(4E)-4-methyl-5-(4-methylphenyl)pent-4-enal

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November to 11 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 407 without any deviation.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September to 09 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 404 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 24 April 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 10, 2012/ signed on November 30, 2012)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK.
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.20-2.65 kg
- Housing: Animals were individually housed in suspended cages.
- Diet: Food (2930C Teklad Global Certified Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Humidity: 30-70 %
- Air changes: 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 25 September to 09 October 2012

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration: For the purpose of the study the test item was used as supplied.

Duration of treatment / exposure:

4 h

Observation period:

1, 24, 48 & 72 h and 7 and 14 days after the removal of the patch

Number of animals:

3 males

Details on study design:

PRETRAEATMENT
- On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

TEST SITE
- Area of exposure: On the day of the test a suitable test site was selected on the back of each rabbit.
- Type of wrap if used: A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
- Immediately following removal of the patches and approximately 1, 24, 48 & 72 h and 7 and 14 days after the removal of the patch

SCORING SYSTEM:
- Method of calculation: Test sites were examined for evidence of primary irritation and scored according to the Draize scale, as described in OECD Guideline No. 404

OTHERS:
- Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Remarks:

due to the persistence of the reaction

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

probability of mild irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Remarks:

due to the persistence of the reaction

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

- Very slight erythema and very slight or slight oedema was noted at all treated skin sites immediately after patch removal. Very slight erythema with or without very slight oedema was noted at all treated skin sites one hour after patch removal. Well-defined erythema and very slight or slight oedema was noted at all treated skin sites at the 24 hour observation with well-defined erythema and slight oedema noted at the 48 and 72 hour observations. Very slight erythema and very slight oedema was noted at one treated skin site at the 7-Day observation.
- Loss of skin elasticity was noted at all treated skin sites at the 48 and 72 hour observations. Moderate desquamation was noted at all treated skin sites at the 7-Day observation. Small superficial scattered scabs and hardened light brown coloured scab were noted at two treated skin sites at the 7-Day observation. Scab lifting to reveal glossy skin was also noted at one treated skin site at the 7-Day observation. Adverse reactions prevented evaluation of erythema at one treated skin site at the 7-Day observation. Glossy skin was noted at two treated skin sites at the 14-Day observation.
- One treated skin site appeared normal at the 14-Day observation.

Other effects:

All animals showed expected gain in bodyweight during the study.

Table 7.3.1/1: Mean irritant/corrosive response data for each animal at each observation time

Score at time point	Erythema (Animal no 1 / 2 / 3) Max. score 4	Oedema (Animal no 1 / 2 / 3) Max. score 4
Immediately	1 / 1 / 1	2 / 1 / 2
1 h	1 / 1 / 1	1 / 0/ 1
24 h	2 / 2 / 2	2 / 1 / 2
48 h	2 Le / 2 Le / 2 Le	2 / 2 / 2
72 h	2 Le / 2 Le / 2 Le	2 / 2 / 2
7 days	1SsSpD / 0 D /?eSsSpSgD	1 / 0 / 0
14 days	0G / 0 /0G	0 / 0 / 0
Mean 24, 48 and 72 h	2.0 / 2.0 / 2.0	2.0 / 1.7 / 2.0

Le = Loss of skin elasticity

Ss = Small superficial scattered scabs

Sp = Hardened light brown coloured scab

Sg = Scab lifting to reveal glossy skin

D = Moderate desquamation

G = Glossy skin

?e = Adverse reactions prevent evaluation of erythema

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions of this study, the test item is classified as irritant (Category 2) according to Regulation (EC) No 1272/2008 (CLP) and to GHS based on the persistence of inflammation until the end of the 14 -day observation period in 2 out of 3 animals.

Executive summary:

In a dermal irritation study performed according to the OECD Guideline No. 404, and in compliance with GLP, 0.5 mL of test item was applied on the clipped skin of the dorsal/flank area (back) of 3 male New Zealand White rabbits. Test sites were covered with a semi-occlusive dressing for 4 h. Skin irritation was assessed and scored according to the Draize scale at 1, 24, 48, 72 h and 7 and 14 days after the removal of the patch.

