 0.05) below control). A statistically significant higher mean testes organ/body weight ratio was seen for the HD group (~30 % (p < 0.001) above control). A statistically significant higher mean kidney weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the HD group (~25 % (p < 0.001), ~62 % (p < 0.001) and ~29 % (p < 0.001), respectively, above control) and a mean organ/body weight ratio was seen for the MD group (~21% (p < 0.001) above control). A statistically significant lower mean pituitary gland weight and mean organ/brain weight ratio was seen for the HD group (~27 % (p < 0.01) and ~25 % (p < 0.01) below control). A statistically significant lower mean heart weight and mean organ/brain weight ratio was seen for the HD group (~19 % (p < 0.001) and ~17 % (p < 0.01) below control). A statistically significant lower mean epididymides weight and mean organ/brain weight ratio was seen for the HD group (~24 % (p < 0.01) and ~22 % (p < 0.01) below control). A statistically significant lower mean prostate weight and mean organ/brain weight ratio was seen for the HD group (~30 % (p < 0.001) and ~28 % (p < 0.001) below control). A statistically significant higher mean liver weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the MD and HD groups (~20 % (p < 0.001), ~26 % (p < 0.001) and ~19 % (p < 0.01), ~35 % (p < 0.001), ~75 % (p < 0.001) and ~39 % (p < 0.001), respectively, above control) (Annex 1).

In female animals statistically significant higher mean spleen weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the HD group (~21 % (p < 0.01), ~31 % (p < 0.001) and ~22 % (p < 0.01), respectively, above control) and a mean organ/body weight ratio and a mean organ/brain weight ratio was seen for the MD group (~16 % (p < 0.05) and ~15% (p < 0.05) above control). A statistically significant higher mean kidney weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the MD and HD group (~12 % (p < 0.001), ~ 14 % (p < 0.01), ~14 % (p < 0.001), ~14 % (p < 0.001), ~24 % (p < 0.001) and ~16 % (p < 0.001), respectively, above control) and a mean weight and a mean organ/brain weight ratio was seen for the LD group (~8 % (p < 0.05) and ~11 % (p < 0.05) above control). A statistically significant lower mean adrenal gland weight was seen for the MD and HD group (~14 % (p < 0.05) and ~ 18 % (p < 0.01) below control) and lower mean organ/brain weight ratio was seen for the HD group (~16 % (p < 0.05) below control). A statistically significant higher mean liver weight, mean organ/body weight ratio and mean organ/brain ratio was seen for the LD, MD and HD group (~15 % (p < 0.01), ~14 % (p < 0.01), ~18 % (p < 0.05), ~28 % (p < 0.001), ~31 % (p < 0.001), ~31 % (p < 0.001), ~56 % (p < 0.001), ~69 % (p < 0.001) and ~58 % (p < 0.001), respectively, above control). (Annex 1).

All values were within the range of the historical control data except for the liver and kidneys mean weight in male HD group and the liver mean weight in female MD and HD groups which were above


Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of the animals at necropsy listed in male animal no. 31 (HD group) a depressed, dark, glandular mucosa surface of the stomach, in male animal no. 19 (LD group) an abnormal dark red color in the thymus, in male animal no. 26 (MD group) abnormal spotted color in the thymus, in female animals no. 67 (MD group) a diaphragmal herniation in the liver, in animal no. 71 (HD group) an abnormal white color in the ovaries, in female animal no. 50 (C group) a fluidfilled uterus .

The female animal no. 72 (HD group) euthanized on study day 14 listed a dark red fluid filled thoracic cavity and a cyst (0.2 – 0.6 cm) in the uterus (Annex 1).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Refer to Annex 3 for figures. Tables are presented below.

Liver
Half of the HD male rats and one female presented minimal foci of single cell death often associated with multifocal, hepatocellular fatty change. The fatty change was multifocal and characterized by hepatocytes presenting variable-sized, clear, well-defined vacuoles within the cytoplasm (Table 3). Hepatocellular fatty change was also detected in the control dose and intermediate LD and MD groups from both sexes, but not associated with further degenerative changes in hepatocytes.

Several HD males and females also presented multifocal, well-circumscribed, non-encapsulated, small (up to 500 µm in diameter) eosinophilic foci, where clusters of hepatocytes showed an enlarged, light, pale pink cytoplasm. Multifocally, most of the HD males and one female at presented enlarged nuclei (minimal to slight karyomegaly), with coarse chromatin and prominent nucleoli.

Most of the HD males and females presented with a proliferation of bile ducts (hyperplasia) at portal spaces which exceeded in severity and frequency the presence of this finding in the control group. This finding in MD males and females was similar in severity and frequency to the control group.

Several treated HD males and females showed induced centrilobular hepatocellular hypertrophy, characterized by hepatocytes with enlarged and homogeneous cytoplasm and compression of sinusoidal spaces.

Thyroid gland
The thyroid gland from all the HD males and most of the females presented diffuse, bilateral, areas of follicular cell hypertrophy together with hypercellularity and increase in number of follicles (follicular hyperplasia), with a predominance of follicles in reabsorption phase (Table 5). The follicular epithelium presented intracytoplasmic vacuolation, variable height (up to 10 µm), with occasional cellular crowding and minimal papillary protrusion into the lumen. This lesion was accompanied by a colloid alteration: paler, basophilic colour of the colloid with pyknotic clumps. The association of follicular hypertrophy with hyperplasia led to include them in the same diagnosis. These findings were not present in the LD and MD males and females.

Kidney
The kidneys of the following groups were analysed with an optimized immunohistochemistry technique: all control, LD, MD and HD males, all control and HD females.

Anti-alpha2u-globulin IHC was validated by specific positive immunolabeling in the positive control slide (Figure 9); in contrast to the lack of positive immunolabelling for the negative IHC control slide (Figure 10). There was a very mild, unspecific, background immunolabeling observed in the glomeruli, apical border of the renal tubules, interstitium and some vessels from all the animals/groups.

In most of the males from the control group and all treated groups, there were large deposits of an amorphous, acellular, bright, eosinophilic, up to 40 µm, proteinaceous deposits (hyaline droplets), which were distorting the cytoplasm from the tubular cells of the proximal segment in the renal cortex or intraluminal. In addition, in males from all treated groups, there were abundant, smaller (up to 5 µm), eosinophilic, parallel, round to elongated, proteinaceous bodies (eosinophilic bodies) at the basal levels of the renal epithelium in the cortex, which exceeded in frequency and severity the background levels shown in control group (Figure 6 to Figure 8, and Figure 11 to Figure 15, Table 4). These proteinaceous deposits were not observed in any female group (Figure 16, Figure 17, Table 4). All HD males presented an increase in mineralization in the tubular lumen, mostly in the outer medulla, compare to the control group. Tubular basophilia in the renal cortex exceeded background levels from the control group in males exposed to ≥ LD and HD females. The presence of intratubular casts appeared with more frequency or severity in LD and MD male groups compared to females, and to males from control or high dose groups.

Immunohistochemistry results showed positive immunolabelling against alpha2u-globulin in all males from the control and treated groups, whereas no specific immunolabelling was observed in females. The positive alpha2u-globulin immunolabelling was greater at LD and MD compared to the control group and high dose groups. The alpha2u-globulin positive signal in the intermediate dose groups was found targeting most of the proteinaceous deposits: the hyaline droplets and elongated, eosinophilic bodies. In high dose group, most of the hyaline droplets were alpha2u-globulin positive whereas a large fraction of the elongated proteinaceous eosinophilic bodies was not alpha2u-globulin immunoreactive (Figure 15).

The individual image analysis data are presented in Table 6 and the group arithmetic means are summarized in Table 7.

The “Mean % Positive alpha2u-globulin Area (%)” values from the groups followed a normal distribution and unpaired T-test was applied to investigate the statistical significance level. Medium dose group was the only exception were values were not following a normal distribution, where Mann-Whitney test was applied (Table 8-Table 10). Male groups presented specific positive immunolabelling against alpha2u-globulin, higher at LD and MD, followed by the control and then the high dose group. The increase in alpha2u-globulin positive vs Control group was statistically significant in LD males but was statistically non-significant in MD males (Table 10). The decrease in alpha2u-globulin immunolabelling of the high dose group vs control was also considered statistically significant. Female control and high dose group did not show positive specific immunolabelling against alpha2u-globulin, and the detection of the positive signal were associated with background/non-specific stain. The changes in males were considered characteristic early changes associated with alpha2u-globulin nephropathy.

Illustrative images showing the difference between the groups can be observed in Figures 20-24.



Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
25 other: mg/kg bw/day; In males only (alpha-2u-globulin-mediated nephropathy)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no

Table 3: Incidence and mean severity of relevant microscopic findings in the Liver, only survivors

Findings in the Liver Treatment Period Dose (mg/kg bw/day) 
0 25 80 250
Microscopic findings Affected / Mean Grade
Number of animals  10 (M) 10 (F) 10 (M) 10 (F) 10 (M) 10 (F) 10 (M) 9 (F)
Fatty change 5/1.4 4/1.0 9/1.0 9/1.0 8/1.1 5/1.2 8/1.4 5/1.2
Single cell death, hepatocell. - - - - - - 5/1.0 1/1.0
Karyomegaly, hepatocellular - - - - - - 8/1.3 1/1.0
Hyperplasia, bile duct 2/1.0 2/1.0 - - 2/1.0 1/1.0 9/1.2 7/1.3
Eosinophilic foci, hepatocell. - - - - - - 3/1.0 5/1.4
Hypertrophy, hepatocellular - - - - - - 8/1.6 8/1.5

Table 4: Incidence and mean severity of microscopic findings in the kidneys, only survivors.

