Indeno[4,5-d]-1,3-dioxin, 4,4a,5,6,7,8,9,9b-octahydro-7,7,8,9,9-pentamethyl-

Endpoint summary

Description of key information

In an oral (gavage) 28-day repeated dose toxicity study in rats (according to OECD guideline 407 and GLP) the mid-dose level of 150 mg/kg bw/day was a NOAEL. A number of minimal mainly adaptive effects on liver and kidney were observed at 1000 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2003 and 08 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD TG 405 under GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA Environmental Protection Agency (EPA) health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health and Welfare (MHW) Guidelines 1986 for a twenty-eight days repeated dose oral toxicity study as required by Japanese Chemical Substances Control Law 1973 of the Ministry of International Trade and Industry (MITI), amended.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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 old

- Weight at study initiation:
At the start of treatment the males weighed 155 to 191 g, the females weighed 129 to 173 g

- Fasting period before study:
No

- Housing:
Groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper

- Diet:
A pelleted diet (Rodent 5LF2 (Certified) Diet, International Product Supplies Ltd., Northants, UK was used (ad libitum)

- Water:
Mains drinking water was supplied from polycarbonate bottles attached to the cage (ad libitum)
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study

- Acclimation period:
Nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21 ± 2 °C

- Humidity (%):
55 ± 15 %

- Air changes (per hr):
At least fifteen air changes per hour

- Photoperiod (hrs dark / hrs light):
Low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES:
From: Day 1 To: Day 28

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.

DIET PREPARATION
- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
Not applicable

- Concentration in vehicle:
3.75, 250 mg/mL

- Amount of vehicle (if gavage):
4 mL/kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of 2-Eyed Musk in the test material formulations was determined by gas chromatography (GC) using an external standard technique. The test material formulations were sampled and analysed for up to fourteen days. The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Frequency of treatment: 7 days/week

Remarks:

Doses / Concentrations:
0, 15, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 15 mg/kg/day
Male and Female: 5 animals per sex at 150 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Random

- Rationale for animal assignment (if not random):
Not applicable

- Rationale for selecting satellite groups:
Not applicable

- Post-exposure recovery period in satellite groups:
Not applicable

- Section schedule rationale (if not random):
Not applicable

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Immediately before dosing and one and five hours after dosing during the working week.
Animals were observed immediately before dosing and one hour after dosing at weekends and public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends or on public holidays). All observations were recorded.

- Cage side observations included:
signs of toxicity, ill-health or behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends and public holidays. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends or on public holidays). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28 and, in the case of recovery group animals, on Days 35 and 42. Bodyweights were also recorded at terminal kill.

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

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: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Not applicable
- Time schedule for examinations:
Not applicable
- Dose groups that were examined:
Not applicable

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
On all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42).

- Anaesthetic used for blood collection:
No

- Animals fasted:
No

- How many animals:
All non-recovery test and control group animals and on all recovery group animals.

- Parameters examined:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH),
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PL T)
Reticulocyte count (Retic) - Cresyl blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by 'Hepato Quick' and Activated partial thromboplastin time (APTT) was assessed by 'Preci Clot' using samples collected into sodium citrate solution (0. 11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
At the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42).

- Animals fasted:
No

- How many animals:
All non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42).

- Parameters examined:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K +)
Chloride (CI-)
Calcium (Ca++)
Inorganic phosphorus (P)
Gamma glutamyltranspeptidase (yGT)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Triglycerides (Tri)
Total cholesterol (Chol)
Total bilirubin (Bili)

URINALYSIS: Yes
- Time schedule for collection of urine:
All non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6

- Metabolism cages used for collection of urine:
Yes

- Animals fasted:
Animals were maintained under conditions of normal hydration during collection but without access to food.

- Parameters examined:
The following parameters were measured on collected urine:
Volume
Specific Gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Reducing Substances
Blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Prior to the start of treatment and on Days 7, 14, 21 and 22, all animals were observed for signs of functional / behavioural toxicity. Functional perfomance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

Observations were carried out from approximately two hours after dosing on each occasion.

