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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1999-08-16 to 1999-12-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A GLP compliant study performed in line with standardised guidelines (OCED 407 and EU method B.7) with a sufficient level of detail to assess the quality of the presented data. The test material in this study is the aldehyde of 3-methyl-5-phenylpentanol and as such is considered similar enough to be representative of the sub-acute oral toxicity and to allow read-across to address the endpoint.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
55066-49-4
EC Number:
611-227-5
Cas Number:
55066-49-4
IUPAC Name:
55066-49-4
Constituent 2
Reference substance name:
-
EC Number:
433-900-0
EC Name:
-
IUPAC Name:
433-900-0
Constituent 3
Reference substance name:
3-methyl-5-phenylpentan-1-al
IUPAC Name:
3-methyl-5-phenylpentan-1-al
Test material form:
other: liquid (not specified)
Details on test material:
- Physical state: liquid, colourless
- Storage condition of test material: room temperature, in the dark until 20 August 1999, thereafter room temperature, in the dark under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Five to seven weeks old
- Weight at study initiation: 141 to 182 g for males and 128 to 152 g for females
- Housing: Groups of five, by sex in polypropylene grid-floor cages
- Diet: Rat and Mouse SQC, Expanded Diet No. 1, Special Diet Services Limited, Witham, Essex, UK ad libitum
- Water: Mains water ad libitum supplied in polycarbonate bottles
- Acclimation period: Eight days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was prepared based on the most recent bodyweight and was adjusted at weekly intervals. Control animals were dosed with 2 mL/kg of Arachis oil BP only.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test material formulation were analysed for the concentration of the test substance. The results indicated that the formulations were within ± 6 % of the nominal concentration.

In brief, the concentration of the test substance was determined using high performance liquid chromatography (HPLC) using an external standard technique.
The test material formulations were extracted with acetonitrile to give a final theoretical concentration of 0.1 mg/mL (approx.). The analysis was run under the following conditions:
- HPLC system: Hewlett-Packard 1050, incorporating autosampler and workstation
- Column: Luna 5 µ C18 (250 x 4.6 mm i.d.)
- Mobile phase: acetonitrile:water (75:25 v/v)
- Flow rate: 1.0 mL/min
- UV detector
- Wavelength: 220 nm
- Injection volume: 25 µL
Retention time: ~ 7 minutes

The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom in triplicate. The detector response was shown to be linear up to 0.15 mg/mL. Analysis of the solvent and a blank Arachis oil BP (control) produced no signal that interfered with the signal when assessing specificity. The analytical method was considered to be sufficiently accurate for the purpose of the study. The test samples were not corrected for recovery.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 15, 150 and 500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 7.5, 75 and 250 mg/mL
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the fourteen day range finder study.

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed for overt signs of toxicity, ill-health, behavioural abnormalities immediately before, one and five hours after dosing during the working week. Observations were performed immediately before and one hour after dosing at weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded on days 0, 7, 14, 21 and 28, and also at terminal kill.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/week: Yes. Food consumption was recorded for each cage group at weekly intervals.

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 (if drinking water study): Yes
- Time schedule for examinations: Daily visual inspections of water bottles during week 2 revealed possible intergroup differences. Water consumption was measured from each cage group from day 15 onwards (excluding day 25 for 500 mg/kg bw/day males due to a technical error).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 from the lateral tail vein (where necessary repeat samples were obtained by cardiac puncture at termination)
- Animals fasted: No
- How many animals: All animals from each group
- Parameters checked:
Haemoglobin (Hb); Erythrocyte count (RBC); Haematocrit (Hct); Total leucocyte count (WBC); Platelet count (PLT);
Erythrocyte indices: mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC);
Differential leucocyte count: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos) and basophils (Bas);
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: Day 28 from the lateral tail vein (where necessary repeat samples were obtained by cardiac puncture at termination)
- Animals fasted: No
- How many animals: All animals from each group
- Parameters checked:
Urea; Glucose; Total protein (Tot.Prot.); Albumin; Albumin/Globulin (A/G) ratio by calculation; Sodium (Na+); Potassium (K+); Chloride (Cl-); Calcium (Ca++); Inorganic phosphorous (P); Aspartate aminotransferase (ASAT); Alanine aminotransferase (ALAT); Alkaline phosphatase (AP); Creatinine (Creat); Total cholesterol (Chol); Total bilirubin (Bili)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observations were performed prior to treatment and on days 6, 13, 21 and 26. On day 26 functional observations were also performed with a sensory assessment. Observations were performed approximately two hours post dosing on each occasion.
- Dose groups that were examined: All animals.