Findings in the Kidneys Treatment Period Dose (mg/kg bw/day)
0 25 80 250
Microscopic findings Affected / Mean Grade
Number of animals  10 (M) 10 (F) 10 (M) 10 (F) 10 (M) 10 (F) 10 (M) 9 (F)
Hyaline droplets 9/1.4 - 10/1.5 - 10/1.7 - 10/1.2 -
Eosinophilic bodies 3/1.0 - 10/2.6 - 10/2.8 - 10/2.5 -
Cast, intratubular - - 3/1.0 - 2/2.0 1/1.0 1/1.0 -
Tubular basophilia 7/1.1 4/1.0 9/1.6 5/1.0 8/1.6 5/1.0 10/2.0 8/1.6
Mineralization, tubular 2/1.5 - 2/1.0 - 1/1.0 1/1.0 10/1.4 1/1.0

Table 5: Incidence and mean severity of relevant microscopic findings in the thyroid gland, only survivors

Findings in the Thyroid Gland Treatment Period Dose (mg/kg bw/day) 
0 25 80 250
Microscopic findings Affected / Mean Grade
Number of animals  10 (M) 10 (F) 10 (M) 10 (F) 10 (M) 10 (F) 10 (M) 9 (F)
Hypertrophy/hyperplasia, follicular cell - - - - - - 10/2.9 8/3.0

Table 6: Individual sample data of kidney cortex area, positive alpha2u-globulin

area and percentage positive alpha2u-globulin area of the kidney cortex area.

Group Name Sex Animal No.  Kidney Cortex Area (µm²) Positive alpha2u-globulin Area (µm²)  Positive alpha2u-globulin
Area (%) 
Control  Male 1 38728731.8 9460.1 0.02%
2 52790768.2 432766.31 0.82%
3 51295307.1 13129.93 0.03%
4 17877970.9 53187.85 0.30%
5 27275907.3 73469.01 0.27%
6 46696084 139710.65 0.30%
7 48381523.7 462873.03 0.96%
8 55406655.5 386263.34 0.70%
9 45352378.5 197341.13 0.44%
10 57575647.3 1051877.6 1.83%
Low Dose  Male 11 54669618.5 1462280.3 2.68%
12 53195422.6 1908886.6 3.59%
13 65788617.6 174459.87 0.27%
14 64408455.5 644023.49 1.00%
15 46226033.1 423896.27 0.92%
16 46034508.8 432162.91 0.94%
17 52474357.4 872893.22 1.66%
18 51542463.2 539665.45 1.05%
19 47355426.3 1353946 2.86%
20 38237244.3 644802 1.69%
Medium Dose  Male 21 62940651.3 1988745.4 3.16%
22 51948876.4 293246 0.56%
23 54768511.8 1306948.2 2.39%
24 62477755.5 676534.26 1.08%
25 52889692 257855.32 0.49%
26 62401241 311629.43 0.50%
27 68802853.2 218075.31 0.32%
28 57998574.7 341818 0.59%
29 56781173.9 430029.16 0.76%
30 49987778.4 481610.5 0.96%
High Dose  Male 31 57686591.3 58869.62 0.10%
32 47696395.4 50816.16 0.11%
33 59245111.5 53404.84 0.09%
34 52794586.5 36237.71 0.07%
35 58219756.4 43742.97 0.08%
36 52971758.8 10305.23 0.02%
37 54976253.3 62035.04 0.11%
38 46124817.6 24826.58 0.05%
39 52240314.1 56701.6 0.11%
40 57686893.9 78374.18 0.14%
Control  Female 41 29202716.6 11024.73 0.04%
42 26448723.3 10097.75 0.04%
43 27261753.3 2310.78 0.01%
44 32978699.7 8205.73 0.03%
45 34184512.4 9166.97 0.03%
46 37767933.9 40661.3 0.11%
47 30729441.8 23543.66 0.08%
48 28784938 22148.44 0.08%
49 37395198.3 10817.25 0.03%
50 37017844.9 1157.29 0.00%
High Dose  Female 71 34213482.7 16202.09 0.05%
72 34646437.5 7581.4 0.02%
73 37407498.3 11913.64 0.03%
74 34578003.1 20338.27 0.06%
75 31793076.9 4351.27 0.01%
76 43829677.2 11788.01 0.03%
77 35070895.3 10406.11 0.03%
78 44861389.7 10874.36 0.02%
79 31183736 29033.18 0.09%
80 37159933 14548 0.04%
Negative control Male na 48854635.3 6334.65 0.01%
Positive control Male na 33208218.5 112874.04 0.34%

 Na= not applied

Table 7: Summarized group arithmetic mean of kidney cortex area,

positive alpha2u-globulin area and percentage positive alpha2u-globulin

area of the kidney cortex area.

Group Name Sex Animal No.  Mean Kidney Cortex Area (µm²) Mean Positive anti-α2 globulin Mean % Positive anti-α2 globulin Standard deviation
Area (µm²) Area (%) 
Male 1-10 44138097.42 282007.892 0.57% 0.54%
LD Male 11-20 53726750.24 770748.68 1.44% 1.05%
MD Male 21-30 580997108.1 6306491.56 1.08% 0.94%
HD  Male 31-40 539642478.8 475313.92 0.09% 0.03%
Female 41-50 51717268.53 38906.14 0.08% 0.03%
HD  Female 71-40 36474412.97 13703.63 0.04% 0.02%

Table 8: Column Statistics for Normality Test of Male Groups.

Group C LD  MD HD
Number of values 10 10 10 10
Minimum 0,02400 0,2650 0,3170 0,01900
25% Percentile 0,2083 0,9335 0,4963 0,06525
Median 0,3670 1,355 0,6730 0,09600
75% Percentile 0,8543 2,721 1,409 0,1100
Maximum 1,827 3,588 3,160 0,1360
Range  1,803 3,323 2,843 0,1170
Mean 0,5652 1,664 1,081 0,08740
Std. Deviation 0,5435 1,055 0,9385 0,03395
Std. Error of Mean 0,1719 0,3336 0,2968 0,01074
Lower 95% CI of mean 0,1764 0,9093 0,4092 0,06311
Upper 95% CI of mean 0,9540 2,419 1,752 0,1117
Sum 5,652 16,64 10,81 0,8740
Shapiro-Wilk normality test C LD  MD HD
W 0,8613 0,9145 0,7488 0,9546
P value 0,0791 0,3135 0,0034 0,7235
Passed normality test (alpha≥0.05)? Yes Yes No Yes

Table 9:  Column Statistics for Normality Test of Female Groups.

Group C HD 
Number of values 10 10
Minimum 0,003000 0,01400
25% Percentile 0,02075 0,02350
Median 0,03350 0,03100
75% Percentile 0,07700 0,05000
Maximum 0,1080 0,09300
Range  0,1050 0,07900
Mean 0,04300 0,03870
Std. Deviation 0,03365 0,02307
Std. Error of Mean 0,01064 0,007297
Lower 95% CI of mean 0,01893 0,02219
Upper 95% CI of mean 0,06707 0,05521
Sum 0,4300 0,3870
Shapiro-Wilk normality test C HD
W 0,9008 0,8542
P value 0,2234 0,0651
Passed normality test (alpha≥0.05)? Yes Yes

Table 10: P values.

Groups compared: Control - High dose p-value Significance
Control Male-Low dose male 0,0090 Yes
Control Male-Medium dose male 0,0753 No
Control Male-High dose male 0,0125 Yes
Control female- High dose Female 0,7428 No
Conclusions:
Under the conditions of this study and based on the pathology evaluation, the NOAEL for a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)] -1 - (2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in male and female Wistar rats, can be established at 80 mg/kg bw/day.
Executive summary:

In a repeated dose toxicity study (191870), [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one was administered to Wistar Crl: WI(Han) rats (10/sex/dose) by gavage in corn oil at dose levels of 0, 25 (LD), 80 (MD) and 250 (HD) mg/kg bw/day daily for 90 days.

 

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

The body weight of HD male animals was statistically significantly reduced from study day 8 until the end of treatment. The body weight change of HD male animals was statistically significantly reduced compared to the control group. The body weight of HD female animals was statistically significantly reduced from study day 29 and 36 only. The body weight change of HD female animals was not statistically significantly reduced compared to the control group. The mean body weights of male and female HD animals were within the range of the historical control data.

No treatment related or toxicologically relevant effects on body weight gain, food consumption, clinical observations, functional observation battery, ophthalmology, haematological parameters, blood coagulation, clinical biochemistry, urine, thyroid hormones or reproductive organs were observed during the treatment period. The male HD PLT count, male and female MD and HD TBIL and male HD mean kidney weight and male HD and female MD and HD mean liver weights were above the historical control data and are considered to be test item related.

