Cyclohexadecen-1-one

Endpoint summary

Description of key information

28-d repeated dose toxicity, oral, rat, NOAEL is >1000 mg/kg bw/d

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: males: 35 days; females: 35 days
- Weight at study initiation: males: 130.4 - 158.9 g; females 112.8 - 142.5 g
- Housing: single housing
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 55 +/- 15 %
- Photoperiod: 12/12 (hrs dark / hrs light)

Route of administration:

oral: gavage

Vehicle:

other: 0.8 % aqueous hydroxypropylmethyl-cellulose gel

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: daily

VEHICLE
- Concentration in vehicle: according to dose level
- Amount of vehicle: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

main study: 5 males and 5 females for each dose group
recovery: additional 5 males and 5 females in control and high dose group

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: based on 7-day dose range finding study
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale: random

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before administration and once in a week thereafter (1, 2, 4, 8 and 24 hours after administration)
- detailed clinical observations were made outside the home cage in a standard arena and at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: by visual eye inspection

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Prior to start of administration, at the end of test week 4 and at the end of recovery period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination or end of recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: haemoglobing content (HGB); erythrocytes (RBC); leucocytes (WBC); differential blood count (relative); differential blood count (absolute); reticulocytes (Reti); platelets (PCT); haematocrit (HCT); thromboplastin time (TPT); activated partial thromboplastin time (ePTT); mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see haematology
- Animals fasted: Yes, see haematology
- How many animals: see haematology
- Parameters checked: albumin; globulin; albumin/globulin ratio; bilirubin (total); cholesterol (total); creatinine; glucose; protein (total); urea (in blood); clacium; chloride; potassium; sodium; alanine aminotransferas (ALAT); alkaline phosphatase (aP); aspartate aminotransferase (ASAT); lactate dehydrogenase (LDH)

URINATION: Yes
- Time schedule for collection of urine: When an animal is removed from its cage, the pan beneath the animal's cage is examined while returning the animal to its cage. The signs of urination are evaluated on a scale of 0 (lacking) to 5 (polyurea).
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked: volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in test week 4; approx. 1 - 2 hours after dosing and before blood sampling
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength (MEYER), motor activity and sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli, based on GAD)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (organs preserved: adrenal gland (2), aorta abdominalis, bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla /pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: left and right ventricle, septum), intestine, large (colon, rectum), intestine, small (duodenum, Jejunum, ileum, incl. Peyer's patches; Swiss roll method), 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, nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate, salivary glands (mandibular, sublingual and parotid gland), skin (left flank), spinal cord (3 levels: cervical, midthoracic, lumbar), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids) tissue masses or tumours, (including regional lymph nodes) trachea (incl. larynx) urinary bladder, uterus (incl. cervix and oviducts), vagina


HISTOPATHOLOGY: Yes (all organs listed above were histopathologically evaluated in the control and high dose group)

Statistics:

The test item groups 2 - 4 were compared with the control group 1. The following statistical methods were used:

STUDENT´s t-test : All numerical functional tests
Multiple t-test based on DUNNETT : body weight, food consumption, haematology, clinical biochemistry, relative and absolute organ weights
FISHER´s Exact test : Histopathology

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/d:
None of the rats revealed any changes of behaviour or external appearance.

300 mg/kg bw/d:
4 of 5 males and 4 of 5 females revealed slight salivation starting 2-3 min p.a. lasting up to 10 minutes starting on test day 8 for the males and on test day 10 for the females. This clinical sign was observed on 3 to 14 test days in the males and on 2 to 11 test days in the females.

1000 mg/kg bw/d:
Slight salivation in the males and slight to moderate salivation in the females was observed in all animals. The symptom occurred immediately to 3 min p.a. lasting up to 30 min starting on test day 8. This clinical sign was noted on 2 to 17 test days in the males and on 10 to 19 test days in the females.

Recovery period:
None of the rats previously treated with 1000 mg/kg b.w./day revealed any changes of behaviour or external appearance during the recovery period.

The faeces of all animals were of normal consistency throughout the experimental period.

