3,7-dimethyloct-6-enenitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethyloct-6-enenitrile

EC Number:

257-288-8

EC Name:

3,7-dimethyloct-6-enenitrile

Cas Number:

51566-62-2

Molecular formula:

C10H17N

IUPAC Name:

3,7-dimethyloct-6-enenitrile

Details on test material:

- Name of test material: Citronellyl nitrile
- Batch number : R7467
- Description : Clear colourless liquid
- Purity : 99.8% (sum of isomers)
- Storage conditions : room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Approximately five to eight weeks
- Weight at study initiation: males: 147 to 183 g, females: 131 to 162 g
- Housing: In groups of three or four by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK) and cardboard fun tunniels (Datesand Ltd., Cheshire, UK).
- Diet: Pelleted diet (Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: eight days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Weekly and stored at 4°C in the dark.

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The concentration of Citronellyl Nitrile CAS # 51566-62-2 in the test material formulations was determined by gas chromatography (GC) using an external standard technique. The test material formulations were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/mL. The standard and sample solutions were analysed by GC using the following conditions: GC system Agilent Technologies 5890, incorporating autosampler and workstation, column: DB-5 (30 m X 0.25 mm id X 0.25 µm film), oven temperature program: initial 100 °C for 0 min, rate 10°C/min; temp 150°C for 0 min, rate 70°C/min; final 325°C for 3 mins, injection temperature: 250°C, Flame ionisation detector temperature: 300°C, injection volume: 1µL, retention time ~4.2 mins. The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
- Stability determinations: The test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days.
- Verification of test material formulation concentrations: The test material formulations were sampled and analysed within three days of preparation.
- Results show the formulations to be stable for at least fourteen days. The results indicate that the prepared formulations were within acceptable limits of the nominal concentration.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 30, 100, and 300 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on previous toxicity data.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before and after dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.
- Behavioural assessments: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, Skin colour, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation.

DETAILED CLINICAL OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before and after dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

BODY WEIGHT: Yes
- Individual bodyweights were recorded on day 1 and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during week 12). Examinations included observation of the anterior structures of the eye, pupillary and comeal blink reflex. Following pupil dilation with 0.5% "Tropicamide" solution (Alcon Laboratories (UK) Ltd., Imperial Way, Watford, Hertfordshire), detailed examination of the intemal structure of the eye using a direct ophthalmoscope was performed.

LABORATORY INVESTIGATIONS: Yes
- Haematological, blood chemical and urinalysis investigations were perfomed on all animals from each test and control group during week 7 and at the end of the study (day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on day 91. Animals were not fasted prior to sampling.

HAEMATOLOGY: Yes
- The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices: (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), total leucocyte count, differential leucocyte count: (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, prothrombin time.

CLINICAL CHEMISTRY: Yes
- The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: urea, inorganic phosphorus, glucose, aspartate aminotransferase, total protein, alanine aminotransferase, albumin, alkaline phosphatase, albumin/globulin (AG) ratio (by calculation), creatinine, sodium, total cholesterol, potassium, total bilirubin, chloride, calcium.

URINALYSIS: Yes
- The following parameters were measured on collected urine: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional performance tests: Motor activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The time in seconds each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period). Forelimb/hindlimb grip strength: an automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
- Sensory Reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli: grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex, blink reflex.

OESTROUS CYCLES: Yes
During week 6 and 7 and week 12 and 13 a vaginal smear was taken daily and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of oestrous was recorded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full extemal and intemal examination, and any macroscopic abnormalities were recorded.
- Organ Weights: The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus.

HISTOPATHOLOGY: Yes
- Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, gross lesions, heart, ileum (including Peyer's patches), jejunum, kidneys, liver, lungs (with bronchi, inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), mammary glands, muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, sciatic nerve, salivary glands (submaxillary), seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus.
- Samples of the following tissues were removed from all animals and preserved in Bouins fluid and then in 70% IMS after forty-eight hours: right epididymis, right testis.
- Eyes were fixed in Davidson's fluid.
- All tissues were despatched to Propath UK Ltd for processing (Principal Investigator: N Carless). All tissues from control and 300 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesion were also processed. Since there were indications of treatment-related bone marrow and liver changes, examination was subsequently extended to include similarly prepared sections from all animals in the other treatment groups. Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
- Semen assessment: At necropsy the following evaluations were performed on all males: The left testis and epididymis were removed, dissected from connective tissue and weighed, for the testis, the tunica albuginea was removed and numbers of homogenisation resistant spermatids was analysed, luminal fluid of the epidymis was assessed for sperm motility and sperm morphology, morphometric analysis of semen was performed, the cauda epididymis was separated from the body of the epididymis, and analysed for homogenisation resistant spermatids.