Macroscopic findings during necropsy were considered to be incidental and not test item related.

Under the conditions of this study, after a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)] -1 - (2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in Wistar rats, there were induced findings in the liver, thyroid and kidney in HD males and females. In the liver, the fatty change associated with single cell death observed in some HD males and one female, was considered an adverse finding caused by the Test Item. In the intermediate doses (LD and MD), there was also a subtle increase in severity or frequency levels of fatty changes compare to control group but in absence of other degenerative changes, therefore it was considered an adaptative finding at these dosages. The karyomegaly observed in most of the males and in one HD female was also deemed to be a treatment induced adaptative finding. Karyomegaly is considered often a reflection of hepatocyte polyploidy that occurs when there is duplication of nuclear material in the absence of cytokinesis. Also, in several HD males and females, the presence of well-defined, eosinophilic foci of hepatocellular alteration and the increase in frequency and severity of bile duct hyperplasia were deemed to be induced by the treatment. The eosinophilic foci are considered to represent a localized proliferation of hepatocytes that are phenotypically different from surrounding hepatocyte parenchyma and could represent preneoplastic lesion. Hepatocellular hypertrophy observed in some HD males and females was deemed to be an adaptative Test Item induced effect, which would probably recover after withdrawn of the treatment. This often occurs secondary to increase in microsomal enzymes following exposure to enzyme inducing xenobiotics.

In the thyroid gland, the follicular hypertrophy associated with alterations in the colloid in HD males and females only was considered a test item-related change. These findings were not present in the LD and MD males and females. In rats, liver changes (hepatocellular hypertrophy) may induce an increase in thyroid hormones degradation and an increased in TSH leading to thyroid gland hypertrophy. This phenomenon is more rat specific and of less toxicologic relevance in humans. Both, follicular cell hyperplasia and hypertrophy are expected to return to normal if the stimulus is withdrawn. A statistically significant decrease in T3 (p < 0.01) and T4 (p < 0.001) was found in HD male animals only. However, all values were within the range of the historical control data. There were no statistically significant changes in the level of TSH in male or females at any dose and no statistically significant changes in (para)thyroid weight in males or females at any dose group.

In the kidney of the males from all the groups, the hyaline droplets and the elongated, eosinophilic bodies at the basal level of the renal epithelium were observed. Both were considered proteinaceous deposits accumulated in the cytoplasm from renal tubular epithelial cells. On haematoxylin and eosin (HE) pathology evaluation, there was an increase (in frequency or severity) of proteinaceous deposits in all treated groups compare to the control group, especially for the elongated eosinophilic bodies at the basal area. This was considered a Test Item induced effect. Tubular basophilia in HD females, in absence of further degenerative changes, was considered in this case most likely an adaptative response.

An indirect immunohistochemistry optimized method using the anti-alpha2u-globulin (primary antibody) and BOND Polymer Refine Detection system (containing secondary Rabbit anti mouse IgG, HRP conjugated anti-rabbit Polymer-IgG and the substrate chromogen, 3,3’-Diaminobenzidine tetrahydrochloride hydrate (DAB)) was performed on the kidneys from all the male rats and from females of the control and high dose group using Leica Bond-III system, automated immunohistochemistry stainer. Anti-alpha2u-globulin IHC was validated by specific positive immunolabeling in the positive control slide; in contrast to the lack of positive immunolabelling for the negative IHC control slide.

With immunohistochemistry, alpha2u-globulin positive signal was detected on the proteinaceous deposits (i.e. hyaline droplets and the elongated, eosinophilic bodies) in all the male groups, which demonstrated the accumulation of this protein in renal tubules. After image analysis evaluation, the increase in the percentage of positive alpha2u-globulin signal in the intermediate doses compared the controls, revealed an induced increase in accumulation of alpha2u-globulin related to the treatment at these dosages, however these differences were found to be statistically different only at the LD compared to control group. In comparative evaluation, image analysis showed that the HD male group presented a decrease in the presence of alpha2u-globulin-positive signal compare to the control group and intermediate doses. The most likely hypothesis is that the induced hepatic damage at the high dose, may impair the hepatic production of alpha2u-globulin decreasing progressively the circulating level, and therefore decreasing the renal accumulation. There may also be an overloading of the metabolic pathway and therefore a decrease in the reduction of MCK and subsequent conjugation with glucuronic acid and/or alpha2u-globulin. The glucuronic pathway can be overloaded for non-acidic substances (Howell et al., 1986); possibly the alpha-2u pathway can become overloaded also.

Alpha2u-globulin is a circulating low molecular protein which is synthesized in the liver of male rats only, under androgen regulation on physiological conditions. This explains the absence of accumulation of alpha2u-globulin in the female groups and the presence of alpha2u-globulin in the male control group. Furthermore, alpha2u-globulin accumulation is a male- and rat- specific entity, which will not be translated to humans. Many xenobiotics bind to circulating alpha2u-globulin and decrease effectiveness of lysosomal degradation in the proximal tubules resulting in greater accumulation. The increase in tubular basophilia in LD and MD males exceeded the background levels from the control group. This, and the presence of tubular hyaline casts in few treated males were considered characteristic early changes associated with alpha2u-globulin nephropathy. This effect is not relevant for the human health risk assessment.

Under the conditions of this study and based on the pathology evaluation, the NOAEL for a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)] -1 - (2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in male and female Wistar rats, can be established at 80 mg/kg bw/day.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Dose Range Finding Study for Repeated dose 90 day oral toxicity study in rodents (OECD 408)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 May 2019 - 29 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: DRF study in rats for OECD 408
Qualifier:
according to guideline
Guideline:
other: DRF study in rats for OECD 408
Version / remarks:
The details in the study report results relevant to study number 191870 (90 day Repeated Dose Oral Toxicity Study in Wistar Rats with [3R-(3alpha,3abeta,7beta,8aalpha)]-1- (2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl)ethan-1-one).
GLP compliance:
no
Remarks:
DRF study; GLP not required
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hangzhou Grascent Co., Ltd.; 0100179438
- Expiration date of the lot/batch: 28 February 2021
- Purity: ≥ 75 % (GLC); ≥ 77 % (ketone-test) impurities were taken into account for calculation of final formulation (based on 80 % ketone content according to certificate of analysis)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
Species:
rat
Strain:
other: Wistar rats, Crl: WI(Han) (Full Barrier)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: males: 195 – 215 g (mean: 206 g, ± 20 % = 165 – 247 g); females: 153 – 176 g (mean: 162 g, ± 20 % = 129 – 194 g)

- Housing: The animals were kept in groups of 3 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: at least five days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food and water are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 3 °C
- Humidity (%): 55 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared freshly on each administration day before the administration procedure. Thereafter, the test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. Formulated were kept under magnetic stirring during the daily administration

VEHICLE
The vehicle used in this study was corn oil.

- Amount of vehicle (if gavage): The application volume for all groups was 4 mL/kg body weight
- Lot/batch no. (if required): Sigma Aldrich; MKCH1635
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
125 mg/kg bw/day without consideration of a.i. content
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
312.5 mg/kg bw/day without consideration of a.i. content
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
625 mg/kg bw/day without consideration of a.i. content
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
937.5 mg/kg bw/day without consideration of a.i. content
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
1250 mg/kg bw/day without consideration of a.i. content
No. of animals per sex per dose:
3 males and 3 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The animals were dosed at concentrations based on active ingredient content within the test item and re-calculated to doses of test item without consideration of active ingredient content (Table 2).
- Fasting period before blood sampling: Overnight fasting for haematology and clinical chemistry.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All Animals were observed for clinical signs during the entire treatment period of 28 days.

General clinical observations were made at least once a day, approximately at the sametime each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).

The body weight was recorded once before assignment to the experimental groups and on study days 1, 8, 15, 22 and 28 during the treatment period as well as on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured on study days 1, 8, 15, 22 and 28 for each animal (except of female groups 1-3, where food weights were not determined on day 28).

HAEMATOLOGY: Yes; Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals (Table 3).

CLINICAL CHEMISTRY: Yes; Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals (Table 4).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; On study day 29, all surviving animals of the study were sacrificed using anaesthesia (ketamine/xylazin) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. All macroscopic findings were recorded and organs showing gross abnormalities and all organs listed in Table 5 were preserved in 4 % neutral-buffered formaldehyde. All animals found dead and/or intercurrently sacrificed were subjected to a gross necropsy.

Statistics:
All results were reported in tabular form (summarised in mean or summary tables).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Annex 1.

All males (3/3) of group 5 (750 mg/kg bw/day) and 3/3 males of group 6 (1000 mg/kg bw/day) were euthanized due to animal welfare reasons on study day 4. On the day of sacrifice they all showed similar clinical symptoms such as apathy, abnormal recumbency, prone position, reduced spontaneous activity, muscular hypotonia, dehydration, half eyelid closure, diarrhoea and discoloured (yellow) faeces.

All males (3/3) of group 4 (500 mg/kg bw/day) were euthanized in a moribund condition on study day 12. The males were observed with similar clinical signs of reduced spontaneous activity, muscular hypotonia, wasp waist, dehydration, piloerection and half eyelid closure.

