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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 March 2012 - 11 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline No 422 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
other: audited draft report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name of the test item (as cited in the study report): NOPYL ACETATE
IUPAC Name of the test item: (1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate
Synonyms: (-) Nopyl Acetate; 6,6-dimethyl -(1R,5S)-bicyclo[3.1.1]hept-2-ene-2-ethanol-2-acetate
Substance type: monoconstituent
Batch No.: 119307
Purity: 99.2%
This composition is within the specifications of the substance identity profile agreed within the SIEF.
Colour: colourless – slightly amber
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 13 June 2013

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: Charles River (UK) Limited, Margate, Kent, UK
Age at study initiation: approximately nine weeks
Weight at study initiation: males (390-447 g); females (233-294 g)
Housing: all animals during the pre-mating phase, all mated males, recovery group females and toxicity phase females were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids. During the mating phase, one male and one female within each dose group were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Diet (e.g. ad libitum): ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK), ad libitum
Water (e.g. ad libitum): mains drinking water, ad libitum
Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
Temperature: 21 ± 2 °C
Humidity: 55 ± 15 %
Air changes: at least 15 air changes/h
Photoperiod: 12 h dark / 12 h fluorescent light

IN-LIFE DATES:
from 09 March 2012 to 11 May 2012

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
Rate of preparation of diet (frequency): dietary admixtures were prepared prior to the first treatment, and twice monthly thereafter.

Mixing appropriate amounts with (basal laboratory diet - Rodent PMI 5002): a known amount of test item was initially mixed with 2 % corn oil and subsequently a small amount of basal laboratory diet was incorporated until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1, in a Hobart H800 mixer.

Storage temperature of food: diet was stored in labelled, double plastic bags in labelled, covered plastic bins at room temperature.

Stability: dietary admixtures were found to be stable for three weeks at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method: the concentration of test item in the dietary admixtures was determined by gas chromatography (GC) using an external standard technique.

Conditions:
GC system: Agilent Technologies 5890, incorporating autosampler and workstation;
Column: DB-5 (30 m x 0.53 mm id x 5 µm film)

Sampling: a representative sample of the dietary admixtures was accurately weighed and extracted in a suitable volume of acetonitrile. The extract was diluted using acetonitrile to give a final, theoretical test item concentration of approximately 50 mg/kg.

Results: the mean concentrations achieved for each dose mix were within specification at a range of 96-99 %.
Duration of treatment / exposure:
Main phase: males were dosed daily during premating and mating periods and up to 42 days; females were dosed up to 63 consecutive days (including a 3 week maturation phase, pairing, gestation and early lactation).

Toxicity phase: 42 days

Recovery phase: treated for 42 days and then maintained without treatment for a further 14 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1000, 3000 and 9000 ppm (equivalent to a mean achieved dosage of 0, 56.5, 180.2 and 478.5 mg/kg bw/day respectively)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 56.5, 180.2 and 478.5 mg/kg bw/day
Basis:
other: equivalent to mean achieved dosages
No. of animals per sex per dose:
Main phase: 10 males and 10 females/dose (except for males from control and top dose groups: 5 males/dose)
Toxicity phase: 5 females/dose
Recovery phase: 5 rats/sex/dose at 0 and 9000 ppm
Control animals:
other: basal laboratory diet with 2 % corn oil added
Details on study design:
Dose selection rationale: dose levels were chosen based on the results of previous toxicity work (Project Number 41103357).

Rationale for animal assignment: animals were allocated to dose groups using a randomisation procedure based on stratified body weights.

Post-exposure recovery period in satellite groups: 14 days
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
Time schedule: once daily
Cage side observations: overt signs of toxicity, ill-health and behavioural change

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and for main phase males, toxicity phase females and recovery animals once weekly thereafter. Observations were also performed on main phase females weekly during the pre-mating phase and then on days 0, 6, 13 and 20 post coitum and on days 1 and 7 of lactation. Functional performance tests were also performed in the first five main phase males per dose group and in toxicity phase females once during the final week of treatment.
- Parameters of behavioural assessments: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation
- Parameters of functional performance tests: motor activity and forelimb/hindlimb grip strength

NEUROBEHAVIOURAL EXAMINATION: yes
Time schedule: once during the final week of treatment extensive functional observations were performed on five selected main phase males from each dose group and for all (five) toxicity females.
Parameters:
Sensory reactivity: grasp response, touch escape, vocalisation, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex and finger approach

BODY WEIGHT: yes
Time schedule for examinations: individual body weights were recorded on day 1 and then weekly for main phase males and toxicity phase females until termination. For main phase females, individual body weights were recorded on day 1 and then weekly until pairing. Mated females were weighed on days 0, 6, 13 and 20 post coitum and on days 1, 4 and 7 post-partum. Recovery animals were weighed on day 1 and then weekly until termination.