- Dose groups that were examined:
All

- Battery of functions tested: sensory activity, grip strength, motor activity
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex·
Blink reflex
Startle reflex was measured using the ST l 058 Startle Test Meter (Benwick Electronics). Each animal was placed on the force platform and allowed to settle. An audible tone was activated and any change in the force exerted by the animal on the force platform, as a result of startle induced tremor, was measured. The percentage average response, root of the mean square and peak response were calculated for each animal and two consecutive trials were performed.

OTHER:
Behavioural assessments:
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

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 sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour 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 reactlvlty to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex*
Blink reflex

(* Startle reflex was measured using the ST l 058 Startle Test Meter (Benwick Electronics). Each animal was placed on the force platform and allowed to settle. An audible tone was activated and any change in the force exerted by the animal on the force platform, as a result of startle induced tremor, was measured. The percentage average response, root of the mean square and peak response were calculated for each animal and two consecutive trials were performed.)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ weights:
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation: Adrenals, Brain, Heart, Kidneys, Liver, Ovaries, Spleen and Testes

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, Caecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, Ileum, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord, Spleen, Stomach, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Muscle (skeletal), Testes, Thymus, Thyroid/parathyroid, Trachea, Urinary bladder and Uterus

All tissues were despatched to Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford for processing. The following preserved tissues from all non-recovery and 1000 mg/kg/day dose group animals were prepared as paraffin wax blocks, sectioned at nominal thickness of 5 µmand stained with haematoxylin and eosin for subsequent microscopic examination:

Since there were indications of treatment-related changes in the liver, kidneys, thyroids and stomach, examination was subsequently extended to include similarly prepared sections of these tissues from all animals in the other treatment groups.

Other examinations:

Mortality

Bodyweight
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 1 4, 21 and 28 and, in the case of recovery group animals, on Days 35 and 42. Bodyweights were also recorded at terminal kill.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative urinalytical data, functional performance and sensory reactivity data were assessed for dose response relationships for non-recovery groups where appropriate, by 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. In the case of recovery group data, the analysis performed was a two-tailed t-test incorporating Levene's test for homogeneity of variance. Where Levene's test showed unequal variances among either nonrecovery or recovery group data, the affected parameters were analysed using non-parametric methods: Kruskal-Wallis ANOV A and mann-Whitney 'U' test.

The haematology variable basophils were not analysed since consistently greater than 30 % of the data were recorded as the same value.

Probability values (P) are presented as follows:
p < 0.001 ***
pP < 0.05 *
p ≥ 0.05 (not significant)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See results

Mortality:

mortality observed, treatment-related

Description (incidence):

See results

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See results

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See results

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths during the study.

During the final week of treatment hunched posture and tiptoe gait were observed in females treated with 1000 mg/kg/day. All but hunched posture regressed in recovery 1000 mg/kg/day animals on cessation of treatment with hunched posture resolving by Day 33. No treatment-related clinical observations were detected for animals of either sex treated with 15 or 150 mg/kg/day.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related effects on bodyweight. Test animals showed weight gains similar to those of the controls throughout the study period.

The statistically significant intergroup differences detected was considered to be of no toxicological importance.

FOOD CONSUMPTION AND COMPOUND INTAKE
There was no adverse effect on food consumption throughout the study.

FOOD EFFICIENCY
Food efficiency in test animals was similar to that of controls.

WATER CONSUMPTION AND COMPOUND INTAKE
Daily visual inspection of water bottles throughout the study period revealed no intergroup differences.

OPHTHALMOSCOPIC EXAMINATION
Not applicable

HAEMATOLOGY
No treatment-related effects were detected in the haematological parameters measured.

Statistical analysis revealed no significant intergroup differences.

CLINICAL CHEMISTRY
Animals of either sex treated with 1000 mg/kg/day showed statistically significant increases in plasma cholesterol concentrations (males; p<0.01, females; p<0.001) and bilirubin (males; p<0.05, females; p<0.01 accompanied in 1000 mg/kg/day males only by a slight increase (p<0.05) in alanine aminotransferase. Individual values for these parameters were invariably within the expected normal ranges so the toxicological significance is dubious but cannot be entirely disregarded. The remaining statistically significant intergroup differences detected were considered to be of no toxicological importance.

URINALYSIS
No toxicologically important abnormalities were detected in any of the urinalysis parameters measured.

