(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-08-2001 to 12-02-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Reference: Commission Directive 96/54/EC

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: US EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, (2000).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000 ; signature: April 2000

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-120-6

EC Name:

-

Cas Number:

166432-53-7

Molecular formula:

C14H22O

IUPAC Name:

2-methyl-4-(2,2,3-trimethylcyclopent-3-en-1-yl)pent-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: room temperature in the dark, under nitrogen
- Other: clear, colourless liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD) IGS BR

Details on species / strain selection:

The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 5 to 7 weeks
- Weight at study initiation: males 168 – 225 g and females 158 – 192 g; individuals were randomly allocated to treatment groups using a randomization procedure.
- Fasting period before study: None
- Housing: polypropylene grid-floor cages with wooden chew blocks for environment enrichment ; group housed (5 per group) by sex. During locomotor investigations were housed individually.
- Diet (e.g. ad libitum): ground rodent diet number 1, certified (recognised supplier), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: ground rodent diet, certified (recognised supplier) – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. A known amount of test item was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a QE200 mixer. 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 H800 mixer. Dietary admixtures were prepared prior to treatment. The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use. The test item was incorporated into the diet at concentrations of 225, 2250 and 15000 ppm

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ±15 %
- Air changes (per hr): > 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 19-03-2001 to 17-04-2001

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. A known amount of test item was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a QE200 mixer. 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 H800 mixer. Dietary admixtures were prepared prior to treatment. The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use. The test item was incorporated into the diet at concentrations of 225, 2250 and 15000 ppm

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Monthly (dietary admixtures were determined to be stable for a period of at least six weeks).
- Mixing appropriate amounts with (Type of food): Basal diet (number 1 Certified) with test item.
- Storage temperature of food: Room temperature, diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use.
- Stability under test conditions: Stable. Dietary admixtures were determined to be stable for a period of at least six weeks. The stability and homogeneity of the test material formulations were determined. Results indicated that the mean prepared admixture concentrations were within ± 10% of the nominal concentration.
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. See above and note that the test item was demonstrated to be stable and homogenous when formulated in diet.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The formulated diet analysis consisted of GC FID analysis with external calibration. The method was calibrated by using calibration standards of the test item response between nominal concentrations of 225 ppm and 15000 ppm within a dedicated dietary formulation analysis report attached to the full study report. The dietary admixtures were extracted with methanol to give a final, theoretical test item concentration of approximately 0.01 mg/mL. Standard solutions of test item were prepared in methanol at a nominal concentration of 0.01 mg/mL. These were then subjected to analysis by GC FID. The analytical method was validated (details available within the full study report). With linearity = 0.997 between 0.005 mg/mL and 0.015 mg/mL. Accuracy and precision was confirmed and mean procedural recovery was 111% (n=2) at ca. 225 ppm and 100% at ca. 15000 ppm.
- The homogeneity and stability was confirmed in formulated diets of the test item in diet. Dietary admixtures were determined to be stable for a period of at least six weeks. The stability and homogeneity of the test material formulations were determined. Results indicated that the mean prepared admixture concentrations were within ± 10% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

225 ppm

Remarks:

Low – Group

Doses / concentrations 3

Dose / conc.:

2 250 ppm

Remarks:

Intermediate – Group

Doses / concentrations 4

Dose / conc.:

15 000 ppm

Remarks:

High – Group

Doses / concentrations 5

Dose / conc.:

23 mg/kg bw/day (actual dose received)

Remarks:

Low – Group ; mean achieved dose

Doses / concentrations 6

Dose / conc.:

215 mg/kg bw/day (actual dose received)

Remarks:

Intermediate – Group ; mean achieved dose

Doses / concentrations 7

Dose / conc.:

1 212 mg/kg bw/day (actual dose received)

Remarks:

High – Group ; mean achieved dose

No. of animals per sex per dose:

5 per sex per dose (5 male / 5 female)

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 225, 2250 and 15000 ppm) were chosen based upon the results obtained in a 14 day preliminary study (available in the full study report) which utilised dose levels as follows: 0 (control) mg/kg (0 ppm) and estimated 1000 mg/kg (15000 ppm).
- Rationale for animal assignment (if not random): Randomly assigned.
- Rationale for selecting satellite groups: Not applicable.
- Post-exposure recovery period in satellite groups: Not applicable.
- Section schedule rationale (if not random): Random

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily. Prior to the start of treatment and on Days 6, 14, 21 and 27. Detailed functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes.
- Body weight gain % was determined.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No.
- How many animals: All animals
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium, high fluorescence), Leukocyte count, total, Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count, Methemoglobin Heinz bodies, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals
- Animals fasted: No.
- How many animals: All animals
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin, total Cholesterol, Alanine aminotransferase, Alkaline phosphatase, Creatine, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein, total Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- organs weighed: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Kidneys, Liver, Ovaries