FOOD CONSUMPTION: yes
Time schedule for examinations: during the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. Weekly food consumption values were calculated for each cage of toxicity phase females and recovery group females throughout the study period. Weekly food consumption values were calculated for recovery group males during the pre-pairing period, after the mating phase and during the recovery period.

FOOD EFFICIENCY: yes
Time schedule for examinations: food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for main phase and recovery males prior to and after pairing, for toxicity and recovery phase females during the recovery period where applicable, and for main phase females prior to pairing.

WATER CONSUMPTION: yes
Time schedule for examinations: daily from day 11 onwards (with the exception of the pairing phase)

HAEMATOLOGY AND CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: main phase males and toxicity phase females (5/dose) on day 42; recovery phase (all animals) on day 56
- Blood samples were obtained from the lateral tail vein. When necessary repeat samples were taken by cardiac puncture at termination
- Animals fasted: no
- Haematology parameters: haemoglobin, erythrocyte count (RBC), haematocrit, erythrocyte indices [mean corpuscular haemoglobin (MCH), mean corpuscular volume (mcv), mean corpuscular haemoglobin concentration (MCHC)], total leucocyte count (WBC), differential leucocyte count [neutrophils, lymphocytes, monocytes, eosinophils, basophils], platelet count, reticulocyte count, prothrombin time and activated partial thromboplastin time
- Blood Chemistry parameters: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma glutamyl transpeptidase, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine, total cholesterol, total bilirubin and bile acids
Sacrifice and pathology:
SACRIFICE
Adult main phase males and toxicity phase females were killed by intravenous overdose of a barbiturate agent followed by exsanguination on day 43.
Adult main phase females and recovery group animals were killed in a similar manner on day 7 post-partum and on day 57, respectively.

GROSS NECROPSY
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs, removed from main phase males, toxicity phase females and recovery phase animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides (left and right), heart, kidneys (left and right), liver, lungs, ovaries (left and right), pituitary, prostate, seminal vesicles, spleen, testes (left and right), thymus, thyroid (weighed post-fixation with parathyroid) and uterus (weighed with cervix and oviducts).

The following organs, removed from main phase females that were killed at the end of the study, were dissected free from fat and weighed before fixation: liver, ovaries (left and right) and uterus (weighted with cervix and oviducts).

Samples of the tissues listed in table 7.5.1/1 were removed from main phase males, toxicity phase females and recovery animals and preserved in buffered 10 % formalin, except where stated.
Statistics:
The following parameters were subjected to statistical anlysis: quantitative functional performance data, body weight and body weight change, food consumption and water consumption, haematology, blood chemistry, absolute and body weight-relative organ weights.

Data for males and females prior to pairing and functional performance test data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No unscheduled deaths or treatment-related clinical signs were noted. One main phase female treated with 9000 ppm had fur loss between days 29 and 51. One control main phase female also had fur loss between days 11 and 24. One main phase male treated with 3000 ppm had a scab formation from day 36. Observations of this nature are commonly observed during the gestation period or in group housed animals and were considered to be of no toxicological significance.
- No treatment-related effects were noted on behavioural, sensory reactivity and functional performance parameters.

BODY WEIGHT (PARENTAL ANIMALS)
- Reduced overall body weight gain was evident in animals of either sex treated with 9000 ppm (-14 % males, -48 % females). Statistically significant reductions in body weight gain were achieved for males during Week 1 and in females during weeks 2 and 3.
- Non-statistically significant reduction in body weight gain for main phase females treated with 9000 ppm was also evident during gestation and the first four days of lactation.
- No such effects were detected in animals of either sex treated with 3000 or 1000 ppm.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- At 9000 ppm, mean food consumption for females was lower than control during the first week of the study (-23 %) and was considered to reflect an initial reluctance to eat the diet admixture due to its low palatability.
- No such effects were detected in animals of either sex treated with 3000 or 1000 ppm.

FOOD EFFICIENCY (PARENTAL ANIMALS)
- Food efficiency was intermittently adversely affected in animals of either sex treated with 9000 ppm and was considered to be the result of reduced body weight gain in these animals.
- No such effects were detected in animals of either sex treated with 3000 or 1000 ppm.