Males and females treated with 1000 mg/kg/day micturated an increased volume of urine (p<0.05) of low specific gravity achieving statistical significance (p<0.05) in males compared with that produced by controls. In view that there was no convincing evidence of associated microscopic or blood chemical changes, this increased urine output was considered to be of dubious toxicological significance and probably associated with a physiological response to the increased salivation.

No similar changes were apparent in either sex treated with 150 or 15 mg/kg/day or for recovery 1000 mg/kg/day animals at the end of the fourteen day recovery period.

NEUROBEHAVIOUR
Hunched posture and tiptoe gait were observed in a number of females treated with 1000 mg/kg/day during open field assessments at Week 3 and Week 4, which correlated with the clinical observations. These changes were considered not to be of neurotoxicological importance but more likely an indirect effect of abdominal discomfort associated with the irritancy of the test material formulation. No such observations were reported for males treated with 1000 mg/kg/day or in animals of the remaining treatment groups.

There were no treatment-related changes detected in the functional performance parameters measured. Statistical analysis of the quantitative data revealed no significant intergroup differences.

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. Statistical analysis of the quantitative data revealed no significant intergroup differences.

ORGAN WEIGHTS
A statistically significant increase (p<0.001 ) in liver weight both absolute and relative to bodyweight was observed in either sex treated with 1000 mg/kg/day, with an increase (p<0.05) in the relative weight of this organ detected in males treated with 150 mg/kg/day. Males treated with 1000 mg/kg/day also showed an increase (p<0.05) in absolute and relative kidney weight. No such observations were reported for 150 or 15 mg/kg/day animals.

The remaining statistically significant intergroup differences detected were considered to be of no toxicological importance.

GROSS PATHOLOGY
Three males treated with 1000 mg/kg/day showed enlarged and pale kidneys. No other treatment related macroscopic findings were observed.

The other incidental findings detected were of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: Treatment-related centrilobular hepatocyte enlargement was seen for female rats only dosed at 1000 mg/kg/day. One female rat receiving 150 mg/kg/day of the test material was similarly affected but hepatocyte enlargement is occasionally seen among untreated control animals as a spontaneous change and an association with treatment at this dose level cannot be reliably established.

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, as evidenced by regression of the condition among Recovery
1000 mg/kg/day group animals following an additional fourteen days without treatment.

Kidneys: Globular accumulations of eosinophilic material were observed in the tubular epithelium of three male rats dosed at 1000 mg/kg/day. This finding is consistent with the presence 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. Higher grades of severity of groups of basophilic renal tubules were also observed among 1000 mg/kg/day rats, associated in some instances with tubular dilatation. Globular accumulations of eosinophilic material were also seen in relation to treatment for male rats dosed at 150 mg/kg/day but probably not at 15 mg/kg/day; the condition is occasionally seen as a spontaneous entity among control rats. All conditions were observed to have regressed among Recovery 1000 mg/kg/day male rats compared with Recovery Control animals following completion of the recovery period.

Thyroids: Higher grades of severity of follicular cell hypertrophy were observed for rats of either sex dosed at 1000 mg/kg/day. A treatment-related higher incidence of the condition was also seen for female rats at this dose level. A similar treatment-related response was not observed at any of
the remaining dose levels. Follicular cell hypertrophy was observed to have regressed among Recovery 1000 mg/kg/day group animals following an additional fourteen days without treatment.

Stomach: Acanthosis and hyperkeratosis of the forestomach were observed for two female rats dosed at 1000 mg/kg/day. Although this condition is seen occasionally among untreated control rats the possibility of an association with treatment cannot be excluded in this instance. Animals from the remaining dose levels were unaffected and there was no evidence of similar changes among recovery group animals.

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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Minor mostly adaptive changes were seen at 1000 mg/kg bw. In view of the study being a 28-day study these effects were taken into account for the derivation of the NOAEL.