HISTOPATHOLOGY: Yes
- Organs and tissues examined and/or preserved in neutral buffered 10% formalin (where appropriate): Adrenals, Aorta (thoracic), Oesophagus, Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Colon, Salivary glands (submaxillary), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, Eyes, Skin (hind limb), Gross lesions, Spinal cord (cervical), Heart, Spleen, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, Liver, Thyroid/parathyroid, Lungs (with bronchi), Trachea, Lymph nodes (cervical and mesenteric), Urinary bladder, Muscle (skeletal), Uterus.
- Other: Additional investigations of liver, kidney and thyroid were made in all individuals in all treatment groups.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
The following statistical methods were used to analyse: Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight):
• dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance
• where homogenous variances observed: homogenous pairwise comparisons were conducted using Dunnett's test
• Where the Levene’s test showed unequal variances: non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm and 2250 ppm: no (adverse) clinical signs of toxicity were noted during the observation period. There were no treatment-related changes in behavioural assessments, functional performance or sensory reactivity.

At 15000 ppm: No clinical signs of toxicity were detected until the final two days of treatment when diuresis together with associated findings of staining of the external body fur and fur wetting were observed in males/females. These findings were accompanied in two females on Day 28 by hunched posture.

Within behavioural assessments: Hunched posture and tiptoe gait were detected in one female treated with 15000 ppm during Week 4 assessment. No other abnormalities were detected.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm and 2250 ppm: No adverse effect on bodyweight was detected (any changes may have had statistical significance but were discounted based on minimal intergroup differences.

At 15000 ppm: A reduction in bodyweight gain was detected for males/females during Week 1 of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm and 2250 ppm: No adverse effect on dietary intake or food efficiency was detected.

At 15000 ppm: A reduction in dietary intake was detected for males/females. Food efficiency was also reduced during Week 1.

Applicant assessment indicates that these observations at 15000 ppm may be speculated to be palatability of diet related unless correlated with other systemic toxicity observations.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See body weight and weight changes and food consumption sections.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in the haematological parameters measured.

At 15000 ppm: males dosed with 15000 ppm showed a statistically significant reduction in haemoglobin compared with controls. The level of significance achieved was minimal (p<0.05), all of the values were within the normally expected ranges for the rat strain and age used. In the absence of any histopathological correlates or any other evidence of anaemia, this reduction was considered to have arisen fortuitously.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm and 2250 ppm: No adverse effect on blood chemistry was detected. Males treated showed statistically significant reductions in alanine aminotransferase levels (p<0.05) when compared with controls. Individual values were entirely within the normal range for rat strain and age.

At 15000 ppm: Plasma bilirubin was elevated for males/females compared with controls achieving statistical significance (p<0.001) for females only. In addition, males at this dose level showed elevated plasma alkaline phosphatase levels. Males treated showed statistically significant reductions in alanine aminotransferase levels (p<0.01) when compared with controls. Individual values were entirely within the normal range for rat strain and age.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm and 2250 ppm: There were no treatment-related changes in behavioural assessments, functional performance or sensory reactivity.

At 15000 ppm: Within behavioural assessments: Hunched posture and tiptoe gait were detected in one female treated with 15000 ppm during Week 4 assessment. No other abnormalities were detected. There were no treatment-related changes in functional performance or sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm and 2250 ppm: No treatment related changes were detected.

At 15000 ppm: Statistically significant increases in liver (p<0.001) and kidney weight (males, p<0.01, females, p<0.05), relative to terminal bodyweight, were detected for males/females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm and 2250 ppm: No treatment related changes were detected.

At 15000 ppm: males treated showed pale kidneys

Incidental findings were considered to be of no toxicological significance, due to no dose-responses and being consistent with findings commonly seen post-mortem.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no significant treatment-related changes in behavioral assessments, functional performance or sensory reactivity.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 225 ppm: No findings were considered treatment related.

At 2250 ppm: See below, similar findings of different severity were observed.

At 15000 ppm:
- Liver: Treatment-related centrilobular hepatocyte enlargement was observed for males/females. 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.
- Kidneys: Globular accumulations of eosinophilic material were observed in the tubular epithelium of males. This finding is consistent with the appearance of hydrocarbon nephropathy, which results from the excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells. α2-Microglobulin is found only in the proximal tubular epithelium of adult male rats. Applicant assessment indicates that this finding has no relevance to human health since a2-Microglobulin is found only in the proximal tubular epithelium of adult male rats.
- Thyroids: An increased incidence and severity of treatment-related follicular cell hypertrophy was observed in relation to treatment for males.

It was considered the treatment-related effects observed at 2250 ppm or 15000 ppm were predominantly adaptive liver and thyroid changes and the male rat specific condition hydrocarbon nephropathy. Without relevance for human health.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities detected.