WATER CONSUMPTION (PARENTAL ANIMALS)
- Males treated with 9000 and 3000 ppm showed an increase in water consumption throughout the treatment period (+14 %, +10 % respectively).
- Females treated with 9000 ppm showed a slight increase in water consumption throughout the treatment period (+8 %).
- Main phase females treated with 9000 and 3000 ppm also showed an increase in water consumption during gestation. Statistical significance was achieved throughout gestation.
- A slight increase (+20 %) in water consumption remained evident in recovery phase males during the treatment free period.
- Females treated with 1000 ppm also showed an increase in water consumption during the final week of gestation.
- No such effects were detected in males treated with 1000 ppm or recovery phase females.
- Increase in water consumption would be considered not to represent an adverse effect of treatment therefore the intergroup differences were considered not to be of toxicological importance.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Mean achieved dosages for animals of either sex at 1000, 3000 and 9000 ppm were 56.2, 180.2 and 478.5 mg/kg bw/day, respectively.
- Mean weekly achieved dosage was fairly consistent at all dose levels.
- Mean achieved dosage during gestation and lactation was increased when compared to the maturation phase for main phase females. This increase was to be expected due to the increased physical demand of pregnancy and nursing of offspring.

HAEMATOLOGY (PARENTAL ANIMALS)
- No treatment-related significant changes were detected.
- Main phase males treated with 9000 ppm and recovery phase males showed statistically significant reductions in haemoglobin and haematocrit. Main phase males treated with 9000 ppm also showed a statistically significant reduction in activated partial thromboplastin time. Main phase males from all treatment groups showed a statistically significant reduction in total leucocyte count. Main phase males treated with 3000 ppm also showed a reduction in lymphocyte count. The majority of individual values were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were considered to be of no toxicological importance.

BLOOD CHEMISTRY (PARENTAL ANIMALS)
- Blood chemical investigations revealed increases in urea, glucose, gamma glutamyl transpeptidase and creatinine in main phase males treated with 9000 ppm. Although some of the individual values were outside of the normal ranges for rats of the strain and age used, these findings were most likely to be associated with the adaptive metabolic changes.
- Main phase males treated with 9000 ppm showed a statistically significant increase in chloride concentration and a statistically significant reduction in calcium concentration. Toxicity phase females treated with 9000 ppm showed a statistically significant increase in inorganic phosphorous. Toxicity phase females from all treatment groups showed a statistically significant increase in albumin/globulin ratio. Toxicity phase females treated with 3000 ppm also showed a statistically significant increase in chloride concentration. All individual values were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were considered to be of no toxicological significance.
- Following fourteen days without treatment the recovery group 9000 ppm males showed a statistically significant reduction in alanine aminotransferase. Recovery group 9000 ppm females also showed a statistically significant increase in potassium concentration and a statistically significant reduction in bilirubin. The majority of individual values were within the normal ranges for rats of the strain and age used, and in the absence of similar findings detected in main phase males or toxicity phase females at the end of the treatment period the intergroup differences were considered to be of no toxicological significance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Main phase and recovery phase males treated with 9000 ppm showed an increase in kidney and liver weight both absolute and relative to terminal body weight.
- Toxicity and main phase females treated with 9000 ppm also showed an increase in liver weight both absolute and relative to terminal body weight.
- No such effects were detected in main phase males, toxicity phase females or main phase females treated with 3000 or 1000 ppm or in recovery 9000 ppm females following the treatment free period.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- There was no adult macroscopic finding considered to be related to test item toxicity.
- Post mortem examinations did not reveal any treatment-related macroscopic findings for interim death or terminal kill offspring.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The following treatment related microscopic abnormalities were detected:
- Liver: minimal to slight diffuse hepatocellular hypertrophy was evident in all main phase males and main phase females treated with 9000 ppm. In these females, associated glycogen depletion was also evident. In toxicity phase females treated with 9000 ppm, minimal to slight centrilobular to diffuse hepatocellular hypertrophy was evident. The results of the recovery groups showed that all the liver findings were fully reversible. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature and does not represent an adverse health effect.
- Thyroid: minimal diffuse follicular cell hypertrophy was evident in a few toxicity phase females treated with 9000 ppm. This finding was fully reversible and generally regarded as adaptive in nature and of limited human relevance.
- Kidneys: minimal focal bilateral tubular degeneration/regeneration in the renal cortical tubules was evident in one main phase male treated with 3000 ppm. Minimal to marked multifocal bilateral tubular degeneration/regeneration in the renal cortical tubules was also evident in the majority of main phase males treated with 9000 ppm and in one of these males, associated slight multifocal interstitial fibrosis was also evident. In all main phase males treated with 9000 ppm, minimal to marked hyaline droplets were present in the proximal convoluted tubules (severity: 2.4) versus minimal to slight hyaline droplets in two controls (severity: 1.0) and three low dose males (severity: 1.3). At 3000 ppm, hyaline droplets occurred in four main phase males at a mean severity of 1.8. Minimal to slight tubular dilation and moderate tubular granular casts (inner cortex) were associated with the tubular degeneration/regeneration in three main phase males treated with 9000 ppm. These kidney findings were partially reversible.
The kidney effects detected in males from all treatment groups were considered to represent an effect of the test item. However, the tubular degeneration/regeneration, hyaline droplets and granular casts diagnosed in main phase and recovery males are compatible with alpha 2u-globulin nephropathy, which are well documented changes that are peculiar to the male rat in response to treatment with some xenobiotics. These kidney effects are not found in immature rats, females rats or humans and therefore are considered to be of no relevance to man. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for males can be established based on the observed adverse effects excluding those related to alpha 2u-globulin nephropathy.