Statistics:

- Data were processed to give group mean values and standard deviations where appropriate.
- All data were sumrnarised in tabular form. Where appropriate, quantitative 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 homogeneiiy of means assessed using ANOVA or ANCOVA and Bartlett'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).
- Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes: Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater. Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY: There were no unscheduled deaths. No clinically observable signs of toxicity were detected in test or control animals throughout the study period. Incidental findings of increased salivation were evident in 300 mg/kg/day animals throughout the treatment period. Incidents of increased salivation were also evident in 100, 30 and 10 (males only) mg/kg/day animals. Observations of this nature are often reported following oral administration of an unpalatable or slightly initant test material formulation and considered not to be an indication of systemic toxicity.

BEHAVIOURAL ASSESSMENT: There were no toxicologically significant changes in the behavioural parameters measured. All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

BODY WEIGHT AND WEIGHT GAIN: No test substance related effect on bodyweight/bodyweight gain of toxicological importance was detected.

FOOD CONSUMPTION/FOOD EFFICIENCY: There was no adverse effect on food consumption during the study period. Food efficiency (the ratio of bodyweight gain to dietary intake) was similar to that of controls.

WATER CONSUMPTION AND COMPOUND INTAKE: Daily visual inspection of water bottles revealed no intergroup differences.

OPHTHALMOSCOPIC EXAMINATION: There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of this age and strain.

HAEMATOLOGY: There were no toxicologically significant changes in the haematological parameters measured.

CLINICAL CHEMISTRY: During the week 7 assessments, a statistically significant increase in total protein was evident for females treated with 300 mg/kg/day. Increases in total protein were still evident during week 13 assessments for females treated with 300 mg/kg/day. Males from this treatment group also show a statistically significant increase in albumin levels. No such effects were detected in animals of either sex treated with 100, 30 or 10 mg/kg/day.

URINALYSIS: No treatment-related effects were detected in the urinalytical parameters measured.

NEUROBEHAVIOUR: There were no toxicologically significant changes in the functional perfomance parameters measured. There were no treatment-related changes in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used, and was of no toxicological importance.

OESTROUS CYCLE ASSESSMENTS: There were no treatment-related effects on female oestrous cycles or on the type or proportion of females with anomalous oestrous cycles.

ORGAN WEIGHTS: Animals of either sex treated with 300 mg/kg/day showed a statistically significant increase in liver weight both absolute (128% males and 114% females vs control) and relative (122% males and 115% females vs control) to terminal bodyweight. A statistically significant effect on liver weight also extended to males treated with 100 mg/kg/day (109% absolute and 108% relative vs control). No significant effect was detected in females treated with 100 mg/kg/day or in animals of either sex treated with 30 or 10 mg/kg/day. No other toxicological significant changes were observed.

SPERM ANALYSIS: There were no treatment-related effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts.

GROSS PATHOLOGY: No toxicologically significant macroscopic abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
- LIVER: Marginal centrilobular hepatocyte enlargement was observed among animals of either sex treated with 300 mg/kg/day (p <0.05). Two instances of centrilobular hepatocyte enlargement were seen among males and one female treated with 100 mg/kg/day. It is possible that the effect of treatment extended to animals at this dose level, although hepatocyte enlargement is occasionally observed as a spontaneous change among untreated control rats. 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.
- BONE MARROW: A higher incidence of lower grades of severity of adipose infiltration of the marrow, indicative of increased marrow cellularity, was seen for females only treated with 300 mg/kg/day but not at any other treatment level. This was considered to be a marginal effect that did not attain statistical significance.
- All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were observed in the 30 and 10 mg/kg/day dose groups.

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified