Octa-1,7-diene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-Oct-2015 to 07-Jan-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octa-1,7-diene

EC Number:

223-054-9

EC Name:

Octa-1,7-diene

Cas Number:

3710-30-3

Molecular formula:

C8H14

IUPAC Name:

octa-1,7-diene

Test material form:

liquid

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Strain: Rat / CD® / Crl:CD(SD)
- Number and sex: 80 animals (40 males and 40 females)
- Breeder: Charles River Laboratories
- Body weigth: Males 251 - 287.8 g, Females 196.1 - 228.5 g
- Age: 61 days at 1st administration

ENVIRONMENTAL CONDITIONS:
- Diet: a certified commercial diet (ssniff® R/M-H V1534) ad libitum
- Water: tab water ad libitum
- Housing singly in MAKROLON cages (type III plus)
- Temperature: 22°C ± 3°C
- Rel. Humidity: 55% ± 15%
- Light/dark period: 12/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was administered orally at a constant volume per kilogram body weight (2 mL) once daily for 90 days.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CLINICAL SIGNS:
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness, and additionally regularly throughout the working day. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

NEUROLOGICAL SCREENING:
At the end of the treatment period, screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad5), as well as the assessment of grip strength (Meyer6) and motor activity assessment was conducted for all animals.
In detail the test battery included testing of righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, piloerection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.

MORTALITY:
Checks on mortality were made early in the morning and again in the afternoon of each day to look for dead or moribund animals.

BODY WEIGTH:
The weight of each rat was recorded at group allocation (test day -7), on test day 1 (before first administration), and once a week thereafter always on the same day of the week throughout the experimental period (test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90).

FOOD CONSUMPTION:
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

DRINKING WATER CONSUMPTION:
The drinking water consumption was recorded weekly by weighing the water bottles when filled and the residues upon removal at the end of the test week. Any residue was discarded. Weekly mean values per animal were calculated.

LABORATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The blood samples were collected at the end of test week 13 (on test day 91, before necropsy) and were used for haematological investigations, coagulation tests, clinical chemistry tests, and for bile acid determination.

Haematological parameters:
Haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), reticulocytes (Reti), platelets (PLT), haematocrit value (HCT), absolute and relative differential blood count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

Coagulation parameters:
Thromboplastin time (TPT), activated partial thromboplastin time (aPTT).

Clinical chemistry parameters:
Albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol, creatinine, glucose, protein, triglycerides, urea (in blood), calcium, chloride, potassium, sodium, alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH)

Urinalysis:
Urine samples were collected from all animals fasted overnight on test day 91 (test week 13). Investigated parameters were volume, pH and specific gravity. The analytes of qualitative urinalyis were protein, glucose, bilirubin, urobilinogen, ketones, heamoglobin and nitrite. A microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, cassts), crystalluria.

OPHTHALMOLOGICAL AND AUDITORY EXAMINATIONS:
Examinations were performed pre-dose (test day 1, prior to first dosing) and in test week 13 (end of the treatment period, test day 90). The eyes were examined with a HEINE ophthalmoscope. Prior to examination, mydriasis was produced after instillation of STULLIN®10 eye drops into the conjunctival sacs.
The following ocular structures were examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
The auditory acuity was checked with a simple noise test.

Sacrifice and pathology:

SACRIFICE:
Starting on test day 91 (approx. 24 hours after the last administration), the animals were dissected following a randomisation scheme. Animals not dissected on test day 91, were dosed again on test day 91 and dissected on test day 92. The animals were euthanized by carbon dioxide (CO2), exsanguinated by cutting the aorta abdominalis, weighed, dissected, and inspected macroscopically.The weights of the following organs of all animals were determined before fixation: adrenal gland, kidney, spleen, uterus (incl. cervix), brain, liver, testicle, prostate and seminal vesicles (with coagulating glands as a whole), epididymis, ovary, thymus, heart, pancreas. The paired organs (adrenal glands, gonads and kidneys) were identified as left or
right and weighed individually.
The organs or parts of organs of all animals were fixed in 7% buffered formalin. Eyes were preserved in Davidson's solution and testes in Bouin's solution for optimum fixation.

HISTOPATHOLOGY:
The organs listed below of all animals of groups 1 and 4 (control and high dose) were examined histologically after preparation of paraffin sections and haematoxylin-eosin (H & E) staining:
Adrenal gland (2)
Aorta abdominalis
Bone (os femoris with joint)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Epididymis (2)
Eye with optic nerve (2)
Gross lesions observed
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and bronchioles, preserved by inflation with fixative and then immersion)
Lymph node (1, cervical)
Lymph node (1, mesenteric)
Mammary gland
Muscle (skeletal, leg)
Nerve (sciatic)
Oesophagus
Ovary and oviducts (2)
Pancreas
Pituitary
Prostate and seminal vesicles with coagulating glands
Salivary glands (mandibular, parotid, sublingual)
Skin (left flank)
Spinal cord (3 sections)
Spleen
Stomach
Testicle (1)
Thymus
Thyroid (2) (incl. parathyroids)
Tissue masses or tumours (including regional lymph nodes)
Trachea (incl. larynx)
Urinary bladder
Uterus (incl. cervix)
Vagina

In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined microscopically, and the stomach and the kidneys (2) of the animals of group 2 (low dose) and group 3 (intermediate dose) were examined microscopically following H & E) staining.
Furthermore, a detailed histomorphological examination was performed on one testicle and one epididymis of all male animals of groups 1 to 4 (control and all dose groups) following H & E and PAS staining (with special emphasis on the qualitative stages of the spermatogenesis and the histopathology of the interstitial testicular structure). Sperm count an viabilty was assessed and sperm morphology was examined for all male animals.