All females (3/3) of group 5 (750 mg/kg bw/day) and 3/3 females of group 6 (1000 mg/kg bw/day) were euthanized due to animal welfare reasons on study day 3 with showing clinical signs of wasp waist, piloerection, reduced spontaneous activity, dehydration, half eyelid closure, diarrhoea, discoloured (yellow) faeces and dehydration on their respective day of sacrifice.

Females of group no. 4 (500 mg/kg bw/day) were euthanized on treatment day 8 (1/3) and treatment day 11 (2/3). They were observed with clinical signs of apathy, abnormal recumbency, prone position, reduced spontaneous activity, muscular hypotonia, dehydration, half eyelid closure, diarrhoea and discoloured (yellow) faeces on the day of sacrifice.

The clinical sign of moving the bedding was observed transiently in 3/3 males and 1/3 females of group 3 (250 mg/kg bw/day), 3/3 males and 3/3 females of group 4 (500 mg/kg bw/day). The clinical sign of increased salivation was seen in 1/3 males and 1/3 females of group 2 (100 mg/kg bw/day), 2/3 males and 1/3 females of group 3 (250 mg/kg bw/day), 3/3 males of group 4 (500 mg/kg bw/day). As both signs were observed in short timely relation to test item administration or in anticipation thereof, they were considered to be a sign of discomfort or a local reaction to the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
For a detailed description of the findings see Annex 1.

One female animal (animal no. 29) of dose group 4 (500 mg/kg bw/day) was euthanised for animal welfare reasons on study day 8, the remaining two females (animals no. 28, 30) were euthanised on study day 11 and all three male animals (animals no. 10-12) from group 4 were euthanised for animal welfare reasons on study day 12.

From dose group 5 (750 mg/kg bw/day) and dose group 6 (1000 mg/kg bw/day), all female animals (animals no. 31-36) were euthanised on study day 3 and all three males from groups 5 and 6 (animals no. 13-18) were euthanised for animal welfare reasons on study day 4.

No deaths were observed in the control group 1 or in dose group 2 (100 mg/kg bw/day) and group 3 (250 mg/kg bw/day).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Annex 1.

Treatment with the test item was shown to affect body weights and body weight development.

Males of the test item treated groups showed lower mean body weights from the second week of treatment onwards with a trend towards dose-dependent lower mean body weights (day 8: 4 % deviation in group no. 2 and 5 % deviation in group no. 3, day 15: 1 % deviation in group no. 2 and 7 % deviation in group no. 3, day 22: 1 % deviation in group no. 2 and 6 % deviation in group no. 3, day 28: 3 % deviation in group no. 2 and 13 % deviation in group no. 3). Corresponding to lower mean body weights in test item treated groups, males showed lower mean body weight gain almost throughout the whole study period. In the last week of treatment, males of group no. 3 showed a mean body weight loss of 7 g in comparison to mean body weight gain of 13 g in the control group.

Females of group 3 (250 mg/kg bw/day) were observed with lower mean body weight compared to control females on treatment day 28 (9 % deviation from control). Corresponding to lower mean body weights in group no. 3 on treatment day 28, females of this group showed a body weight loss in the last week of treatment (mean body weight loss was 12 g compared to mean body weight gain of 8 g in the control group).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
For a detailed description of the findings see Annex 1.

Food consumption showed no trend towards lower food consumption in test item treated animals compared to their corresponding controls. Differences between the groups followed no dose-dependency or were within the normal range of variation and thus were not considered adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Annex 1.

In both, male and female animals, mean WBC level showed a dose-dependent trend towards higher levels in test item treated groups which survived the scheduled study period compared to the corresponding controls (male group 2: 53 % deviation from control, male group 3: 60 % deviation from control, female group 2: 72 % deviation from control, female group 3: 78 % deviation from control). Based on the dose dependency and observation of this effect in both genders higher WBC levels may indicate an inflammatory reaction which is possibly related to treatment with the test item.

Mean PLT levels showed a dose-dependent trend towards higher mean levels in test item treated males compared to control males (male group 2: 19 % deviation from control, male group 3: 37 % deviation from control) and were higher in test item treated females compared to control females (female group 2: 31 % deviation from control, female group 3: 31 % deviation from control). It cannot be excluded that higher PLT levels in test item-treated animals were caused by treatment with the test item.

Other haematological parameters including RBC, HGB, and HCT showed no test itemrelated changes in either sex.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Annex 1.

Possible test item-related changes in parameters of clinical biochemistry examined at the end of the treatment period were observed in the parameters crea and urea.

In test item treated males, dose-dependently higher mean levels of crea (group no. 2: 13 % deviation from control, group no. 3: 19 % deviation from control) and urea (group no. 2: 39 % deviation from control, group no. 3: 104 % deviation from control) were observed compared to the controls. Females showed dosed-dependently higher mean urea levels in test item-treated groups compared to female controls (group no. 2: 15 % deviation from control, group no. 3: 21 % deviation from control).

Liver parameters ALAT and ASAT were higher in some test item-treated male and female groups compared to their respective controls, however, this was not considered adverse.

Other parameters showed no toxicologically relevant differences in either sex when comparing test item treated groups to their corresponding controls.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Annex 1.

Macroscopic findings that could be attributed to treatment with the test item and were observed in preliminary euthanized animals of group 4 (1/3 males and 2/3 females) and animals of group 6 (2/3 males and 2/3 females) consisted of an abnormal coloured stomach (white spotted).

Males (2/3) of group 2 (100 mg/kg bw/day) and 2/3 males of group 3 (250 mg/kg bw/day) were observed with pale kidneys and 1/3 males of group 3 showed a pale liver at the time point of necropsy. Females showed no macroscopic findings at necropsy in any of the groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
dose level: 0, 25, 80, and 250 mg/kg bw/day for main study
Effect level:
0 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Dose levels for main test identified
Critical effects observed:
no
Conclusions:
Based on the data generated from this dose range finding study, dose levels of 0, 25, 80, and 250 mg/kg bw/day (dose formulations prepared without consideration of active ingredient content) are suggested for the subsequent main OECD 408 study with [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one.
Executive summary:

In a dose range finding study (191871) for a repeated dose 90 day oral toxicity study in rats (191870), [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one was administered to Wistar Crl: WI(Han) rats (3/sex/dose) by gavage in corn oil at dose levels of 0, 100, 250, 500, 750, 1000 mg/kg bw/day (based on active ingredient content)/0, 125, 312.5, 625, 937.5, 1250 mg/kg bw/day (without consideration of active ingredient content) daily for 28 days.  

Test item-related mortality occurred in all male and female animals of groups number 4 (500 mg/kg bw/day), 5 (750 mg/kg bw/day), and 6 (1000 mg/kg bw/day). All female animals from groups number 5 and 6 were euthanized in a moribund condition on study day 3 and all male animals from groups number 5 and 6 were euthanized in a moribund condition on study day 4. All males of group no. 4 were euthanized due to animal welfare reasons on study day 12 and all females of group no. 4 were euthanized in a moribund condition on study days 8 and 11.  

Severe clinical findings in groups 4-6 caused by treatment with the test item were apathy, abnormal recumbency, prone position, reduced spontaneous activity, muscular hypotonia, dehydration, half eyelid closure, diarrhoea and discoloured (yellow) faeces. No mortality or clinical symptoms indicating systemic toxicity were observed at dose levels of 100 and 250 mg/kg body weight/day.

Treatment with the test item was shown to affect body weights and body weight development of males in groups 2 and 3 showing lower body weight gain compared to controls throughout the study period and a body weight loss during the last week of treatment. Females in group 3 also showed a body weight loss during the last week of treatment.  Treatment with the test item had no considerable effect on food consumption in any of the test item-treated groups.

Mean WBC level showed was observed to be higher in test item treated male and female animals which was considered test item-related based on the dose dependency and observation of this effect in both genders. Mean PLT levels showed a dose-dependent trend towards higher mean levels in test item treated males compared to control males and were higher in test item treated females compared to control females. Possible test item-related changes in parameters of clinical biochemistry were observed in the parameters crea and urea. In test item treated males dose-dependently higher mean levels of crea and urea were observed compared to the controls. Females showed dosed-dependently higher mean urea levels in test item-treated groups compared to female controls.

Gross pathological changes which may be caused by treatment with the test item were an abnormal coloured stomach (white spotted) in 1/3 males and 2/3 females of group no. 4 (500 mg/kg bw/day) and 2/3 males and 2/3 females of group no. 6 (1000 mg/kg bw/day). Further possible findings which could be related to treatment with the test item were pale kidneys in several males of groups 2 and 3 and a pale liver in one animal in one male of group 3. Females showed no gross pathological changes.  