Very slight erythema and very slight or slight oedema was noted at all treated skin sites immediately after patch removal. Very slight erythema with or without very slight oedema was noted at all treated skin sites one hour after patch removal. Well-defined erythema and very slight or slight oedema was noted at all treated skin sites at the 24 hour observation with well-defined erythema and slight oedema noted at the 48 and 72 hour observations. Very slight erythema and very slight oedema was noted at one treated skin site at the 7-Day observation. Loss of skin elasticity was noted at all treated skin sites at the 48 and 72 hour observations. Moderate desquamation was noted at all treated skin sites at the 7-Day observation. Small superficial scattered scabs and hardened light brown coloured scab were noted at two treated skin sites at the 7-Day observation. Scab lifting to reveal glossy skin was also noted at one treated skin site at the 7-Day observation. Adverse reactions prevented evaluation of erythema at one treated skin site at the 7-Day observation. Glossy skin was noted at two treated skin sites at the 14-Day observation. One treated skin site appeared normal at the 14-Day observation.

The individual scores for each animal within 3 scoring times (24, 48 and 72 h) were 2.0 / 2.0 / 2.0 for erythema and 2.0 / 1.7 / 2.0 for oedema.

Under the experimental conditions of this study, the test item is classified as irritant (Category 2) according to Regulation (EC) No 1272/2008 (CLP) and to GHS based on the persistence of inflammation until the end of the 14 -day observation period in 2 out of 3 animals.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 3 October 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 10, 2012 / signed on November 30, 2012)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-4-methyl-5-(4-methylphenyl)pent-4-enal

Cas Number:

1226911-69-8

Molecular formula:

C13H16O

IUPAC Name:

(4E)-4-methyl-5-(4-methylphenyl)pent-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Pale yellow liquid
- Storage condition of test material: Approximately 4 °C in the dark under nitrogen

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST strain

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK.
- Age at study initiation: ca. 6-8 weeks
- Weight at study initiation: Males: 160-187 g, females: 138-163 g
- Housing: Animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet: Pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories UK Limited, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 ºC
- Humidity: 30-70 %
- Air changes: 15 changes/ h
- Photoperiod: 12 h dark/ 12 h light

IN-LIFE DATES: 13 November to 11 December 2012

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.
The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly during the treatment period and stored at approximately 4 ºC in the dark under nitrogen.

VEHICLE
- Concentration in vehicle: 7.5, 75 and 187.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day

DOSE VOLUME: 4 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of ST 01 C 11. The concentration of ST 01 C 11 in the test item formulations was determined by gas chromatography (GC) using an external standard technique.The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the 7 day range finding study, the test item was administered by gavage to three groups, each of three male and three female Wistar Han™:HsdRccHan™:WIST strain rats, for up to seven consecutive days, at dose levels
of 250, 500 and 1000/750 mg/kg/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.Two males treated with 1000 mg/kg bw/day died in this study and there was some early indication (bodyweight) that the remaining animals from this test group were also affected by treatment. Overall the evidence from treatment at 1000 mg/kg bw/day indicated that the test item was toxic at this dosage. In addition the test item was shown to be slightly irritant producing gastric inflammation and sloughing of the stomach lining in both decedents and weight losses, increased salivation and high water consumption in the 1000 mg/kg bw/day females. However, following the dose level reduction to 750 mg/kg bw/day there was no further deterioration in the condition of the surviving animals from this treatment group. Animals treated at 250 and 500 mg/kg showed dose tolerance.
For these reasons 1000 mg/kg bw/day is considered to be slightly too high for use in longer term exposure studies. However, 750 mg/kg bw/day was indicated to be tolerated and therefore shown to be suitable for use in 41200604.
Based on the above considerations the following dose levels are recommend for use in the 28 Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat (Project No. 41200604): High : 750 mg/kg bw/day; Intermediate dose : 300 mg/kg bw/day;
Low dose : 30 mg/kg bw/day; Control : Vehicle only (Arachis Oil BP)
- Rationale for animal assignment: Animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