- Battery of functions tested:
Behavioral assessment:
Gait; Tremors; Twitches; Convulsions; Bizzare/abnormal/stereotypic behaviour; Salivation; Pilo-erection; Exophthalmia; Lachrymation; Hyper/hypothermia; Skin colour; Respiration; Palpebral closure; Urination; Defecation; Transfer arousal; Tail elevation

Functional performance tests:
a) Motor activity (Using twenty, 44 infra-red beam automated activity monitors)
b) Forelimb/hindlimb grip strength (Using an automated grip strength meter)

Sensory reactivity:
Grasp response; Vocalisation; Toe pinch; Tail pinch; Finger approach; Touch escape; Pupil reflex; Startle reflex (measured with a ST1058 Startle Test Meter); Blink reflex
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Animals were sacrificed by an intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination.

ORGAN WEGHTS: Yes. Adrenals, kidneys, testes, brain, liver, thymus, epididymides, ovaries, heart and spleen

HISTOPATHOLOGY: Yes.
The following tissues were removed from all animals and preserved in buffered 10 % formalin: aorta (thoracic), bone and bone marrow (femur including stifle joint), eyes, muscle (skeletal), oesophagus, pancreas, pituitary, salivary glands (submaxillary) and skin (hind limb).

In addition to being preserved in buffered 10 % formalin the following tissues from all control and 500 mg/kg bw/day animals were prepared as paraffin blocks section at 5 µg and stained with haemotoxylin and eosin for microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 15 and 150 mg/kg bw/day dose group animals: adrenals, bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides, gross lesions, heart, ileum, jejenum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesentric), ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

Lungs were inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative.

As there were indications of treatment related liver and thyroid gland changes, examination was extended to include prepared sections of these tissues from all animals.
Statistics:
Data were processed where appropriate to give group mean values and standard deviations. Haematological, clinical chemistry, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships 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. Where Levene’s test showed unequal variances the data were analysed using non-parametric 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.

Probability values (p) were:
P < 0.001***
P < 0.01**
P < 0.05 *
P ≥ 0.05 (not significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths occurred during the study. Post dosing, increased salivation was noted in animals from the 500 and 150 mg/kg bw/day treatment groups from day 4. This was attributed to the unpleasant taste or locally irritant test substance formulation as it accompanied administration and not considered to be of toxicological importance. On day 8, animals treated with 500 mg/kg bw/day showed further clinical signs including fur loss, an isolated incident of hunched posture and prolonged increased salivation together with staining and wetting of the external body surface. These observations were not detected in the 15 mg/kg bw/day animals. Isolated incidents of fur staining or scab formation were detected in both a control and a 500 mg/kg bw/day female. These observations were not considered to be toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN
A slight reduction in bodyweight form males treated with 500 mg/kg bw/day during week 1 only was recorded; however, statistical significance was not reached. No effect in bodyweight gain was seen in females from this group or in animals of either sex from the other dose groups. Bodyweight gain for all female treatment groups was significantly reduced during week 2 of the study. However, these differences were attributed as a consequence of an elevated control group mean and were therefore deemed of no toxicological significance.

FOOD CONSUMPTION
A slight reduction in dietary intake was noted for animals of both sex at the 500 mg/kg bw/day dosing level during week 1. All other dose groups were found to be unaffected.

FOOD EFFICIENCY
A reduction in food efficiency for females from all treatment groups during week 2 was not attributed to treatment and was considered to be related to the elevated control group during this period.

WATER CONSUMPTION
An increase in water consumption for the 500 mg/kg bw/day animals during the final two weeks of treatment was recorded (when compared with controls). This was more severe in females. Animals from the 150 and 15 mg/kg bw/day groups were not affected.

HAEMATOLOGY
No treatment related effects were noted. Statistical analysis of haematological data did not indicate any significant intergroup differences when compared to controls.