Based on the data generated from this dose range finding study, dose levels of 0, 25, 80, and 250 mg/kg body weight per day (dose formulations prepared without consideration of active ingredient content for calculation of respective concentrations) are suggested for the subsequent main OECD 408 study with [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study was an OECD 408/GLP compliant study and was assigned a Klimisch score of 1. The overall quality of the database is high.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 September 1999 to 29 April 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to OECD Guideline 411 and GLP but incorrect dermal data presentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Michigan, USA.
- Age at study initiation: 60-66 days
- Weight at study initiation: Male rats: 255-348 g; Female rats: 175-242 g.
- Housing: Housed individually in suspended, stainless-steel cages
- Diet (e.g. ad libitum): Certified rodent diet (No. 8728C Harlan Teklad) ad libitum. Animals fasted for clinical pathology.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days before initiation of treatment. Animals collared for 6 hours/day for 3 days to acclimate to collars.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C-25°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: End date: 13/01/2000
Type of coverage:
other: Gauze dressing over the application site, secured with nonirritating tape, covered with elastic bandage, and overwrapped with elastic tape
Vehicle:
water
Remarks:
reverse osmosis
Details on exposure:
TEST SITE
- Area of exposure: Dorsal trunk area
- % coverage: 5cm x 5cm
- Type of wrap if used: Gauze dressing over the application site, secured with nonirritating tape, covered with elastic bandage, and overwrapped with elastic tape.
- Time intervals for shavings or clipplings: Fur clipped from dorsal trunk area before 1st dose and as needed after.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of each exposure, the area was wiped gently with a dosposable paper towel moistened with reverse osmosis water to remove any remaining test material.
- Time after start of exposure: At the end of each exposure period i.e. after 6-7 hrs exposure per day.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50, 150, 300 mg/kg bw/day
- Constant volume or concentration used: No. 0.050mL; 0.150mL; .300mL for 0, 50, 150, 300 mg/kg bw/day respectively.
Dose preparations were dispensed approximately weekly.


VEHICLE
- Amount(s) applied (volume or weight with unit): 0 mg/kg/day as 0.300 mL/kg bw/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes. Flexible plastic collars during exposure period.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test material was dosed as supplied, dose analysis was not done.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6-7 hours per day , 7 days per week
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
30 animals (15 male and 15 female) per dose

Five animals/sex/group were designated as recovery animals and were observed for approximately 4 weeks post-treatment.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:Not provided in this test report
- Rationale for animal assignment (if not random): Randomised
- Rationale for selecting satellite groups: Five animals/sex/group were designated as recovery animals and were observed for approximately 4 weeks
post-treatment.
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): Randomised

Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Once daily for 2 weeks and weekly thereafter during treatment, on the day of terminal necropsy (all animals), daily during recovery, and on the day of recovery sacrifice, detailed observations were made for each animal; abnormal findings or an indication of normal was recorded.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Dermal irritation will be scored daily immediately before each application (Table 1), on the day of terminal necropsy (all animals), daily during recovery, and on the day of recovery sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight data were recorded on the first day of treatment and weekly thereafter.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption data (grams of food) were recorded weekly for males and females during treatment and recovery.

FOOD EFFICIENCY: Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to treatment and during Weeks 13 and 17 an examination was performed by a veterinarian on all animals using an indirect ophthalmoscope. The eyes were dilated with a mydriatic agent prior to examination.
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from each animal during Weeks 13 and 17.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes; overnight
- How many animals: All animals
- Parameters checked in Table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from each animal during Weeks 13 and 17.
- Animals fasted: Yes; overnight
- How many animals: All animals
- Parameters checked in Table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No




Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A necropsy was done on each animal that was sacrificed or found dead at an unscheduled interval. After Week 13, 10 animals/sex/group (based on survival) were necropsied. After 13 weeks of treatment and 4 weeks of recovery, all surviving recovery animals were necropsied. The necropsy included a macroscopic examination of the extemal features of the carcass; application site; extemal body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Organ weights were also recorded (see Table 4).


HISTOPATHOLOGY: Yes (see Table 5)
Tissues from each animal in the control and high-dose groups and each animal that died or was sacrificed at an unscheduled interval were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Macroscopic lesions and the lung, liver, kidney, and skin (treated and untreated) were also examined microscopically from each animal in the low- and mid-dose groups.
Other examinations:
Estrous Cycle.
Beginning on Day 71 and continuing until Day 91, all females had daily vaginal smears done. Beginning on Day 100 and continuing until Day 120, all recovery females had daily vaginal smears done. Vaginal smears were prepared and examined to evaluate the stage of the estrous cycle.

Male Reproductive Assessment.
Male reproductive capacity was evaluated for 10 males/group at the terminal sacrifice. Sperm morphology, motility and total count were assessed.
Statistics:
Variance homogeneity: Levene's test. (Levene, 1960)

Body weights, body weight changes, food consumption, continuous clinical pathology values, organ weight data : ANOVA; Dunnett's t-test (Dunnett, 1964) .

Group comparisons: two tailed t- test (p<0.05)
Clinical signs:
no effects observed
Description (incidence and severity):
See details on results
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Mortality:
no mortality observed
Description (incidence):
See details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See details on results
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
See details on results
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
See details on results
Haematological findings:
no effects observed
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
See details on results
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
See details on results
Gross pathological findings:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Details on results:
CLINICAL SIGNS AND MORTALITY
Two females in control group died at day 33 (no adverse reason identified). One female in mid-dose sacrificed at day 63 as moribund (due to spontaneous fibrosarcoma). All other animals survived and no test material-related clinical observations noted.

DERMAL IRRITATION
Dermal irritation observations noted during the treatment phase included erythema, edema, atonia, desquamation, and fissuring for animals treated with the test material. One female given 50 mg/kg bw/day had exfoliation. Generally, incidence and severity increased in a dose-related manner and severity ranged from slight to severe or marked. In general, the onset of observations occurred during Weeks 1 to 2 (the earliest observations were noted on Day 4.). Most observations disappeared or the severity was only slight prior to the terminal sacrifice during Week 14. Erythema, edema, atonia, and desquamation were still noted during recovery in animals treated with test material; however, the severity was only slight, which indicates that animals were improving. Most of the observations noted during the recovery period disappeared during Weeks 14 and 15.

BODY WEIGHT AND WEIGHT GAIN
There were no test material-related effects on mean body weights. Body weight changes were statistically significantly lower during Week 1 for females given 50 mg/kg bw/day, during Week 10 for males given 50 or 300 mg/kg bw/day, and during Week 13 for males given 150 mg/kg bw/day. Body weight changes were higher for females during Weeks 15 and 16, significantly during Week 15 for females given 50 or 150 mg/kg bw/day (133% and 200% of controls, respectively). Throughout the duration of the recovery phase (Weeks 14 to 18), body weight changes for the females in all dose groups were remarkably higher, 187.5 to 250% of the control females while body weight changes for the males were 92 to 96% of the control males. The reason for this difference is probably due to the difference in growth rate for males and females. The sporadic significant differences in body weight gains were considered within normal biologic variation.

FOOD CONSUMPTION
Food consumption was statistically significantly higher (7 to 9%) during Weeks 1, 3, and 7 for females given 50 mg/kg bw/day compared with controls, and during Weeks 1 and 2 for females given 300 mg/kg bw/day compared with controls. During Week 4, food consumption was statistically significantly decreased by 8% for females given 150 or 300 mg/kg bw/day. These changes in food consumption were probably due to normal variation and are not considered to be adverse.

OPHTHALMOSCOPIC EXAMINATION
Animals selected for the study had no lesions at the prestudy examination. One incidence of focal retinal degeneration in the right eye was noted during the Week 13 examination for a male given 150 mg/kg bw/day. This finding was considered due to biological variation and not test material-related. No test material-related ophthalmic observations were noted at the Week 17 examination.


HAEMATOLOGY and CLINICAL CHEMISTRY
Administration of Acetyl Cedrene had no obvious effects on clinical pathology test results. Of uncertain relationship to administration of the Acetyl Cedrene was slightly, but statistically, higher activated partial thromboplastin time for males treated with 300 mg/kg bw/day. The difference in the mean values for the control males and the males treated with 300 mg/kg bw/day was less than 1 second, and females were not similarly affected.

ORGAN WEIGHTS, GROSS PATHOLOGY, HISTOPATHOLOGY: NON-NEOPLASTIC
At the terminal sacrifice, test material-related increases in kidney-to-body weight percentages were noted in Group 3 and 4 males. In the kidneys of the males given 300 mg/kg bw/day, hyaline droplet formation was noted in the tubular epitheum. This finding may be unique to the renal tubular biochemistry of the male rat. By the recovery sacrifice, this finding had resolved. At the terminal sacrifice, mild chronic inflammation and acanthosis/hyperkeratosis was noted in the treated skin from all dose groups and both sexes. Again, these changes had completely resolved by the recovery sacrifice.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
One female in mid-dose sacrificed at day 63 as moribund (due to spontaneous fibrosarcoma).

OTHER FINDINGS
Sperm Analysis Report
Mean percent motility, total sperm count, and morphology were not affected by treatment with Acetyl Cedrene at dosage levels of 50,150, and 300 mg/kg bw/day. No biologically meaningful differences were observed between the study groups.

Estrous Cycle
There were no test material-related effects during the treatment or recovery periods on the estrous cycle of females given 0,50,150, or 300 mg/kg bw/day of Acetyl Cedrene.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: not appplicable
Critical effects observed:
not specified
Conclusions:
Daily dermal administration of Acetyl Cedrene to male and female Crl:CD(SD)IGS BR rats for 13 weeks with a 4-week recovery was associated with test material-related observations in the treated skin of treated animals at all dose levels. However, these findings were resolved by the recovery sacrifice. Based on the findings in this study, the NOAEL is considered to be 300 mg/kg bw/day. The LOAEL (local) is 50mg/kg bw/day.