Critical effects observed:

not specified

Oral administration of the test material, by gavage, for a period of 28 consecutive days resulted in treatment-related but non-adverse changes in either sex at a dose level of 1000 mg/kg/day and in males treated with 150 mg/kg/day. Rat specific hydrocarbon nephropathy in males at 150 and 1000 mg/kg/day with associated increased kidney weight in males and macroscopic renal changes in males treated with 1000 mg/kg/day. Elevated liver weight in animals of either sex treated with 1000 mg/kg/day and in males treated with 150 mg/kg/day. Histological evidence of gastric irritation in females dosed at 1000 mg/kg/day and minimal clinical observations, adaptive liver and thyroid changes in both sexes at 1000 mg/kg/day resulted in the final conclusion that the No-Adverse-Effect-Level is considered to be 150 mg/kg bw.

The following differences detected between treated and control animals were considered not to be

toxicologically significant:

Clinical Observations

- Animals treated with 1000 mg/kg/day showed transient episodes of increased salivation from Day 6 onwards together with associated signs of excessive salivation detected one hour after dosing, noisy respiration and wet/soiled fur. Excessive salivation of short duration is often reported following oral gavage administration and the daily occurrence of this finding around the time of dosing, was considered attributable to the unpalatability or locally irritant nature of the test material rather than an indication of systemic toxicity. Similarly, signs of hunched posture and tiptoe gait that developed in females treated with 1000 mg/kg/day from Day 21 were considered also to be associated with abdominal discomfort and gastric irritation.

Behavioural Assessments

- Hunched posture and tiptoe gait were observed in a number of females treated with 1000 mg/kg/day during open field assessments at Week 3 and Week 4. These findings correlated with the daily clinical observations and were considered associated with abdominal discomfort and therefore considered not to be of neurotoxicological importance.

Bodyweight

- Recovery 1000 mg/kg/day males showed a slight but statistically significant increase (p<0.05) in bodyweight gain during Week 6 of the study, however, an isolated increase in bodyweight gain is unlikely to represent an adverse effect on health and was therefore considered of no toxicological relevance.

Blood Chemistry

- Females treated with 1000 mg/kg/day showed increased plasma levels of creatinine, calcium (both p<0.05) and inorganic phosphorus (p<0.01) with reductions in potassium (p<0.01) and chloride (p<0.05) compared with controls. In the absence of histological evidence of a change in renal function or any similar changes in males, these differences were considered not to be toxicologically significant.

- Females treated with 1000 mg/kg/day showed an increase in total protein (p<0.01) and reduction in plasma glucose (p<0.05) and an apparent reduction in the albumin/globulin ratio (p<0.05). The individual values for glucose in these animals were within the normally expected range (122 - 170 mg/dl) and the intergroup difference was considered to be acceptable. The protein changes indicate an elevation in globulin levels and, in the absence of any associated effects indicative of injection or inflammatory changes, this was considered not to be toxicologically important.

- A statistically significant reduction (p<0.01) in aspartate aminotransferase was detected in females treated with 1000 mg/kg/day in comparison with controls. However, a reduction in this parameter is unlikely to be associated with chemical injury and this finding was

therefore considered to be of no toxicological relevance.

- Recovery 1000 mg/kg/day females showed minimal increases in calcium (p<0.05) and cholesterol (p<0.05) levels compared to controls. However, in the absence of histological evidence of associated histopathological changes such as renal function or bile duct

proliferation, these findings were considered not to be of toxicological significance.

Urinalysis

- Males and females treated with 1000 mg/kg/day micturated an increased volume of urine (p<0.05) of low specific gravity achieving statistical significance (p<0.05) in males compared with that produced by controls. In view that there was no convincing evidence

of associated microscopic or blood chemical changes, this increased urine output was considered to be of dubious toxicological significance and probably associated with a physiological response to the increased salivation.

Necropsy

- Macroscopic examination revealed a liver abnormality involving a dark red and firm caudal lobe in one recovery 1000 mg/kg/day male (subsequently identified histologically as necrotic). A dark red colouration of the thymus (affecting approximately half the

tissue) was observed in one further recovery 1000 mg/kg/day male. The former abnormality in isolation was considered to represent a spontaneously arising condition of no toxicological relevance while the latter was considered to represent a low incidence

finding possibly associated with the exsanguination of animals at study termination.

Histopathology

- Heart: Focal myocarditis was observed in several control and treated rats and is a common background entity in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.

- Liver: Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.

- Spleen: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits.