Statistics:

Data for toxicology and pathology were captured, as far as possible, using the departmental computerized systems (Provantis® Integrated preclinical software, version 9.4.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs. The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used for the data captured with the Provantis system: Multiple t-test based on DUNNETT, Exact Test of R.A. Fisher.
The following statistical methods were used for the data not captured with the Provantis system: STUDENT's t-test, chi²-test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant changes in comparison to the control animals were noted for forelimb and hindlimb grip strength in test week 13. However, all changes are considered to be coincidental effects; the slight change in comparison to the control group is within the normal range of biological variation and is considered to be without toxicological relevance and thus not to be related to the test item.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In all dose groups, the food consumption was statistically significantly (at p ≤ 0.05 or p ≤ 0.05) increased compared to the control group in various test weeks. However, all changes are considered coincidental as the slight differences to the control are in the normal range of biological variation.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

The drinking water consumption of the male animals treated with 1000 mg/kg/day was constantly and always statistically significantly (at p ≤ 0.01) increased by up to 39% compared to the control group in test weeks 1 to 13. This effect is considered to be test item-related.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Occassionally, statistically significant differences in haematological parameters compared to the control group were noted on test day 91 are not considered to be test item-related, because chagnes were only slight and within the normal range of biological variation, and/or changes were not dependent on dose. Changes consits of increase in basophilic granulocytes count (Baso) in males of the low-dose group, decrease in aPTT as well as increase on MCHC in males of the high-dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The plasma levels of total cholesterol and of triglycerides were increased in the male and female animals treated with 1000 mg/kg/day compared to the control group. These changes are considered to be test item-related.

Occassionally, statistically significant differences in clinical biochemistry parameters compared to the control group were noted on test day 91 are not considered to be test item-related, because chagnes were only slight and within the normal range of biological variation, and/or changes were not dependent on dose. Changes consits of increase in ASAT in females of the low-dose group, and decrease in glucose, blood urea, chloride and sodium levels in males or females of the high-dose group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The relative urine volume of the male animals appeared to be increased by 82% at the mid-dose group and by 92% at the high-dose group compared to the control group on test day 91 (statistically significant at p ≤ 0.01). However, these apparent increases are due to the very low value of only 10.52 mL/kg
b.w./24 h observed for the control group and are considered not test item-related. Furthermore, the slight increase by 6% (statistically significant at p ≤ 0.05) noted for the pH value of the urine of the female high-dose animals compared to the control group is considered as coincidental and not test item-related.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the high dose of 1000 mg/kg b.w./day, statistically significantly (at p ≤ 0.01) increased relative and absolute kidney weights were noted for male and female animals, which can be correlated with the histopathological findings of chronic progressive nephropathy and tubule hypertrophy in the high-dose group. Further, the relative and absolute liver weights were slightly increased by up 26% at the high dose, the males (statistical significance at p ≤ 0.05 or p ≤ 0.01) being more affected than the females (no statistical significance).
The increased kidney and liver weights are considered to be test item-related.

Slightly increased relative and absolute kidney weights partly attaining statistical significance (at p ≤ 0.05 or p ≤ 0.01) that were noted in low- and mid-dose groups are not considered to be test item-related because of the following reasons:
- No corresponding histopathological findings in the kidney were noted for any animal of groups 2 and 3.
- At the low-dose group, the marginal alterations (without statistical significance) in comparison to the control group are within the normal range of biological variation. The statistically significant (at p ≤ 0.05) increase by 10.7% compared to the control noted for the relative weight of the right kidney of the female group 2 animals is considered to due to the slightly reduced average body weight of these animals in comparison to the control and not to be test item related.
- At the mid-dose group, the increases noted for the relative kidney weights (up to 18%, statistically significant at p ≤ 0.01) of the male animals compared to the control were much smaller than the respective absolute kidney weight changes. The changes are considered to be due to the higher average body weight of these animals in comparison to the control and not to be test item related. The differences in kidney weights (up to 16% compared to the control, mostly statistically significant at p ≤ 0.05) noted for the female animals of group 3 are considered to be due to 2 to 3 animals with relatively high kidney weights related to higher body weights compared to the control. Therefore, the effect in group 3 is also regarded as coincidental and not test item-related.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

For the high-dose animals, significant histopathological changes were observed for the kidney (chronic progressive nephropathy, tubule hypertrophy) and the stomach (non-glandular: hyperkeratosis, squamous cell hyperplasia), being slightly more severe in the male animals compared to the female animals. No corresponding findings were noted for the kidneys and the stomach of the animals of the low- and mid-dose groups. These kind of histopathological changes are either generally reversible (hyperplasia and hyperkeratosis) after discontinuation of dosing or are a male rat-specific spontaneous age-related disease that may be exacerbated by chemicals, but a rodent disease of no relevance for extrapolation in human risk assessment.

The coincidental findings in various organs in a small number of control and test item-treated animals are considered to be spontaneous organ changes and are thus not test item-related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No influence was noted on sperm count and sperm motility of male animals in all groups. The examination of the sperm morphology did not reveal any test item-related differences between the control group and the test item-treated groups in test week 13. These results are supported by the fact that the microscopic evaluation did not reveal any test item related effects on the male reproductive organs at the histomorphological level.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

The test substance produces adverse effects on the kidney in rats (chronic progressive nephropathy, tubule hypertrophy) after consecutive treatment with 1,7-Octadiene at a dose level of 1000 mg/kg b.w./day for 90 days. The NOAEL is at 300 mg/kg b.w./day.