Executive summary:

In a repeated dose dermal toxicity study (Covance 7018 -100), Methyl Cedryl Ketone was applied to the shaved skin of four groups of Crl:CD(SD)IGS BR male and female rats (15/sex/group) at dose levels of 0, 50, 150, 300 mg/kg bw/day, 6 -7 hours/day for 7 days/week during a 13 week period, with a 4 week recovery period.

There were no test material-related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology.

At the terminal sacrifice, test material-related increases in kidney-to-body weight percentages were noted in Group 3 and 4 males. In the kidneys of the males given 300 mg/kg bw/day, hyaline droplet formation was noted in the tubular epitheum. This finding may be unique to the renal tubular biochemistry of the male rat. By the recovery sacrifice, this finding had resolved.

Dermal irritation observations noted during the treatment phase included erythema, edema, atonia, desquamation, and fissuring for animals treated with the test material. In general, the effects were dose dependent and were evident beginning from weeks 1 -2, but were resolved upon sacrifice or were almost fully resolved during weeks 14 -15 of the recovery phase. The NOAEL is 300 mg/kg bw/day. The LOAEL (local) is 50 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was an OECD 411/GLP study and was assigned a Klimisch score of 2. It is a supporting study as ECHA concludes that the dermal route of exposure is not the most appropriate route of exposure in the meaning of Annex IX, 8.6.2., column 2 (CCH-D-2114453248-46-01/F). The overall quality of the database is medium.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 September 1999 to 29 April 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: According to OECD Guideline 411 and GLP but incorrect dermal data presentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Michigan, USA.
- Age at study initiation: 60-66 days
- Weight at study initiation: Male rats: 255-348 g; Female rats: 175-242 g.
- Housing: Housed individually in suspended, stainless-steel cages
- Diet (e.g. ad libitum): Certified rodent diet (No. 8728C Harlan Teklad) ad libitum. Animals fasted for clinical pathology.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days before initiation of treatment. Animals collared for 6 hours/day for 3 days to acclimate to collars.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C-25°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: End date: 13/01/2000
Type of coverage:
other: Gauze dressing over the application site, secured with nonirritating tape, covered with elastic bandage, and overwrapped with elastic tape
Vehicle:
water
Remarks:
reverse osmosis
Details on exposure:
TEST SITE
- Area of exposure: Dorsal trunk area
- % coverage: 5cm x 5cm
- Type of wrap if used: Gauze dressing over the application site, secured with nonirritating tape, covered with elastic bandage, and overwrapped with elastic tape.
- Time intervals for shavings or clipplings: Fur clipped from dorsal trunk area before 1st dose and as needed after.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of each exposure, the area was wiped gently with a dosposable paper towel moistened with reverse osmosis water to remove any remaining test material.
- Time after start of exposure: At the end of each exposure period i.e. after 6-7 hrs exposure per day.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50, 150, 300 mg/kg bw/day
- Constant volume or concentration used: No. 0.050mL; 0.150mL; .300mL for 0, 50, 150, 300 mg/kg bw/day respectively.
Dose preparations were dispensed approximately weekly.


VEHICLE
- Amount(s) applied (volume or weight with unit): 0 mg/kg/day as 0.300 mL/kg bw/day

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes. Flexible plastic collars during exposure period.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test material was dosed as supplied, dose analysis was not done.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6-7 hours per day , 7 days per week
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
30 animals (15 male and 15 female) per dose

Five animals/sex/group were designated as recovery animals and were observed for approximately 4 weeks post-treatment.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:Not provided in this test report
- Rationale for animal assignment (if not random): Randomised
- Rationale for selecting satellite groups: Five animals/sex/group were designated as recovery animals and were observed for approximately 4 weeks
post-treatment.
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): Randomised

Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Once daily for 2 weeks and weekly thereafter during treatment, on the day of terminal necropsy (all animals), daily during recovery, and on the day of recovery sacrifice, detailed observations were made for each animal; abnormal findings or an indication of normal was recorded.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Dermal irritation will be scored daily immediately before each application (Table 1), on the day of terminal necropsy (all animals), daily during recovery, and on the day of recovery sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight data were recorded on the first day of treatment and weekly thereafter.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption data (grams of food) were recorded weekly for males and females during treatment and recovery.

FOOD EFFICIENCY: Yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to treatment and during Weeks 13 and 17 an examination was performed by a veterinarian on all animals using an indirect ophthalmoscope. The eyes were dilated with a mydriatic agent prior to examination.
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from each animal during Weeks 13 and 17.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes; overnight
- How many animals: All animals
- Parameters checked in Table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from each animal during Weeks 13 and 17.
- Animals fasted: Yes; overnight
- How many animals: All animals
- Parameters checked in Table 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No




Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A necropsy was done on each animal that was sacrificed or found dead at an unscheduled interval. After Week 13, 10 animals/sex/group (based on survival) were necropsied. After 13 weeks of treatment and 4 weeks of recovery, all surviving recovery animals were necropsied. The necropsy included a macroscopic examination of the extemal features of the carcass; application site; extemal body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Organ weights were also recorded (see Table 4).


HISTOPATHOLOGY: Yes (see Table 5)
Tissues from each animal in the control and high-dose groups and each animal that died or was sacrificed at an unscheduled interval were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Macroscopic lesions and the lung, liver, kidney, and skin (treated and untreated) were also examined microscopically from each animal in the low- and mid-dose groups.
Other examinations:
Estrous Cycle.
Beginning on Day 71 and continuing until Day 91, all females had daily vaginal smears done. Beginning on Day 100 and continuing until Day 120, all recovery females had daily vaginal smears done. Vaginal smears were prepared and examined to evaluate the stage of the estrous cycle.

Male Reproductive Assessment.
Male reproductive capacity was evaluated for 10 males/group at the terminal sacrifice. Sperm morphology, motility and total count were assessed.
Statistics:
Variance homogeneity: Levene's test. (Levene, 1960)

Body weights, body weight changes, food consumption, continuous clinical pathology values, organ weight data : ANOVA; Dunnett's t-test (Dunnett, 1964) .

Group comparisons: two tailed t- test (p<0.05)
Clinical signs:
no effects observed
Description (incidence and severity):
See details on results
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Mortality:
no mortality observed
Description (incidence):
See details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See details on results
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
See details on results
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
See details on results
Haematological findings:
no effects observed
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
See details on results
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
See details on results
Gross pathological findings:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Details on results:
CLINICAL SIGNS AND MORTALITY
Two females in control group died at day 33 (no adverse reason identified). One female in mid-dose sacrificed at day 63 as moribund (due to spontaneous fibrosarcoma). All other animals survived and no test material-related clinical observations noted.

DERMAL IRRITATION
Dermal irritation observations noted during the treatment phase included erythema, edema, atonia, desquamation, and fissuring for animals treated with the test material. One female given 50 mg/kg bw/day had exfoliation. Generally, incidence and severity increased in a dose-related manner and severity ranged from slight to severe or marked. In general, the onset of observations occurred during Weeks 1 to 2 (the earliest observations were noted on Day 4.). Most observations disappeared or the severity was only slight prior to the terminal sacrifice during Week 14. Erythema, edema, atonia, and desquamation were still noted during recovery in animals treated with test material; however, the severity was only slight, which indicates that animals were improving. Most of the observations noted during the recovery period disappeared during Weeks 14 and 15.

BODY WEIGHT AND WEIGHT GAIN
There were no test material-related effects on mean body weights. Body weight changes were statistically significantly lower during Week 1 for females given 50 mg/kg bw/day, during Week 10 for males given 50 or 300 mg/kg bw/day, and during Week 13 for males given 150 mg/kg bw/day. Body weight changes were higher for females during Weeks 15 and 16, significantly during Week 15 for females given 50 or 150 mg/kg bw/day (133% and 200% of controls, respectively). Throughout the duration of the recovery phase (Weeks 14 to 18), body weight changes for the females in all dose groups were remarkably higher, 187.5 to 250% of the control females while body weight changes for the males were 92 to 96% of the control males. The reason for this difference is probably due to the difference in growth rate for males and females. The sporadic significant differences in body weight gains were considered within normal biologic variation.

FOOD CONSUMPTION
Food consumption was statistically significantly higher (7 to 9%) during Weeks 1, 3, and 7 for females given 50 mg/kg bw/day compared with controls, and during Weeks 1 and 2 for females given 300 mg/kg bw/day compared with controls. During Week 4, food consumption was statistically significantly decreased by 8% for females given 150 or 300 mg/kg bw/day. These changes in food consumption were probably due to normal variation and are not considered to be adverse.

OPHTHALMOSCOPIC EXAMINATION
Animals selected for the study had no lesions at the prestudy examination. One incidence of focal retinal degeneration in the right eye was noted during the Week 13 examination for a male given 150 mg/kg bw/day. This finding was considered due to biological variation and not test material-related. No test material-related ophthalmic observations were noted at the Week 17 examination.