CLINICAL CHEMISTRY
Animals of either sex treated with 500 mg/kg bw/day gave a statistically significant increase in plasma alkaline phosphatise and a reduction in cholesterol levels in comparison to controls. Females of this dosing level gave a statistically significant increase in aspartate aminotransferase and alanine aminotransferase levels. No treatment related changes were detected at 150 or 15 mg/kg bw/day. Males treated with 150 mg/kg bw/day had a statistically significant increase in plasma creatinine when compared with controls. However in the absence of a convincing dose response relationship, the increase was considered to be coincidental. Although a statistically significant reduction in urea was noted in all treated females, a reduction in this parameter was considered unlikely to be a toxicological effect.

NEUROBEHAVIOUR
No treatment related changes were noted at 150 and 15 mg/kg bw/day during behavioural assessments. At 500 mg/kg bw/day, increased salivation was observed in all animals and hunched posture was noted in one female. During the functional performance tests, there were no treatment related changes. Females treated with 500 mg/kg bw/day had a statistically significant reduction in percentage total mobile activity. However in the absence of any other evidence suggesting neurotoxicity or any clinical evidence of lethargy, this isolated and minimal (p<0.05) intergroup difference was considered to be coincidental and not toxicologically significant. No treatment related changes were recorded in the sensory reactivity assessments.

ORGAN WEIGHTS
At the 500 mg/kg bw/day dose level, an increase in liver weight both absolute and relative to terminal bodyweight was recorded (compared to controls), male absolute weight did not reach statistical significance. Females in this treatment groups also showed a statistically significant increase in kidney weight both absolute and relative to terminal bodyweight, this was also noted in the relative weights at 150 mg/kg bw/day. No treatment related organ weight changes were detected in animals treated with 15 mg/kg bw/day.

GROSS PATHOLOGY
No macroscopic abnormalities related to treatment were observed at termination. One male in the 500 mg/kg bw/day group had pallor of the kidneys at terminal kill. This was considered to be minor variability in the volume of blood removed during exsanguinations, this was not coupled with any histopathological evidence of a renal effect. This finding was considered to be incidental. On removal damage was sustained to the left eye of one control male.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the liver, centrilobular hepatocyte enlargement and an increased severity of glycogen type vacuolation of hepatocytes was observed in relation to treatment for male and for female rats at 500 mg/kg bw/day. These changes are considered to be adaptive as they are commonly associated with the rodent liver response to oral administration of xenobiotics. In the thyroid glands of females at 500 mg/kg bw/day, follicular cell hypertrophy and associated depletion of colloid was reported. This was not recorded at any other treatment level. In male rats, the group incidence of these conditions was highly variable for male rats and there was no indication of an effect of treatment. All remaining morphological changes recorded in these assessments were commonly associated with this age and strain of rats and laboratory conditions.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
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: Increased liver and kidney weight, and histopathogical changes in the liver.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test substance, when dosed up to 500 mg/kg bw/day via oral gavage in male and female rats for 28 days, resulted in treatment-related changes among animals of either sex treated with 500 mg/kg bw/day and females treated with 150 mg/kg bw/day. The “No Observed Effect Level” (NOEL) was therefore considered to be 150 mg/kg bw/day for males and 15 mg/kg bw/day for females. The increase in kidney weight seen in females dosed with 150 mg/kg bw/day was an isolated change and was not considered to represent and adverse health effect. Therefore the NOAEL was determined to be 150 mg/kg bw/day for either sex.
Executive summary:

A GLP compliant repeated dose, sub-acute toxicity study was conducted in accordance with the standardised guidelines OECD 407 and EU Method B.7. Groups of male and female Sprague Dawley rats were dosed with the test substance at 0, 15, 150 and 500 mg/kg bw/day via oral gavage for 28 consecutive days. The animals were assessed for signs of toxicity during the dosing period, and were examined for any gross or histopathological changes at study termination. Treatment-related changes were observed at 500 mg/kg bw/day (males and females) and 150 mg/kg bw/day (females only). The “No Observed Effect Level” (NOEL) was therefore considered to be 150 mg/kg bw/day for males and 15 mg/kg bw/day for females. The NOAEL was determined to be 150 mg/kg bw/day for either sex.