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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 October to 26 November 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 407 and in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
Revised 3 October 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents
Version / remarks:
July 2000.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected on 23-04-2019 / Signed on 01-08-2019
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(3aα,4β,7β,7aα)-octahydro-4,7-methano-1H-indene
EC Number:
220-585-8
EC Name:
(3aα,4β,7β,7aα)-octahydro-4,7-methano-1H-indene
Cas Number:
2825-82-3
Molecular formula:
C10H16
IUPAC Name:
(1R,2S,6R,7S)-tricyclo[5.2.1.0^{2,6}]decane
Test material form:
liquid
Details on test material:
- Physical appearance: clear liquid
- Storage conditions: stored under nitrogen at ambient temperature (15 to 25°C)

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Age at study initiation: 41 to 47 days.
- Weight at study initiation: Males: 206 - 259 g; Females: 158 - 209 g
- Housing: in groups of 5, in Polycarbonate body with a stainless steel mesh lid cages, changed at appropriate intervals.
- Diet (e.g. ad libitum): Teklad 2014C Diet, ad libitum (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (except during urine collection).
- Acclimation period: 13 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 24°C.
- Humidity (%): 40-70%.
- Air changes (per hr): not reported. Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12.

Other:
Aspen gnawing material: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was chosen to simulate the conditions of potential human exposure
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Diet: Teklad 2014C, powdered diet.
- Correction factor: A correction factor was not required.
- Method of preparation: the test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test item required for the premix was added to an equal amount of plain diet and stirred.
An amount of plain diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added or until the mix appeared dry. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a turbula mixer for 100 cycles.
This premix was diluted with further quantities of plain diet using the doubling up process to prepare the test mixes. Each formulation was mixed using a turbula mixer for 100 cycles.
- Frequency of preparation: Weekly.
- Storage temperature of food: at ambient temperature (15 to 25°C).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: The homogeneity and stability of formulations during storage was confirmed as part of another study, Covance Study No. TL52KY. The suitability of the proposed mixing procedures was determined and specimen formulations at 500 to 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix.
Homogeneity and stability of THDCPD in the diet matrix was demonstrated for up to 24 days when stored at ambient (15 to 25°C) or frozen temperature (-10 to -30°C).
- Achieved concentration: Samples of each formulation prepared for administration during Week 1 of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Group 1 (Control group)
Dose / conc.:
1 500 ppm
Remarks:
Group 2 (Low dose)
Dose / conc.:
5 000 ppm
Remarks:
Group 3 (Mid dose)
Dose / conc.:
15 000 ppm
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dietary concentrations selected for this study were based on the findings from the preliminary toxicity study (Covance study No. BL96QQ) where male and female rats were administered THDCPD via the diet continuously for seven days at doses of 1500, 5000 and 15000 ppm. In the seven-day dose range findings study there was no effect on the clinical condition, body weight gain or average food intake of the animals. At macroscopic examination the absolute and body weight adjusted mean weights of the kidneys in males treated with 5000 or 15000 ppm were high when compared with the organ weights of the low dose group, however there was no dose relationship. All organ weights for the females were similar throughout the groups. There were no findings at macroscopic examination that were associated to treatment with THDCPD.
As none of these findings were dose limiting the same dietary concentrations were selected for this OECD 407 study, the high dietary level is 15000 ppm, with the low and intermediate levels set at 1500 and 5000 ppm.
- Rationale for animal assignment: random.
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
MORTALITY / MORBIDITY: Yes
- Time schedule: twice daily

CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 29 of the study (all main study animals) and week R3 (All recovery animals)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes. Animals were deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 29 of the study (all main study animals) and Week R3 (all recovery animals)
- Animals fasted: Yes. Animals were deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on Day 29 of the study (all main study animals)
- Animals fasted: yes
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On one occasion in the final week of the treatment period.
- Dose groups that were examined: all animals.
- Battery of functions tested: sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2)
Other examinations:
None
Statistics:
Depending on the nature of the data set to be analysed, appropriate tests will be applied as indicated in table 7.5.1/3. Where parametric tests may be appropriate they will be preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions fails a log transformation will be applied before retesting. If the transformation fails, appropriate nonparametric tests will be applied.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behaviour of the animals were unaffected by treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths in the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Overall (Week 1 to 4) body weight gain was low for females receiving 5000 or 15000 ppm, attaining statistical significance (0.62X and 0.70X, control, respectively). As the Week 4 body weight was recorded following overnight deprivation of food, the body weight gain for Week 1 to 3 was also assessed. Body weight gain (Week 1 to 3) for females receiving 5000 or 15000 ppm was low, attaining statistical significance (0.74X and 0.70X of the controls, respectively).
During the recovery phase, the overall body weight gain for females previously given 15000 ppm was higher than control (1.33X), showing evidence of complete recovery.
There was no effect of treatment throughout the study for all male treated groups and females given 1500 ppm
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Overall (Week 1 to 4) group mean food intake for females given 5000 or 15000 ppm were lower than the control group (0.81X and 0.84X of the controls, respectively), although there was no dose relationship. Group mean food intake for females given 1500 ppm was similar to control and considered unaffected by treatment.
Overall (Week 1 to 4) group mean food intake for males was similar throughout the groups and was unaffected by treatment.
Group mean food consumption during recovery for males and females previously receiving 15000 ppm was comparable with the control group, showing complete recovery for females had occurred.