HAEMATOLOGY and CLINICAL CHEMISTRY
Administration of Acetyl Cedrene had no obvious effects on clinical pathology test results. Of uncertain relationship to administration of the Acetyl Cedrene was slightly, but statistically, higher activated partial thromboplastin time for males treated with 300 mg/kg bw/day. The difference in the mean values for the control males and the males treated with 300 mg/kg bw/day was less than 1 second, and females were not similarly affected.

ORGAN WEIGHTS, GROSS PATHOLOGY, HISTOPATHOLOGY: NON-NEOPLASTIC
At the terminal sacrifice, test material-related increases in kidney-to-body weight percentages were noted in Group 3 and 4 males. In the kidneys of the males given 300 mg/kg bw/day, hyaline droplet formation was noted in the tubular epitheum. This finding may be unique to the renal tubular biochemistry of the male rat. By the recovery sacrifice, this finding had resolved. At the terminal sacrifice, mild chronic inflammation and acanthosis/hyperkeratosis was noted in the treated skin from all dose groups and both sexes. Again, these changes had completely resolved by the recovery sacrifice.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
One female in mid-dose sacrificed at day 63 as moribund (due to spontaneous fibrosarcoma).

OTHER FINDINGS
Sperm Analysis Report
Mean percent motility, total sperm count, and morphology were not affected by treatment with Acetyl Cedrene at dosage levels of 50,150, and 300 mg/kg bw/day. No biologically meaningful differences were observed between the study groups.

Estrous Cycle
There were no test material-related effects during the treatment or recovery periods on the estrous cycle of females given 0,50,150, or 300 mg/kg bw/day of Acetyl Cedrene.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: not appplicable
Critical effects observed:
not specified
Conclusions:
Daily dermal administration of Acetyl Cedrene to male and female Crl:CD(SD)IGS BR rats for 13 weeks with a 4-week recovery was associated with test material-related observations in the treated skin of treated animals at all dose levels. However, these findings were resolved by the recovery sacrifice. Based on the findings in this study, the NOAEL is considered to be 300 mg/kg bw/day. The LOAEL (local) is 50mg/kg bw/day.

Executive summary:

In a repeated dose dermal toxicity study (Covance 7018 -100), Methyl Cedryl Ketone was applied to the shaved skin of four groups of Crl:CD(SD)IGS BR male and female rats (15/sex/group) at dose levels of 0, 50, 150, 300 mg/kg bw/day, 6 -7 hours/day for 7 days/week during a 13 week period, with a 4 week recovery period.

There were no test material-related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology.

At the terminal sacrifice, test material-related increases in kidney-to-body weight percentages were noted in Group 3 and 4 males. In the kidneys of the males given 300 mg/kg bw/day, hyaline droplet formation was noted in the tubular epitheum. This finding may be unique to the renal tubular biochemistry of the male rat. By the recovery sacrifice, this finding had resolved.

Dermal irritation observations noted during the treatment phase included erythema, edema, atonia, desquamation, and fissuring for animals treated with the test material. In general, the effects were dose dependent and were evident beginning from weeks 1 -2, but were resolved upon sacrifice or were almost fully resolved during weeks 14 -15 of the recovery phase. The NOAEL is 300 mg/kg bw/day. The LOAEL (local) is 50 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
50 mg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was an OECD 411/GLP study and was assigned a Klimisch score of 2. It is a supporting study as ECHA concludes that the dermal route of exposure is not the most appropriate route of exposure in the meaning of Annex IX, 8.6.2., column 2 (CCH-D-2114453248-46-01/F). The overall quality of the database is medium.

Additional information

Repeated dose toxicity

There is a 14 day DRF oral study in rats, a 90 day repeated dose oral toxicity study in rats and a 90 day repeated dose dermal toxicity in rats available. The dermal study is a supporting study, as ECHA concluded that "the dermal route of exposure is not the most appropriate route of exposure in the meaning of Annex IX, 8.6.2., column 2". (CCH-D-2114453248-46-01/F).

A rat OECD 408/GLP compliant study was conducted, modified to include urinalysis and a full histopathological examination, which included immunohistochemical investigation of renal pathology to determine if the pathology is mediated by alpha-2u globulin nephropathy (CCH-D-2114453248-46-01/F).

Repeated dose toxicity (oral):

Dose range finding study

In a dose range finding study, for a repeated dose 90 day oral toxicity study in rats, [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one

was administered to Wistar Crl: WI(Han) rats (3/sex/dose) by gavage in corn oil at dose levels of 0, 100, 250, 500, 750, 1000 mg/kg bw/day (based on active ingredient content)/0, 125, 312.5, 625, 937.5, 1250 mg/kg bw/day (without consideration of active ingredient content) daily for 28 days.  

Test item-related mortality occurred in all male and female animals of groups number 4 (500 mg/kg bw/day), 5 (750 mg/kg bw/day), and 6 (1000 mg/kg bw/day). All female animals from groups number 5 and 6 were euthanized in a moribund condition on study day 3 and all male animals from groups number 5 and 6 were euthanized in a moribund condition on study day 4. All males of group no. 4 were euthanized due to animal welfare reasons on study day 12 and all females of group no. 4 were euthanized in a moribund condition on study days 8 and 11. Severe clinical findings in groups 4-6 caused by treatment with the test item were apathy, abnormal recumbency, prone position, reduced spontaneous activity, muscular hypotonia, dehydration, half eyelid closure, diarrhoea and discoloured (yellow) faeces. No mortality or clinical symptoms indicating systemic toxicity were observed at dose levels of 100 and 250 mg/kg body weight/day. Treatment with the test item was shown to affect body weights and body weight development of males in groups 2 and 3 showing lower body weight gain compared to controls throughout the study period and a body weight loss during the last week of treatment. Females in group 3 also showed a body weight loss during the last week of treatment.  Treatment with the test item had no considerable effect on food consumption in any of the test item-treated groups.

Mean WBC level showed was observed to be higher in test item treated male and female animals which was considered test item-related based on the dose dependency and observation of this effect in both genders. Mean PLT levels showed a dose-dependent trend towards higher mean levels in test item treated males compared to control males and were higher in test item treated females compared to control females. Possible test item-related changes in parameters of clinical biochemistry were observed in the parameters crea and urea. In test item treated males dose-dependently higher mean levels of crea and urea were observed compared to the controls. Females showed dosed-dependently higher mean urea levels in test item-treated groups compared to female controls. Gross pathological changes which may be caused by treatment with the test item were an abnormal coloured stomach (white spotted) in 1/3 males and 2/3 females of group no. 4 (500 mg/kg bw/day) and 2/3 males and 2/3 females of group no. 6 (1000 mg/kg bw/day). Further possible findings which could be related to treatment with the test item were pale kidneys in several males of groups 2 and 3 and a pale liver in one animal in one male of group 3. Females showed no gross pathological changes.

Based on the data generated from this dose range finding study, dose levels of 0, 25, 80, and 250 mg/kg body weight per day (dose formulations prepared without consideration of active ingredient content for calculation of respective concentrations) are suggested for the subsequent main OECD 408 study with

[3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one

 

OECD 408 main study

In a repeated dose toxicity study (OECD 408/GLP), [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one was administered to Wistar Crl: WI(Han) rats (10/sex/dose) by gavage in corn oil at dose levels of 0, 25 (LD), 80 (MD) and 250 (HD) mg/kg bw/day daily for 90 days.

 

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.

The body weight of HD male animals was statistically significantly reduced from study day 8 until the end of treatment. The body weight change of HD male animals was statistically significantly reduced compared to the control group. The body weight of HD female animals was statistically significantly reduced from study day 29 and 36 only. The body weight change of HD female animals was not statistically significantly reduced compared to the control group. The mean body weights of male and female HD animals were within the range of the historical control data.

No treatment related or toxicologically relevant effects on body weight gain, food consumption, clinical observations, functional observation battery, ophthalmology, haematological parameters, blood coagulation, clinical biochemistry, urine, thyroid hormones or reproductive organs were observed during the treatment period. The male HD PLT count, male and female MD and HD TBIL and male HD mean kidney weight and male HD and female MD and HD mean liver weights were above the historical control data and are considered to be test item related.

Macroscopic findings during necropsy were considered to be incidental and not test item related.

Under the conditions of this study, after a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in Wistar rats, there were induced findings in the liver, thyroid and kidney in HD males and females. In the liver, the fatty change associated with single cell death observed in some HD males and one female, was considered an adverse finding caused by the Test Item. In the intermediate doses (LD and MD), there was also a subtle increase in severity or frequency levels of fatty changes compare to control group but in absence of other degenerative changes, therefore it was considered an adaptative finding at these dosages. The karyomegaly observed in most of the males and in one HD female was also deemed to be a treatment induced adaptative finding. Karyomegaly is considered often a reflection of hepatocyte polyploidy that occurs when there is duplication of nuclear material in the absence of cytokinesis. Also, in several HD males and females, the presence of well-defined, eosinophilic foci of hepatocellular alteration and the increase in frequency and severity of bile duct hyperplasia were deemed to be induced by the treatment. The eosinophilic foci are considered to represent a localized proliferation of hepatocytes that are phenotypically different from surrounding hepatocyte parenchyma and could represent preneoplastic lesion. Hepatocellular hypertrophy observed in some HD males and females was deemed to be an adaptative Test Item induced effect, which would probably recover after withdrawn of the treatment. This often occurs secondary to increase in microsomal enzymes following exposure to enzyme inducing xenobiotics.