Incidental findings were considered to be of no toxicological significance, due to no dose-responses and being consistent with findings commonly seen post-mortem.

Other effects:

not examined

Effect levels

1

Dose descriptor:

NOEL

Remarks:

NOEL determined within the study report

Effect level:

>= 225 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: 215 mg/kg bw/day : actual dose reveived

2

Dose descriptor:

NOAEL

Remarks:

Applicant assessment of effects within the study report

Effect level:

>= 15 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: see "Remarks"

Remarks:

findings are suggestive that NOAEL = 15000 ppm (equivalent to 1212 mg/kg bw/day) on the basis that findings were adaptive liver and thyroid changes and/or kidney findings were male rat specific and no relevance to human health.

3

Dose descriptor:

NOAEL

Remarks:

Applicant assessment of effects within the study report

Effect level:

>= 1 212 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: mean achieved dose

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Applicant assessment of the study report indicates that: whilst the NOAEL was not identified per se within the study, the findings observed are suggestive that the NOAEL may be set at 15000 ppm (equivalent to 1212 mg/kg bw/day or > 1000 mg/kg bw/day) on the basis that the treatment related findings were adaptive liver and thyroid changes and/or kidney findings were male rat specific and no relevance to human health. This comment was included in the full study report. The additional indications on bodyweight and food consumption in week 1 may be palatability related effects to dietary administration.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed effect level (NOEL) was 225 ppm ((equivalent to a mean achieved dose of 23 mg/kg body weight per day). Applicant assessment indicates: the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 15000 ppm (equivalent to a mean achieved dose of 1212 mg/kg body weight per day or > 1000 mg/kg bw/day) on the basis that: the treatment related findings were adaptive liver and thyroid changes and/or kidney findings were male rat specific and no relevance to human health. This comment was included in the full study report. The additional indications on bodyweight and food consumption in week 1 may be palatability related effects to dietary administration.

Executive summary:

The study was performed according the requirements of OECD TG 407 and EU method B.7 guidelines under GLP conditions. Following a previously conducted 14-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day dietary study in Sprague-Dawley Crl:CD (SD) IGS BR strain rats. Three groups, each comprising five male and five female rats, received test item at dietary concentrations of 225, 2250 and 15000 ppm (equivalent to mean achieved dosage of 23, 215 and 1212 mg/kg/day). A control group of five males and five females was dosed with basal diet. Chemical analyses of the prepared diet was conducted during the study to assess accuracy, homogeneity and stability. Analyses confirmed that dietary admixtures were determined to be stable for a period of at least six weeks at room temperature. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated. Gross necropsy and histopathological evaluation were also performed. There was no mortality, functional performance and sensory reactivity abnormalities found during the study. No clinical signs of toxicity were detected until the final two days of the study whereupon hunched posture, diuresis and wet and stained fur were observed in males/females treated with 15000 ppm. In behavioural assessments: hunched posture and tiptoe gait were detected in one female treated with 15000 ppm during Week 4 assessment. No other abnormalities were detected. At 15000 ppm A reduction in bodyweight gain and dietary intake and food efficiency was detected for males/females during Week 1 of the study. There were no treatment-related changes in the haematological parameters measured. At 15000 ppm, plasma bilirubin was elevated for males/females compared with controls achieving statistical significance for females only. In addition, males at this dose level showed elevated plasma alkaline phosphatase levels. Males treated showed statistically significant reductions in alanine aminotransferase levels when compared with controls. Individual values were entirely within the normal range for rat strain and age. At 15000 ppm, statistically significant increases in liver and kidney weight, relative to terminal bodyweight, were detected for males/females along with males indicating pale kidneys during micropathological examination. In histopathology of the liver: Treatment-related centrilobular hepatocyte enlargement was observed for males/females. 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. In the kidneys: Globular accumulations of eosinophilic material were observed in the tubular epithelium of males. This finding is consistent with the appearance of hydrocarbon nephropathy, which results from the excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells. α2-Microglobulin is found only in the proximal tubular epithelium of adult male rats. Applicant assessment indicates that this finding has no relevance to human health since a2-Microglobulin is found only in the proximal tubular epithelium of adult male rats. In the thyroids: An increased incidence and severity of treatment-related follicular cell hypertrophy was observed in relation to treatment for males. Under the conditions of this study, the no-observed effect level (NOEL) was 225 ppm (equivalent to a mean achieved dose of 23 mg/kg body weight per day). Applicant assessment indicates: the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 15000 ppm (equivalent to a mean achieved dose of 1212 mg/kg body weight per day or > 1000 mg/kg bw/day) on the basis that: the treatment related findings were adaptive liver and thyroid changes and/or kidney findings were male rat specific without relevance to human health. The additional indications on bodyweight and food consumption in week 1 may be palatability related effects to dietary administration.