Mortality:

no mortality observed

Description (incidence):

No mortality was noted at 100, 300 or 1000 mg Aurelione/kg b.w./day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain were not influenced in the male and female animals treated with 100, 300 or 1000 mg/kg b.w./day or previously treated with 1000 mg/kg b.w./day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the food consumption of the male and female animals treated with 100, 300 or 1000 mg/kg bw/day or previously treated with 1000 mg/kg bw/day compared to the control group. The slight increase of the food consumption observed for the males treated with 1000 mg/kg bw/day in test week 4 (statistically significant at p < 0.01) was caused by the relative low value noted for the control and is regarded not to be test item-related. The visual appraisal of the drinking water consumption did not reveal any test item-related changes.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Treatment period / Recovery period
No test item-related changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus were noted in the male and female rats treated with 100, 300 or 1000 mg/kg bw/day or previously treated with 1000 mg/kg b.w./day. No changes were noted for the control animals, either.

Haematological findings:

no effects observed

Description (incidence and severity):

Treatment period
No test item-related influence on the haematological parameters was observed for the animals treated with 100, 300 or 1000 mg/kg bw/day.
The slight increase of the haemoglobin value observed in the high dose males on test day 29 (statistically significant at p < 0.01) was within the normal range of biological variability and is regarded to be a spontaneous change and not test item-related.

Recovery period
No test item-related influence on the haematological parameters was observed for the male and female animals previously treated with 1000 mg/kg bw/day.
The statistically significant decrease (at p <. 0.01) on the absolute LUC value in high dose males observed on test day 43 was related to the relative high value observed for the control group and not considered adverse.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Treatment with 100, 300 or 1000 mg/kg bw/day did not cause any test item-related effects on biochemical parameters compared to the control.

The following change in biochemical parameters was noted for the male and female animals treated with 300 or 1000 mg/kg bw/day on test day 29 compared to the control animals: see Table 1 in "any other information on results". The marginal changes observed (increase in cholesterol levels at 300 and 1000 mg/kg bw/day, statistically significant at p < 0.01 in the males at 300 mg/kg bw/day and the females at 1000 mg/kg bw/day) are considered to be non-specific. The statistically significant (at p <, 0.01) decrease on the glucose value and the increased value for creatinine in high dosed males observed on test day 29 were within the normal range of biological variability and are regarded to be spontaneous changes and not test item-related. In addition, all values were within the LPT background data.

Recovery period
No test item-related influence on the biochemical parameters was observed for the male and female animals previously treated with 1000 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment period
The relative organ weight of the liver was increased in the males treated with 100, 300 or 1000 mg/kg bw/day by 16% to 28% in a dose-related way and in the females treated with 1000 mg/kg bw/day by 33% compared to the control (statistically significant at p < 0.01). The absolute organ weight of the liver was increased in the males treated with 100, 300, or 1000 mg/kg bw/day by 17% to 27% (statistically significant at p < 0.01 at 300 mg/kg bw/day) and in the females treated with 1000 mg/kg bw/day by 32% (statistically significant at p < 0.01).

Recovery period
The increase of the relative and absolute organ weight of the liver had subsided in the male animals previously treated with 1000 mg/kg bw/day but was still noted in the females at the end of the recovery period (statistically significant at p < 0.01).
The increased absolute weight of the spleen observed in the female animals at the end of the recovery period (statistically significant at p < 0.01) is regarded to be a spontaneous organ change and thus not test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Treatment period / Recovery period
No test item-related pathological changes were observed in the organs/tissues of the rats treated with 100, 300 or 1000 mg/kg b.w./day for 28 days or previously treated with 1000 mg/kg b.w./day.

Neuropathological findings:

no effects observed

Description (incidence and severity):

The observational screening performed in test week 4 approximately 1-2 hours after dosing did not reveal any test item-related influence on the rats treated with 100, 300 or 1000 mg/kg b.w./day. No influence was noted on the fore-and hindlimb grip strength or spontaneous motility in any of the tested dose levels.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histomorphological examination of rats treated or previously treated with 1000 mg/kg bw/day did not reveal any morphological lesions which are considered to be test item-related.
There was no morphological difference between the main study and recovery animals of the control group 1 and the high dose group 4.