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

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

FOOD CONSUMPTION AND FOOD EFFICIENCY: Yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was performed daily by visual inspection of water bottles with the exception of Week 3 when intake was measured gravimetrically. Water intake was measured and recorded daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No; animals were not fasted prior to sampling
Parameters checked:
- Haematology: Haemoglobin, erythrocyte count, haematocrit, erythrocyte indices (mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, reticulocyte count, prothrombin time and Activated partial thromboplastin.
- Clinical chemistry: Urea, glucose, total protein, albumin, albumin/Globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed for signs of functional/behavioural toxicity. Functional performance 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.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
Adrenals, brain, epididymides, heart, kidneys, prostate and seminal vesicles (with coagulating glands and fluid), spleen, testes, thymus, liver, thyroid/parathyroid (post-fix), ovaries, uterus, cervix and pituitary (post-fixation) were removed from animals that were killed at the end of the study and were dissected free from fat and weighed before fixation.

HISTOPATHOLOGY: Yes; Samples of the tissues mentioned in Table 7.5.1/1 were removed from all animals and preserved in buffered 10 % formalin for microscopic analysis.

Other examinations:

Thyroid Hormone Assessment: At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20 °C. No treatment-relatedeffects on the pituitary-thyroid axis were identified.

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values were appropriate. All data were summarised in tabular form.
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).
Probability values (p) are presented as follows:
p<0.01 **; p<0.05 *; p≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- Treatment-related findings were confined to sporadic transient episodes of increased salivation observed in both sexes at 750 mg/kg bw/day from Day 4 and from Day 8 in animals at 300 mg/kg bw/day. This observation was evident around the time of dosing persisting through to the final week of treatment. Such observations are commonly observed following gavage administration of a test item formulation, and in the absence of associated evidence of toxicity they are considered to be related to the oral administration of an unpleasant tasting or slightly irritant formulation rather than systemic toxicity.
- No clinical signs were observed in control or 30 mg/kg bw/day animals.
CONCLUSION: No adverse effects

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no toxicologically significant adverse effects on body weight development for treated animals when compared to controls. However, body weight development in 750 mg/kg bw/day males was slightly below that of the concurrent controls throughout the treatment period with a similar trend noted at Week 3 and 4 for females from this test group.
- The body weight of 30 and 300 mg/kg bw/day animals remained similar to that of the controls throughout the treatment period.
CONCLUSION: No adverse effects

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related adverse effect on food consumption was detected in any of the test groups in comparison with controls.

Food efficiency:

no effects observed

Description (incidence and severity):

No treatment-related adverse effect on food efficiency was detected in any of the test groups in comparison with controls.

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

effects observed, non-treatment-related

Description (incidence and severity):

- Gravimetric measurement of water intake performed during the third week of treatment revealed males from the 300 and 750 mg/kg bw/day test groups to have been consuming slightly more water than the male controls. A similar response was not evident in the female test groups. Probable compensatory increase in water consumption due to formulation palatability issues.
bw/day.
- Males treated at 30 mg/kg bw/day also showed a slight increase in consumption, but this was unconvincing and more likely attributable to biological variability than associated with exposure to the test item.
CONCLUSION: No adverse effects

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- There were no toxicologically significant changes detected in the haematological parameters examined in this study.
- Incidental findings involved a slight but statistically significant reduction (p<0.05) for mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin
concentration in both sexes treated at 750 mg/kg bw/day in comparison with group mean control values. A reduction (p<0.05) for MCH was also evident for males that received 300 mg/kg bw/day. However, almost all individual values were within the historical ranges and as there was no supporting evidence from the haematological profile to suggest a treatment-related effect these findings were considered to be of no toxicological significance.
- In addition, females treated at 30 and 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in lymphocyte count in comparison with female controls. In the absence of a dose dependant trend this finding was considered to be attributable to biological variability and not to be associated with test item toxicity.
CONCLUSION: No adverse effects