The estimated food scatters per cage were up to 20 g throughout the study (data not reported), however as the scatters were observed in all groups, with the highest scatters seen for males receiving 5000 ppm, there was considered to be no correlation with the diet concentration given
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After four weeks of treatment, high reticulocyte (1.16X) and white blood cell counts (1.14X), mainly due to a high neutrophil count (1.49X), were seen for males given 15000 ppm, compared with control. A high neutrophil count was also observed for males given 1500 ppm (1.51X), although there was no dose relationship. In females, a high large unstained cell count was seen for animals given 15000 ppm (1.60X) compared with control. In addition, in females, slightly lower platelet counts were observed for animals given 5000 or 15000 ppm (0.80X or 0.82X when compared with control). At the end of the recovery period, the large unstained cell count was still higher than control (1.67X), although less individual animals were outside the control range, showing partial recovery. The other parameters were within the control range at the end of the recovery period showing complete recovery.
All other inter-group differences from control, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
Description (incidence and severity):
The biochemical examination of the blood plasma performed after four weeks of treatment indicated, when compared with controls, statistically significantly lower total glucose concentration (0.76X) and statistically significantly higher triglyceride concentration in males receiving 15000 ppm (1.44X) and females receiving 1500, 5000 or 15000 ppm (1.59X, 1.14X or 1.59X, respectively), although there was no dose relationship for triglyceride in the females.
In females, a lower plasma urea concentration and a higher total phosphorus was seen; attaining statistical significance for all treated groups (0.66X, 0.72X or 0.72X, for urea, and 1.12X, 1.10X or 1.20X for phosphorus, respectively). Cholesterol concentration was also high in females receiving 15000 ppm (1.39X) when compared with control.
Following two weeks of recovery, males previously treated with 15000 ppm continued to show lower glucose (0.83X) females previously treated with this dose level had similar concentrations of glucose to the control. In addition, plasma concentrations of phosphorus, urea and cholesterol were comparable with the control group showing complete recovery.
Following the recovery period males and females previously treated with 15000 ppm exhibited lower triglyceride concentrations (0.71X and 0.85X of the controls, respectively); this decrease in triglyceride concentration showed complete recovery from higher triglyceride concentrations during treatment with THDCPD.
All other inter-group differences from control, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The analysis of urine performed after four weeks of treatment revealed that males and females were unaffected by treatment with THDCPD. Therefore, analysis of urine was not extended to recovery animals.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The general appearance and behaviour of the animals were unaffected by treatment.
Sensory reactivity and grip strength were unaffected by treatment.
Motor activity scores during Week 4 of treatment displayed increased total high and low beam activity scores for males given 5000 ppm, and males and females given 15000 ppm, when compared with controls, with the occasional statistical significance attained. The total low beam score for the females given 15000 ppm was also outside the historical control range (649.2 – 1349.6; 10 studies).
During Week 3 of recovery the activity testing revealed that the total group mean activity scores for high and low beams for males and females previously given 15000 ppm remained high compared with the controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The analysis of absolute and adjusted organ weights performed after four weeks of treatment revealed, when compared with controls, high kidney (1.10X) and prostate, seminal vesicles and coagulating gland (1.16X) weights in males given 15000 ppm. In females, following four weeks of treatment with THDCPD, the absolute and bodyweight adjusted uterus and cervix (0.88X) and ovary weights (0.85X) were low in females receiving 15000 ppm.
Following two weeks of recovery, for males previously treated with 15000 ppm, the absolute and adjusted kidney and prostate, seminal vesicles and coagulating gland weights for the males and the uterus and cervix and ovary weights for the females were comparable with control, showing complete recovery.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 4 weeks of treatment and 2 weeks of recovery revealed no test item related findings.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Animals Killed After 4 Weeks of Treatment:
- Treatment Related Findings: changes related to treatment with THDCPD were seen in the kidneys of males given 15000 ppm.
- Kidneys: increased incidence and severity of hyaline droplet accumulation and basophilic tubules were seen in all males given 15000 ppm. Upon Sponsor request, the kidneys of males given 1500 or 5000 ppm were not examined.
Incidental Findings
All other histological changes were considered to be incidental and unrelated to the test item.