Type, incidence and severity of the minimal to moderate lympho-histiocytic or mixed cell inflammatory lesions in various organs were comparable in all groups examined and are considered to be spontaneous organ changes and thus not test item-related.
The involution of the thymus corresponded to the age of the animals.
The incidence and severity of the fatty infiltration in the liver and kidney was within the physiological limits. There was no difference between the control group 1 and the high dose group 4. The coincidental findings in a few control and/or test item-treated animals are considered to be spontaneous organ changes.
Type, incidence and severity of the lesions recorded were not increased in the test item-treated animals compared to the control animals and are thus not considered to be test item-related.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related adverse effects observed at the highest dose tested.

Key result

Critical effects observed:

no

Table 1 Changes in biochemical parameters on test day 29 compared to the control (%)

Parameter	300 mg/kg bw/d		1000 mg/kg bw/d
Cholesterol	Males	females	males	females
	+47**	+39	+35	51**

* * statistically significant at p ^ 0.01

Conclusions:

Under the conditions of this study the, subacute oral NOAEL in the rat was determined to be >1000 mg/kg bw/d since no test item-related adverse effects were observed up to the highest dose tested.

Executive summary:

Subacute oral toxicity of the test item was assessed in the rat according to OECD 407 TG. Five male and 5 female animals were applied per dose group, respectively. In addition 5 male and 5 female animals were applied in the control and high dose group as recovery group. The test item was administered at doses of 0 (vehicle control = 0.8% aqueous hydroxypropylmethyl-cellulose gel), 100, 300, and 1000 mg/kg bw/d. No mortality was noted at any of the dose levels tested. Four of 5 males and 4 of 5 females treated with 300 mg/kg bw/day and all animals treated with 1000 mg/kg bw/day revealed slight or slight to moderate salivation starting immediately to 3 min p.a. lasting up to 30 min. This finding, observed from test day 8 onwards, was more pronounced in the female animals of the high dose group. None of the rats of the recovery group (i.e. previously treated with 1000 mg/kg bw/day) revealed any changes of behaviour or external appearance during the recovery period. The faeces of all animals were of normal consistency throughout the experimental period (treatment and recovery period). The observational screening did not reveal any test item-related changes at any dose level 1 -2 hours after dosing. No influence was noted on the fore- and hindlimb grip strength or spontaneous motility. No test item related changed were noted in regards to body weight and body weight gain, food and drinking water consumption in the course of the study. No test item-related influence on the haematological parameters was observed for the animals at any dose level or in the recovery group. Treatment with 100, 300 or 1000 mg/kg bw/day did not cause any test item-related effects on biochemical parameters compared to the control. A statistically significant change in cholesterol values was noted for the male and female animals treated with 300 or 1000 mg/kg bw/day on test day 29 compared to the control animals (300 mg/kg bw/d: +47** % (males) and +39 % (females); 1000 mg/kg bw/d: +35 % (males) and +51** % (females)). This effect however was reversible, as it was not seen in the recovery group anymore. No test item related changes of the eyes and the optic region were noted. Gross pathology revealed no effects at any of the dose levels tested. The relative organ weight of the liver was increased (statistically significant at p < 0.01) in the males treated with 100, 300 or 1000 mg/kg bw/day by 16% to 28% in a dose-related way and in the females treated with 1000 mg/kg bw/day by 33%. The absolute organ weight of the liver was increased in the males treated with 100, 300, or 1000 mg/kg bw/day by 17% to 27% (statistically significant at p <, 0.01 at 300 mg/kg bw/day) and in the females treated with 1000 mg/kg bw/day by 32% (statistically significant at p < 0.01). The increase of the relative and absolute organ weight of the liver had subsided in the male animals of the recovery group (previously treated with 1000 mg/kg bw/day) but was still noted in the females at the end of the recovery period (statistically significant at p < 0.01). The histomorphological examination of the rats treated or previously treated with 1000 mg/kg bw/day did not reveal any morphological lesions which are considered to be test item-related.