In the thyroid gland, the follicular hypertrophy associated with alterations in the colloid in HD males and females only was considered a test item-related change. These findings were not present in the LD and MD males and females. In rats, liver changes (hepatocellular hypertrophy) may induce an increase in thyroid hormones degradation and an increased in TSH leading to thyroid gland hypertrophy. This phenomenon is more rat specific and of less toxicologic relevance in humans. Both, follicular cell hyperplasia and hypertrophy are expected to return to normal if the stimulus is withdrawn. A statistically significant decrease in T3 (p < 0.01) and T4 (p < 0.001) was found in HD male animals only. However, all values were within the range of the historical control data. There were no statistically significant changes in the level of TSH in male or females at any dose and no statistically significant changes in (para)thyroid weight in males or females at any dose group.

In the kidney of the males from all the groups, the hyaline droplets and the elongated, eosinophilic bodies at the basal level of the renal epithelium were observed. Both were considered proteinaceous deposits accumulated in the cytoplasm from renal tubular epithelial cells. On haematoxylin and eosin (HE) pathology evaluation, there was an increase (in frequency or severity) of proteinaceous deposits in all treated groups compare to the control group, especially for the elongated eosinophilic bodies at the basal area. This was considered a Test Item induced effect. Tubular basophilia in HD females, in absence of further degenerative changes, was considered in this case most likely an adaptative response.

An indirect immunohistochemistry optimized method using the anti-alpha2u-globulin (primary antibody) and BOND Polymer Refine Detection system (containing secondary Rabbit anti mouse IgG, HRP conjugated anti-rabbit Polymer-IgG and the substrate chromogen, 3,3’-Diaminobenzidine tetrahydrochloride hydrate (DAB)) was performed on the kidneys from all the male rats and from females of the control and high dose group using Leica Bond-III system, automated immunohistochemistry stainer. Anti-alpha2u-globulin IHC was validated by specific positive immunolabeling in the positive control slide; in contrast to the lack of positive immunolabelling for the negative IHC control slide.

With immunohistochemistry, alpha2u-globulin positive signal was detected on the proteinaceous deposits (i.e. hyaline droplets and the elongated, eosinophilic bodies) in all the male groups, which demonstrated the accumulation of this protein in renal tubules. After image analysis evaluation, the increase in the percentage of positive alpha2u-globulin signal in the intermediate doses compared the controls, revealed an induced increase in accumulation of alpha2u-globulin related to the treatment at these dosages, however these differences were found to be statistically different only at the LD compared to control group. In comparative evaluation, image analysis showed that the HD male group presented a decrease in the presence of alpha2u-globulin-positive signal compare to the control group and intermediate doses. The most likely hypothesis is that the induced hepatic damage at the high dose, may impair the hepatic production of alpha2u-globulin decreasing progressively the circulating level, and therefore decreasing the renal accumulation. There may also be an overloading of the metabolic pathway and therefore a decrease in the reduction of MCK and subsequent conjugation with glucuronic acid and/or alpha2u-globulin. The glucuronic pathway can be overloaded for non-acidic substances (Howell et al., 1986); possibly the alpha-2u pathway can become overloaded also.

Alpha2u-globulin is a circulating low molecular protein which is synthesized in the liver of male rats only, under androgen regulation on physiological conditions (Hamamura et al., 2017; McInnes, 2012). This explains the absence of accumulation of alpha2u-globulin in the female groups and the presence of alpha2u-globulin in the male control group. Furthermore, alpha2u-globulin accumulation is a male- and rat- specific entity, which will not be translated to humans (Frazier et al., 2012). Many xenobiotics bind to circulating alpha2u-globulin and decrease effectiveness of lysosomal degradation in the proximal tubules resulting in greater accumulation. The increase in tubular basophilia in LD and MD males exceeded the background levels from the control group. This, and the presence of tubular hyaline casts in few treated males were considered characteristic early changes associated with alpha2u-globulin nephropathy (Brändli-Baiocco et al., 2018). This effect is not relevant for the human health risk assessment.

Under the conditions of this study and based on the pathology evaluation, the NOAEL for a repeated exposure to [3R-(3alpha,3abeta,7beta,8aalpha)] -1 - (2,3,4,7,8,8a-hexahydro- 3,6,8,8-tetramethyl- 1H-3a, 7-methanoazulen-5-yl) ethan-1-one by oral gavage over a period of 90 days in male and female Wistar rats, can be established at 80 mg/kg bw/day.

Repeated dose toxicity (dermal):

In a repeated dose dermal toxicity study (OECD 411/GLP), Methyl Cedryl Ketone was applied to the shaved skin of four groups of Crl:CD(SD)IGS BR male and female rats (15/sex/group) at dose levels of 0, 50, 150, 300 mg/kg bw/day, 6 -7 hours/day for 7 days/week during a 13 week period, with a 4 week recovery period.

There were no test material-related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology.

At the terminal sacrifice, test material-related increases in kidney-to-body weight percentages were noted in Group 3 and 4 males. In the kidneys of the males given 300 mg/kg bw/day, hyaline droplet formation was noted in the tubular epitheum. This finding may be unique to the renal tubular biochemistry of the male rat. By the recovery sacrifice, this finding had resolved.

Dermal irritation observations noted during the treatment phase included erythema, edema, atonia, desquamation, and fissuring for animals treated with the test material. In general, the effects were dose dependent and were evident beginning from weeks 1 -2, but were resolved upon sacrifice or were almost fully resolved during weeks 14 -15 of the recovery phase. The NOAEL (systemic) is 300 mg/kg bw/day. The LOAEL (local) is 50mg/kg bw/day.

Conclusion

In relation to the 90-day dermal repeated dose toxicity study, ECHA noted that “both the pathologist and the Research Institute for Fragrance Material (RIFM) expert panel, who reviewed the study, suggest that the findings indicate alpha-2u-globulin-mediated nephropathy” (CCH-D-2114453248-46-01; Belsito et al., 2013). ECHA also noted in their analysis that “adverse effects such as increases in kidney-to-body weight percentages in group 3 and 4 of male rats and hyaline droplet formation in the tubular epithelium at 300 mg/kg bw/day were observed in the kidneys of male rats and not in female rats. The fact that these effects were only observed in male rats may indicate that the registered substance may induce alpha-2u-globulin-mediated nephropathy. ECHA accordingly considers that the kidney is a target organ of the registered substance. Since humans do not excrete alpha-2u-globulin and this mode of action is considered not relevant to humans the involvement of alpha-2u-globulin in the kidney effects is a key parameter for establishing the relevance of the kidney effects for risk assessment. For these reasons, ECHA considers that urinalysis is required to investigate kidney function (which is optional in paragraphs 3, 4 and 37 of OECD TG 408). Additionally, a full histopathological examination (paragraphs 3, 4, 45 and 47 of OECD TG 408), is required, including immunohistochemical investigation of renal pathology to determine if the pathology is indeed mediated by alpha-2u globulin” (CCH-D-2114453248-46-01).

The registrant carried out the oral OECD 408/GLP study with the required modifications as requested. The registrant has concluded, based on the expert pathologist opinion, that the findings in the kidney in male rats treated with the substance relate to alpha-2u-globulin-mediated nephropathy and are therefore not relevant for the human health risk assessment.

Belsito D, Bickers D, Bruze M, Calow P, Dagli ML, Fryer AD, Greim H, Miyachi Y, Saurat JH, Sipes IG (2013). A toxicological and dermatological assessment of alkyl cyclic ketones when used as fragrance ingredients. Food and Chemical Toxicology (62) S1–S44.

Brändli-Baiocco A, Balme E, Bruder M, Chandra S, Hellmann J, Hoenerhoff MJ, et al. Nonproliferative and Proliferative Lesions of the Rat and Mouse Endocrine System. J Toxicol Pathol. 2018;31(3 Suppl):1S-95S.

Frazier KS, Seely JC, Hard GC, Betton G, Burnett R, Nakatsuji S, et al. Proliferative and nonproliferative lesions of the rat and mouse urinary system. Toxicologic pathology Vol 40, Issue 4_suppl, 2012

Hamamura M, Oshikata T, Katoku K, Tsuchitani M, Yamaguchi R. Two types of deposits, hyaline droplets and eosinophilic bodies, associated with α2u-globulin accumulation in the rat kidney. Journal of Toxicologic Pathology. 2017;30(4):275–82.

S. R. Howell, G. A. Hazelton, C. D. Klaassen. Depletion of Hepatic UDP-glucuronic Acid by Drugs That Are Glucuronidated. J Pharmacol Exp Ther. 1986 Mar;236(3):610-4.

McInnes E. Background lesions in laboratory animals. 1st ed. Elsevier; 2012.


Justification for classification or non-classification

Based on the available information in the dossier, the substance

[3R-(3alpha,3abeta,7beta,8aalpha)]-1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a, 7-methanoazulen-5-yl) ethan-1-one

(CAS No. 32388-55-9) does not need to be classified as specific target organ toxicity (repeated exposure) when considering the criteria outlined in Annex I of 1272/2008/EC.