Animals Killed After 2 Weeks of Recovery
- Treatment Related Findings: evidence of recovery from test item-related findings of the kidneys was seen in males previously given 15000 ppm for 4 weeks followed by 2 weeks off-dose period.
- Kidneys: there was evidence of a complete recovery from test item-related hyaline droplet accumulation in males previously given 15000 ppm. However, increased incidence of basophilic tubules was still present. It was considered that a partial recovery had occurred in terms of reduced incidence of basophilic tubules when compared with those seen after treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Estrous cycles:
Estrous cycles were not affected by treatment with THDCPD.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
other: No adverse effects were observed.
Remarks on result:
other: equivalent to 1205 mg/kg/day in males and 1165 mg/kg/day in females.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the No Observed Adverse Effect level (NOAEL) was considered to be 15000 ppm (equivalent to 1205 mg/kg/day in males and 1165 mg/kg/day in females), based on no adverse effects observed at all tested doses. Therefore, the test substance is not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a sub-acute toxicity study performed according to the OECD test guideline No. 407 and in compliance with GLP, test item was administered by dietary admixture to Sprague-Dawley [Crl:CD(SD)] rats (5/sex/group) at concentrations of 1500, 5000 or 15000 ppm. A similarly constituted control group received the untreated basal diet. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for four weeks, followed by a two week period without treatment to assess the potential for any treatment-related change to recover. 


 


During the study detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment only), estrous cycle evaluation, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.


 


The achieved average dosages during the 4-week treatment period were 116, 394 and 1205 mg/kg/day for males and 125, 387 and 1165 mg/kg/day for females at dietary concentrations of 1500, 5000 and 15000 ppm, respectively.


The mean concentrations were within 4% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.


The general appearance, behaviour, estrus cycles, urinalysis, sensory reactivity and grip strength of the animals were unaffected by treatment.


Motor activity testing during Week 4 of treatment revealed increasedtotal high and low beam activity scores for males given 5000 ppm and males and females given 15000 ppm, when compared with controls, with the occasional statistical significance attained. During Week 3 of recovery, the activity testing revealed that the total group mean activity scores for high and low beams for males and females previously receiving 15000 ppm remained high compared with the controls. 


The body weight gain and group mean food intake after 4 weeks of treatment with THDCPD was low in females receiving 5000 or 15000 ppm, respectively, when compared with the controls. After 2 weeks of recovery body weight gain for females previously given 15000 ppm were higher than control, displaying evidence of complete recovery.There was no effect of treatment on body weight gain or food intake for all treated males.


Histopathological analysis revealed treatment-related findings in the kidneys of males given 15000 ppm; there was an increase in hyaline droplet accumulation and basophilic tubules. Hyaline droplet accumulation is frequently seen in the proximal tubules of the kidneys in male rats and is characterized by prominent eosinophilic material, likely composed of alpha-2u-globulin proteins. Their accumulation accounted for the increase in the kidney weights of males receiving 15000 ppm. Following the 2 weeks off-dose period, a complete recovery of the hyaline droplet accumulation and a partial recovery in terms of reduced incidence of basophilic tubules were seen in males previously given 15000 ppm.


Hematological examination after four weeks of treatment revealed high reticulocyte, neutrophil and white blood cell counts in males receiving 15000 ppm, in females receiving 15000 ppma high large unstained cell count was seen and slightly lower platelet counts were observed for females given 5000 or 15000 ppm,when compared with the controls. Large unstained cell counts in females remained high following recovery, however, in the absence of changes in any associated parameters, the toxicological significance of this change is uncertain. All other hematological parameters were within the control range for animals previously treated with 15000 ppm at the end of the recovery period; displaying evidence of complete recovery.


The biochemical examination of the blood plasma performed after four weeks of treatment revealed low total glucose concentration and high triglyceride concentration in males receiving 15000 ppm and females receiving 1500, 5000 or 15000 ppm. In females, a lower plasma urea concentration and a higher total phosphorus was seen; attaining statistical significance for all treated groups. Cholesterol concentration was also high in females receiving 15000 ppm when compared with control. 


Following two weeks of recovery, males previously treated with 15000 ppm continued to show lower glucose concentrations. Plasma concentrations of glucose, phosphorus, urea and cholesterol in females previously given 15000 ppm were comparable with the control group, showing complete recovery. A decrease in triglyceride concentration was seen in both sexes at the end of the recovery period; this demonstrated a complete recovery from the increased triglyceride concentrations seen during treatment with THDCPD.


After four weeks of treatment, high kidney, prostate, seminal vesicles and coagulating gland weights were seen in males receiving 15000 ppm, and low uterus, cervix and ovary weights in females receiving 15000 ppm. Following two weeks of recovery all organ weights were comparable with the control group, showing complete recovery.


It was concluded that dietary administration to Sprague Dawley rats up to 15000 ppm resulted in low bodyweight and food consumption in females at 5000 and 15000 ppm, with an increased incidence and severity of hyaline droplet accumulation and basophilic tubules in the kidney. As these changes, and all of those described in the results above, were minimal and the animals had shown some evidence of recovery, they were considered not adverse.


 


Therefore, under the test conditions, in the absence of any adverse findings the No‑Observed‑Adverse‑Effect‑level (NOAEL) was considered to be 15000 ppm (equivalent to 1205 mg/kg/day in males and 1165 mg/kg/day in females).,


 


This sub-acute toxicity study is acceptable and satisfies the requirement for repeated dose toxicity endpoint.