In conclusion, the aim of this study was to obtain information on the toxicity of the test item when given daily by oral administration via gavage to CD rats for 28 days and to assess the reversibility of any effects after a 2-week recovery period. Rats were treated with 100, 300 or 1000 mg/kg bw/day. None of the animals died prematurely. No test item-related changes of the body weight, body weight gain, food and drinking water consumption, haematological and biochemical parameters, the eyes and the optic region and macroscopic post mortem findings were noted. The observational screening did not reveal any test item-related changes at any dose level. No test item-related influence was noted for the fore- and hindlimb grip strength and spontaneous motility. The histomorphological examination of the rats treated with 1000 mg/kg bw/day did not reveal any morphological lesions which are considered to be test item-related. The marginal changes observed (increase in cholesterol levels and liver weight with no histopathological correlation) are considered to be a non-specific metabolic adaptation caused by the high workload on the liver with the test item. Under the conditions of this test, the NOAEL was >1000 mg/kg bw/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-02-05 to 1997-09-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 408 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

yes (incl. certificate) UK GLP Compliance Program (inspection date: 1996-01-22)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Sprague-Dawley Crl:CD®BR strain
- Source: Charles River (UK) Limited, Manston, Kent- U.K.
- Age at study initiation: six to seven weeks old
- Weight at study initiation: males: 158 to 206 g, females: 142 to 185 g
- Housing: animals were housed in groups of up to four by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet: ad libitum (pelleted diet (Rat and Mouse SQC Expanded Diet No.1, Special Diets Services Limited, Witham, Essex, UK) Analysis certified
- Water: ad libitum (Analysis certified)
- Acclimation period: at least 8 days

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

Route of administration:

oral: gavage

Details on route of administration:

not specified

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentration as a solution in 0.5% carboxymethyl cellulose (vehicle). For each dose level a separate aliquot of test material was weighed into the appropriate container. The vehicle was added and mixed using a Silverson homogeniser to ensure a homogeneous suspension was formed.

VEHICLE
- Justification for use and choice of vehicle: test compound of low solubility in water
- Dose volume: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration was determined by gas chromatography (GC) using an external standard technique. Analytical data indicated that actual dosages were close to nominal concentrations and acceptable.

HOMOGENEITY of test material formulation (nominal concentrations: 10, 50 and 200 mg/mL) were in the range of 92.1-106%, 96.4-103.6 % and 95.5 to 104.5% of the nominal concentrations with mean values of 100.1 %, 98.2 % and 100.5% of nominal concentrations respectively.

STABILITY ANALYSIS: the concentrations found after 11 days of storage were 97%, 97% and 99 % of the nominal concentration for the following nominal concentrations: 10, 50 & 200 mg/mL

CONCENTRATION ANALYSIS: Daily measured concentrations for 10, 50 & 200 mg/mL nominal concentrations were in the range of 91-113%, 90-126% & 93-107 % of nominal concentrations respectively.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily, 7 days a week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15 animals/sex/dose + 10 recovery animals/sex/control and high dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on previous studies. In the 7-day oral gavage dose range finding study in the rat, twenty-four rats of the Crl:CD(SD)BR strain were divided into 4 groups consisting of 3 males and 3 females. Three groups received the test compound, orally by gavage once daily, for 7 days at dose levels of 500, 750 and 1000 mg/kg bw/day. The fourth group received the vehicle 0.5% (w/v) aqueous carboxymethylcellulose and acted as a control. No adverse significant effects (mortality, clinical signs, bodyweight change, food consumption, organ weight, necropsy & histopathology) were measured for any of the concentrations tested. In the 28-day oral gavage toxicity study in the rat, the NOEL was determined to be 1000 mg/kg bw/d.
- Animal assignment: random
- Rationale for selecting satellite groups: control and high dose groups
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (immediately after dosing and 1 and 5 hours after dosing)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily (immediately after dosing and 1 and 5 hours after dosing)