Effect levels

Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
180.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) of nopyl acetate for systemic toxicity was considered to be 3000 ppm (180.2 mg/kg bw/day) for females and for males (when excluding the sex and species, specific kidney effects in male rats are not relevant for human risk assessment).
Therefore nopyl acetate is not classified for repeated dose toxicity according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.
Executive summary:

In a combined repeated dose toxicity study with a reproduction / developmental toxicity screening test conducted according to OECD Guideline No 422 and in compliance with GLP, nopyl acetate was administered by dietary admixture (initially mixed with 2% corn oil to avoid evaporation) to three groups of Sprague-Dawley Crl: CD® BR strain rats, for up to 63 consecutive days (including a three week maturation phase, pairing, gestation and early lactation for females) at dietary concentrations of 0, 1000, 3000 and 9000 ppm (equivalent to a mean achieved dosage of 0, 56.5, 180.2 and 478.5

mg/kg bw/day, respectively). Each dose group was subdivided into two phases: main phase (at 1000 and 3000 ppm: 10 rats/sex/dose; at 0 and 9000 ppm: 5 males and 10 females/dose) and toxicity phase (5 female/dose). A control group was treated with basal laboratory diet (with 2% corn oil). Two recovery groups (5 rats/sex/dose) were treated with 9000 ppm or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food and water consumption, haematology, blood chemistry. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed.

No unscheduled deaths or treatment-related clinical signs were noted. No treatment-related effects were noted on behavioural, sensory reactivity and functional performance parameters. Reduced overall body weight gain was evident in animals of either sex treated with 9000 ppm (-14% in males, -48% in females). Statistically significant reductions in body weight gain were achieved for males during week 1 and in females during weeks 2 and 3. At 9000 ppm, mean food consumption for females was lower than control during the first week of the study (-23%) and was considered to reflect an initial reluctance to eat the diet admixture due to its low palatability. Food efficiency was intermittently adversely affected in animals of either sex treated with 9000 ppm. Increased water consumption was observed in several animals but it would not be considered as an adverse effect to treatment. No treatment-related significant changes were detected after haematology and blood chemistry investigations. Main phase and recovery phase males treated with 9000 ppm showed an increase in kidney and liver weight both absolute and relative to terminal body weight. Toxicity and main phase females treated with 9000 ppm also showed an increase in liver weight both absolute and relative to terminal body weight. Histopathology revealed fully reversible microscopic abnormalities in liver (minimal to slight diffuse hepatocellular hypertrophy in males and females) and thyroid (minimal diffuse follicular cell hypertrophy in females) at 9000 ppm. At 3000 and 9000 ppm, partly reversible changes in kidney (tubular degeneration/regeneration, hyaline droplets and granular casts) were observed in main phase and recovery males. These kidney effects were considered to be related to alpha 2u-globulin nephropathy and of no relevance to humans.

Under the conditions of the test, the No Observed Adverse Effect Level (NOAEL) of nopyl acetate for systemic toxicity was considered to be 3000 ppm (180.2 mg/kg bw/day) for females and for males (when excluding the sex and species, specific kidney effects in male rats are not relevant for human risk assessment). Therefore, nopyl acetate is not classified for repeated dose toxicity according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.