- Kidneys: Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance at the severities or frequencies reported in this study. Similarly focal corticomedullary mineralisation is a

commonly observed background condition amongst female rats. Hydronephrosis was also reported for three rats. This is widely considered to be a condition of congenital origin and in any event is does not arise as a primary toxicological event.

- Lungs: A minimal severity of bronchus associated lymphoid tissue was reported for most animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.

- Bone Marrow: Adipose infiltration of the marrow is an indicator of changes in marrow cellularity and in this study there was no difference between control and treated groups.

- Uterus: Dilatation of the uterine horns is a commonly observed cyclical condition in laboratory maintained female rats.

Conclusions:

The systemic toxicity of the test material was assessed according to OECD Guideline 407. The oral administration of the test material to rats, by gavage, for a period of twentyeight consecutive days resulted in treatment-related effects at 1000 mg/kg bw somewhat more pronounced in males than in females. Though these effects are in general not considered as adverse the NOAEL has been established at 150 mg/kg bw for both sexes in view of the fairly high number of minimal effects and the study being a 28-day study in which the adversity of the effects cannot fully be evaluated.

Executive summary:

A 28 -day repeated dose toxicity study according to OECD TG 407 has been carried out including a 14 -day recovery period. The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from non-recovery high dose and control animals was performed.

There were no deaths during the study. Increased salivation was observed around the time of dosing in either sex treated with 1000 mg/kg/day from Day 6 onwards, this was accompanied as the study progressed by associated instances of excessive salivation detected one hour after dosing, noisy respiration and wet/soiled fur, which was considered attributable to the locally irritant nature of the test material rather than an indication of systemic toxicity. During the final week of treatment hunched posture and tiptoe gait were observed in females treated with 1000 mg/kg/day. All but hunched posture regressed in recovery 1000 mg/kg/day animals on cessation of treatment with hunched posture resolving by Day 33. No treatment-related clinical observations were detected for animals of either sex treated with 15 or 150 mg/kg/day. The hunched posture and tiptoe gait were observed in a number of females treated with 1000 mg/kg/day during open field assessments at Week 3 and Week 4 were considered to be an indirect effect of abdominal discomfort associated with the irritancy of the test material formulation. No such observations were reported for males at this dose or in animals from remaining treatment groups.There were no treatment-related changes in the functional performance parameters measured an there were no changes in sensory reactivity.

All animals showed normal bodyweight, body weight gain, food consumption and food efficiency as well as water consumption compared to the controls. Heamatology, blood chemistry and urinalysis did not show toxicological relevant effects.

Organ Weights. An increase in liver weight both absolute and relative to bodyweight was observed in either sex treated with 1000 mg/kg/day, with an increase in the relative weight of this organ also detected in males treated with 150 mg/kg/day. Males treated with 1000 mg/kg/day also showed an increase in absolute and relative kidney weight. No organ weight changes were reported for 15 mg/kg/day animals of either sex or for 150 mg/kg/day females.

Necropsy. Enlarged and pale kidneys were detected in three males treated with 1000 mg/kg/day. No other treatment-related macroscopic findings were observed. At histopathology centrilobular hepatocyte enlargement was observed in female rats only dosed at 1000 mg/kg/day and in one female rat receiving 150 mg/kg/day of the test material and is considered to be mainly adaptive in nature because no other related effects were seen. In the recovery group of 1000 mg/kg/day animals following an additional fourteen days without treatment these effects had disappeared. In the kidneys slightly higher grades of severity of groups of basophilic renal tubules were also observed among 1000 mg/kg/day male rats, associated in some instances with tubular dilatation. In single female rats this was also sometimes observed. Globular accumulations of eosinophilic material were also seen in relation to treatment for male rats dosed at 150 mg/kg/day which is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of α₂-microglobulin in renal proximal tubular epithelial cells, an effect occurring only in male rats. In the thyroids a higher incidence of follicular cell hypertrophy was seen in relation to treatment for rats of either sex dosed at 1000 mg/kg/day but not at any other dose level. This effects was not seen after the recovery of fourteen days. In the stomach treatment-related acanthosis and hyperkeratosis of the forestomach were observed for two female rats dosed at 1000 mg/kg/day. Animals from the remaining dose level were unaffected and there was no evidence of similar changes among recovery group animals. Therefore the NOAEL in this repeated 28-day study is considered to be 150 mg/kg/day mainly based on liver effects.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two studies are availalbe, one adequate (OECD TG 407) and one supporting (OECD TG 421) both under GLP and the results are supporting each other.