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 (the day before the start of treatment) and at weekly
intervals thereafter. Bodyweights were also recorded at necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: NOT A FEEDING STUDY, food consumption was recorded wherever possible for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE: NOT A DRINKING WATER STUDY, water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination (during week 12)
- Dose groups that were examined: all non-recovery control and 1000 mg/kg bw/day dose animals
- Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex and, following pupil dilation with 0.5% Tropicamide solution ("Mydriacyl") detailed examination of the internal structure of each eye using a direct ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and treatment-free period (Day 90 and on Day 118 for recovery group). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 119.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all surviving animals, and all recovery groups animals
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment (Day 90 and on Day 118 for recovery group). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 119.
- Animals fasted: No
- How many animals: all surviving animals and all recovery group animals
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: performed on all surviving non-recovery animals during Week 13 wherever possible and on all recovery group animals during Week 17. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight
- Parameters checked in table 7.5.1/1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

PATHOLOGY
On completion of the dosing period all surviving animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal, 60 mg/mL; May and Baker Limited, Dagenham, Essex, UK) followed by exsanguination. Animal number 76 was killed in extremis by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS: The following organs were removed from animals killed either at the end of the dosing period or at the end of the treatment-free period, dissected free from fat and weighed before fixation: Adrenals, Brain, Heart, Kidneys, Liver, Lungs (with bronchi), Ovaries/testes, Pituitary, Spleen, Thymus, Thyroid).

HISTOPATHOLOGY: Samples of the main tissues (40) were removed from all animals and preserved in buffered 10% formalin.

GROSS PATHOLOGY: Yes (See table 7.5.1/2)

HISTOPATHOLOGY: Yes (See table 7.5.1/2)
All tissues were despatched to Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK for processing. All preserved tissues from control and 1000 mg/kg/day animals and the 250 mg/kg/day female killed in extremis were prepared as paraffin blocks, sectioned at nominal thickness of 5 micrometer and stained with haematoxylin and eosin for subsequent microscopic ex amination. The kidneys, liver and lungs from all surviving animals in the remaining non-recovery and recovery dose groups were also processed, and examined microscopically. Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE 84 pathology computerisation system for tabulation and report production.

Other examinations:

None

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), quantitative urinalytical and weekly bodyweight gain data, were assessed for dose response relationships using linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Data showing heterogeneous variances were re-analysed using nonparametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U"-test. The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value. Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:

1. Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Analyses were undertaken using the non-recovery control groups for intergroup comparison with non-recovery treatment groups. In the absence of any treatment-related findings, an intergroup compa rison was not conducted between the recovery control and treatment groups. Animal that died during the course of the study were excluded from statistical analysis.

Clinical signs:

no effects observed

Description (incidence and severity):

Males and females treated with 1000 mg/kg bw/day showed increased salivation either before or approximately two minutes after dosing from Days 6 and 5 onwards respectively. Associated signs detected sporadically throughout the treatment period included increased salivation approximately one hour after dosing, red/brown staining of the mouth and fur wetting. Increased salivation and its associated findings are commonly reported following the oral administration of a test material for mulation and are considered to be attributable to an unpleasant tasting or locally irritant formulation rather than an indication of systemic toxicity. One 250 mg/kg bw/day female briefly developed clinical signs on Days 9 and 10 of the study including pallor of the extremities, hunched posture, lethargy, decreased respiratory rate and gasping, laboured and noisy respiration. The lack of dose relationship exhibited by these observations coupled with their transiency suggests that they were most probably a consequence of a slight mal-dose and, accordingly, were considered to be of no toxicological importance. One female treated with 250 mg/kg bw/day developed a large swelling around the lower left abdominal region on Day 43 of the study. By Day 49 this swelling had darkened and the animal was consequently killed in extremis. The remaining incidental signs detected during the study, including generalised fur loss, an open wound, a scab and a swollen ear were minor physical injuries and were of no toxicological importance.

Mortality:

no mortality observed

Description (incidence):

No treatment-related effects were detected.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect detected on bodyweight development during the study. Treated animals showed a bodyweight gain similar to or greater than controls during the study. Recovery 1000 mg/kg bw/day females showed a slight but statistically significant reduction in body weight gain during the first week of the recovery period compared with controls. No such reductions were detected during the treatment period for non-recovery individuals and such a minimal change (p < 0.05) was considered to be of no toxicological significance. These females also showed a slight statistically significant increase in bodyweight gain during the final week of the recovery period but this is unlikely to represent an adverse effect on health.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related effects were detected.

Food efficiency:

no effects observed

Description (incidence and severity):

This was NOT a feeding study, however there was no adverse effect detected on dietary intake during the study. Treated animals showed a dietary intake and food efficiency (the ratio of bodyweight gain to food consumption) similar to or greater than controls during the study.

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

no effects observed

Description (incidence and severity):

This was NOT a feeding study, however there were no overt intergroup differences detected during the study.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ocular effects. The incidental findings recorded were those normally encountered in laboratory maintained rats of the strain and age employed and were confined to pretest observations, having completely resolved by the end of the study.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes detected in the haematological parameters measured. Recovery 1000 mg/kg bw/day animals of either sex showed a statistically significant reduction in monocyte count with males also showing a reduction in neutrophil count in comparison with controls. Similar reductions were not apparent among non-recovery animals following ninety days of treatment and accordingly these intergroup differences were considered to be of no toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes detected in the blood chemical parameters measured. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in plasma triglyceride concentration compared with controls. All of the individual values were within the normally expected range for rats of the strain and age used and in the absence of any changes in the plasma protein profile or any other evidence of hepatic dysfunction which might otherwise account for hypotriglyceridaemia in these individuals, the reduction was considered to have arisen fortuitously. Males treated with 250 mg/kg bw/day showed a statistically significant reduction in plasma alkaline phosphatase compared with controls. In the absence of a similar change in this parameter at the highest dose level a dose-relationship could not be established and in any case a reduction in the activity of this enzyme is unlikely to be of toxicological importance. The remaining statistically significant intergroup differences were confined to increases in plasma alanine amino transferase and creatinine concentration among recovery 1000 mg/kg bw/day females when compared with controls. No such increases were evident among non-recovery animals following ninety days of treatment and as such these differences were considered to be of no toxicological importance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes detected in the urinalytysis parameters measured. All inter and intra group differences were considered to be a result of normal variation for rats of the species and strain used and, therefore, of no toxicological significance.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related changes detected in the organ weights measured. Females treated with 250 mg/kg bw/day showed a statistically significant increase (p< 0.01) in thymus weight, both absolute and relative to terminal bodyweight, compared with controls. A slight but statistically significant increase (p < 0.05) in relative thymus weight was also evident among 1000 mg/kg bw/day females. The severity of the differences was, however, not dose-related and in the absence of any histopathological evidence of thymus changes these intergroup differences were considered to be of no toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic lesions which could convincingly be attributed to test material toxicity. Five males treated with 1000 mg/kg bw/day and one 250 mg/kg bw/day male were reported to have speckled kidneys at terminal kill, however, in the absence of any toxicological correlates, such as an altered relative kidney weight, microscopically observed lesions or blood chemical changes indicative of renal dysfunction, these findings were, in isolation, considered to be of no toxicological importance. The two male decedents from the 1000 mg/kg bw/day treatment group showed pulmonary lesions suggestive of a mal-dose together with normally expected post mortem changes. The remaining macroscopic abnormalities, including a small or large abdominal mass, hydronephrosis of one or both kidneys, pallor of and multiple dark foci on the left and caudal lobes of the lung, a malformed or misshapen spleen and small testes represent congenital or low incidence findings of the type normally encountered among rats of the strain and age used. In particular there was no difference in the incidence or severity of findings between treated animals and controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. There were three unscheduled deaths during the study. Rat number 95 male and 97 male (1000 mg/kg bw/day) died as a probable result of maldosing, although there was no histopathological evidence in support of this or any other cause of death, and rat number 76 female (250 mg/kg bw/day) was killed as a result of a malignant mammary tumour. Mammary adenocarcinomas are relatively common in aged Sprague Dawley CD female rats, and it is not unusual for mammary neoplasia to develop amongst female rats at an earlier age. Although there were indications of an increased incidence of eosinophilic globular accumulations in the proximal renal tubular epithelium of high dose male rats, the difference, which attained statistical significance (p < 0.05), was not sufficiently great, to indicate a relationship to treatment. The between group variation in incidence and severity of the condition was pronounced (p< 0.001) and further precluded an association with treatment. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed. Although group differences in the incidence or severity of lesions occasionally attained statistical significance, none were considered to be related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed up the highest dose tested