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

A 28 -day repeated dose toxicity study according to OECD TG 407 has been carried out including a 14 -day recovery period. The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from non-recovery high dose and control animals was performed.

There were no deaths during the study. Increased salivation was observed around the time of dosing in either sex treated with 1000 mg/kg/day from Day 6 onwards, this was accompanied as the study progressed by associated instances of excessive salivation detected one hour after dosing, noisy respiration and wet/soiled fur, which was considered attributable to the locally irritant nature of the test material rather than an indication of systemic toxicity. During the final week of treatment hunched posture and tiptoe gait were observed in females treated with 1000 mg/kg/day. All but hunched posture regressed in recovery 1000 mg/kg/day animals on cessation of treatment with hunched posture resolving by Day 33. No treatment-related clinical observations were detected for animals of either sex treated with 15 or 150 mg/kg/day. The hunched posture and tiptoe gait were observed in a number of females treated with 1000 mg/kg/day during open field assessments at Week 3 and Week 4 were considered to be an indirect effect of abdominal discomfort associated with the irritancy of the test material formulation. No such observations were reported for males at this dose or in animals from remaining treatment groups.There were no treatment-related changes in the functional performance parameters measured an there were no changes in sensory reactivity.

All animals showed normal bodyweight, body weight gain, food consumption and food efficiency as well as water consumption compared to the controls. Heamatology, blood chemistry and urinalysis did not show toxicological relevant effects.

Organ Weights. An increase in liver weight both absolute and relative to bodyweight was observed in either sex treated with 1000 mg/kg/day, with an increase in the relative weight of this organ also detected in males treated with 150 mg/kg/day. Males treated with 1000 mg/kg/day also showed an increase in absolute and relative kidney weight. No organ weight changes were reported for 15 mg/kg/day animals of either sex or for 150 mg/kg/day females.

Necropsy-macroscopie: Enlarged and pale kidneys were detected in three males treated with 1000 mg/kg/day. No other treatment-related macroscopic findings were observed.

Necropsy-microscopy: In the liver centrilobular hepatocyte enlargement was observed in female rats only dosed at 1000 mg/kg/day and in one female rat receiving 150 mg/kg/day of the test material and is considered to be mainly adaptive in nature because no other related effects were seen. In the recovery group of 1000 mg/kg/day animals following an additional fourteen days without treatment these effects had disappeared. In the kidneys slightly higher grades of severity of groups of basophilic renal tubules were also observed among 1000 mg/kg/day male rats, associated in some instances with tubular dilatation. In single female rats this was also sometimes observed. Globular accumulations of eosinophilic material were also seen in relation to treatment for male rats dosed at 150 mg/kg/day which is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of α₂-microglobulin in renal proximal tubular epithelial cells, an effect occurring only in male rats. In the thyroids a higher incidence of follicular cell hypertrophy was seen in relation to treatment for rats of either sex dosed at 1000 mg/kg/day but not at any other dose level. This effects was not seen after the recovery of fourteen days. In the stomach treatment-related acanthosis and hyperkeratosis of the forestomach were observed for two female rats dosed at 1000 mg/kg/day. Animals from the remaining dose level were unaffected and there was no evidence of similar changes among recovery group animals. Therefore the NOAEL in this repeated 28-day study is considered to be 150 mg/kg/day mainly based on liver effects.

Besides this study also a reproduction/developmental screening test in rats is available. No repeated dose toxicity effects were observed up to and including the highest dose tested (107 and 110 mg/kg bw for male and females, respectively). In view of the absence of effects in the reproduction/developmental screening test compared to the 28 -day repeated dose toxicity test, the NOAEL observed in the 28 -day repeated dose toxicity study will be taken forward to the DNEL derivation.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The repeated dose toxicity study according to OECD TG 407 has been selected because the methodology and the result are adequately fulfilling this endpoint.

Justification for classification or non-classification

Based on the observed effects in the oral repeated dose toxicity study, the test material does not need to be classified according to Directive 67/548/EEC and EU Classification (DSD), Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.