Key result

Critical effects observed:

no

No other information

Conclusions:

Under the test conditions, the "No Observed Adverse Effect level" (NOAEL) was considered to be ≥1000 mg/kg bw/day.

Executive summary:

In a sub-chronic toxicity study performed according to the OECD test guideline No. 408 and in compliance with GLP, the test item diluted in carboxymethylcellulose was administered by gavage to Sprague-Dawley Crl:CD®BR rats (15/sex/group) at 50, 250 or 1000 mg/kg bw/day for 90 days. A control group received the vehicle only at the same volume-dosage (5 mL/kg bw/day). Control and high dose group contained an additional 10 rats/sex/group for evaluation of recovery from any test substancerelated effects, following a 28-day recovery period. There were no treatment-related deaths during the study. Two males treated with 1000 mg/kg bw/day were found dead during the treatment period one each on Days 34 and 85 almost certainly following a mal-dose. A 250 mg/kg bw/day female was killed in extremis on Day 49 after a large mass developed around its abdominal region. No clinically observable signs of toxicity were detected during the study. No adverse effect on bodyweight development, on dietary intake, on water consumption was detected during the study. No treatment-related effects were observed during ophthalmoscopic examination.Haematology, clinical chemistry and urinalysis were normal. At necropsy, no treatment-related macroscopic or microscopic lesions were noticed, organ weight were within normal ranges. Based on these observations, the "No Observed Adverse Effect level" (NOAEL) was considered to be ≥ 1000 mg/kg bw/day.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For justification for type of information please refer to the Read Across Justification attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed up to the highest dose tested

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline study.

Organ:

other: no adverse effects observed at the highest dose tested

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two GLP-compliant guideline studies on repeated dose toxicity are available: one study on the sub-acute oral toxicity of the target substance Aurelione and one study on sub-chronic oral toxicity of the read across source substance Globalide (Oxacyclohexadecen-2-one, CAS No 111879-80-2).

Sub-acute oral toxicity, OECD 407

Subacute oral toxicity of the target substance Aurelione was assessed in the rat according to OECD 407 TG (). Five male and 5 female animals were applied per dose group, respectively. In addition 5 male and 5 female animals were applied in the control and high dose group as recovery group. The test item was administered at doses of 0 (vehicle control = 0.8% aqueous hydroxypropylmethyl-cellulose gel), 100, 300, and 1000 mg/kg bw/d. No mortality was noted at any of the dose levels tested. Four of 5 males and 4 of 5 females treated with 300 mg/kg bw/day and all animals treated with 1000 mg/kg bw/day revealed slight or slight to moderate salivation starting immediately to 3 min p.a. lasting up to 30 min. This finding, observed from test day 8 onwards, was more pronounced in the female animals of the high dose group. None of the rats of the recovery group (i.e. previously treated with 1000 mg/kg bw/day) revealed any changes of behaviour or external appearance during the recovery period. The faeces of all animals were of normal consistency throughout the experimental period (treatment and recovery period). The observational screening did not reveal any test item-related changes at any dose level 1 -2 hours after dosing. No influence was noted on the fore- and hindlimb grip strength or spontaneous motility. No test item related changed were noted in regards to body weight and body weight gain, food and drinking water consumption in the course of the study. No test item-related influence on the haematological parameters was observed for the animals at any dose level or in the recovery group. Treatment with 100, 300 or 1000 mg/kg bw/day did not cause any test item-related effects on biochemical parameters compared to the control. A statistically significant change in cholesterol values was noted for the male and female animals treated with 300 or 1000 mg/kg bw/day on test day 29 compared to the control animals (300 mg/kg bw/d: +47** % (males) and +39 % (females); 1000 mg/kg bw/d: +35 % (males) and +51** % (females)). This effect however was reversible, as it was not seen in the recovery group anymore. No test item related changes of the eyes and the optic region were noted. Gross pathology revealed no effects at any of the dose levels tested. The relative organ weight of the liver was increased (statistically significant at p < 0.01) in the males treated with 100, 300 or 1000 mg/kg bw/day by 16% to 28% in a dose-related way and in the females treated with 1000 mg/kg bw/day by 33%. The absolute organ weight of the liver was increased in the males treated with 100, 300, or 1000 mg/kg bw/day by 17% to 27% (statistically significant at p <, 0.01 at 300 mg/kg bw/day) and in the females treated with 1000 mg/kg bw/day by 32% (statistically significant at p < 0.01). The increase of the relative and absolute organ weight of the liver had subsided in the male animals of the recovery group (previously treated with 1000 mg/kg bw/day) but was still noted in the females at the end of the recovery period (statistically significant at p < 0.01). The histomorphological examination of the rats treated or previously treated with 1000 mg/kg bw/day did not reveal any morphological lesions which are considered to be test item-related.

In conclusion, the aim of this study was to obtain information on the toxicity of Aurelione when given daily by oral administration via gavage to CD rats for 28 days and to assess the reversibility of any effects after a 2-week recovery period. Rats were treated with 100, 300 or 1000 mg/kg bw/day. None of the animals died prematurely. No test item-related changes of the body weight, body weight gain, food and drinking water consumption, haematological and biochemical parameters, the eyes and the optic region and macroscopic post mortem findings were noted. The observational screening did not reveal any test item-related changes at any dose level. No test item-related influence was noted for the fore- and hindlimb grip strength and spontaneous motility. The histomorphological examination of the rats treated with 1000 mg/kg bw/day did not reveal any morphological lesions which are considered to be test item-related. The marginal changes observed (increase in cholesterol levels and liver weight with no histopathological correlation) are considered to be a non-specific metabolic adaptation caused by the high workload on the liver with the test item. Under the conditions of this test, the NOAEL was >1000 mg/kg bw/day.

Sub-chronic oral toxicity, OECD 408

In this study performed according to the OECD test guideline No. 408 and in compliance with GLP, the read across source substance Globalide (Oxacyclohexadecen-2-one, CAS No 111879-80-2) diluted in carboxymethylcellulose was administered by gavage to rats at 0, 50, 250 or 1000 mg/kg bw/day for 90 days. Additional satellite animals were included in the control and high dose group to evaluate the reversibility or persistence of any toxic effects after a post-treatment period of 28 days. There were no treatment-related deaths during the study. Two males treated with 1000 mg/kg bw/day were found dead during the treatment period one each on Days 34 and 85 almost certainly following a mal-dose. A 250 mg/kg bw/day female was killed in extremis on Day 49 after a large mass developed around its abdominal region. No clinically observable signs of toxicity were detected during the study. No adverse effect on bodyweight development, on dietary intake, on water consumption was detected during the study. No treatment-related effects were observed during ophtalmoscopic examination. Haematology, clinical chemistry and urinalysis were normal. At necropsy, no treatmentrelated macroscopic or microscopic lesions were noticed, organ weight were within normal ranges.

Conclusion on repeated dose toxicity via oral route

Based on these observations, the "No Observed Effect level" (NOEL) was considered to be ≥1000 mg/kg bw/day. The sub-chronic toxicity study (Safepharm, 1998, rel. 1) with the read across source substance Globalide (Oxacyclohexadecen-2-one, CAS No 111879-80-2) was considered to be the key study for the repeated dose toxicity endpoint. The results of the sub-acute toxicty study of LPT (2007, rel.1) support this conclusion. Indeed the substance did not produce any toxic effect at dose levels up to 1000 mg/kg bw/day when administered by gavage to rats during 28 days.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint

The study with the longest duration (90-days) was selected. No adverse effect were observed in the two available studies up to 1000 mg/kg bw/d.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item does not require classification for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP), as amended for the fourteenth time in Regulation (EU) 2020/217.