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- There were no toxicologically significant changes detected in the blood chemical profile.
- A slight increase (p<0.05) in sodium levels was identified in high dose (750 mg/kg bw/day) males and an elevation (p<0.05) for plasma phosphorus concentration in females from this test group. The divergence from control was marginal, however, in view there was histopathological evidence of impaired renal function a relationship to treatment for these disruptions in electrolyte levels cannot be wholly discounted.
- All male test groups showed an increase (p<0.05 or p<0.01) in plasma bilirubin levels compared to controls. In addition, males treated with 750 mg/kg bw/day had an elevated albumin/globulin ratio. With few exceptions individual values were within normal ranges for animals of strain and age used and in the absence of histopathological evidence of liver changes these intergroup differences were considered to be more likely attributable to biological variability than associated with test item toxicity.
- Males treated with 30 mg/kg bw/day showed an increase for plasma chloride levels in comparison with controls. The level of significance achieved was minimal (p < 0.05) and as a dose-response relationship could not be established this finding was considered to be fortuitous and not related to test item toxicity.
CONCLUSION: No adverse effects

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- Behavioural Assessments: There were no treatment-related changes in the behavioural parameters measured. All inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and therefore considered to be of no toxicological importance.

- Functional Performance Tests: There were no treatment-related changes in motor activity or grip strength measurements for treated animals when compared to controls. Incidental findings involved a statistically significant decrease (p<0.05) in one of the three independent hind limb tests in all test groups in comparison with controls. However, in the absence of a dose dependent trend or any supporting evidence of neurotoxicity this isolated change was considered attributable to biological variability and therefore to have no association with test item toxicity.

- Sensory Reactivity Assessments: 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 the differences were of no toxicological importance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Males treated with 750 mg/kg bw/day showed increased kidney (p<0.05) and liver (p<0.01) weights (absolute and relative to terminal body weight) when compared to controls.
- In addition, a reduction (p<0.05 or p<0.01) in pituitary weights was identified in males from the 300 and 750 mg/kg bw/day dose groups while females from the 30 mg/kg bw/day test group showed an increase in spleen weight when compared to that of the controls. For all but one female individual litter values were within the historical ranges for these parameter, and in the absence of any supporting histopathological correlates, these finding were considered not to be of toxicological importance.
- Females treated at 750 mg/kg bw/day showed a slight but statistically significant (p<0.05) increase for ovary weights. However, as the individual values for the majority of females were within the anticipated historical ranges and only two values were marginally above the expected range; this finding was considered to have arisen fortuitously and therefore to be of no toxicological consequence.
CONCLUSION: No adverse effects

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic abnormalities were identified in any animal at the terminal necropsy.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Treatment-related findings were identified in the kidneys of both sexes treated at 300 and 750 mg/kg bw/day. These findings were characterised by minimal or mild basophilic epithelium in the collecting tubules of four males and one female at 300 mg/kg bw/day and in all the males and four females at 750 mg/kg bw/day. In the more severely affected animals there was associated single cell necrosis and/or increased mitoses.
- No treatment-related changes were seen in animals given 30 mg/kg bw/day.
CONCLUSION: adverse effects

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Oral administration of ST 01 C 11 to rats for a period of twenty-eight consecutive days at dose levels of 30, 300 and 750 mg/kg bw/day resulted in treatment-related findings in either sex of 300 and 750 mg/kg bw/day test animals.

Clinical findings were confined to transient episodes of increased salivation which developed during the first week of treatment in animals treated at 300 and 750 mg/kg bw/day and persisted (sporadically) through to Day 28 (the final day of treatment). Such observations are commonly observed following gavage administration of a test item formulation, and in the absence of associated evidence of toxicity they are considered to be related to the oral administration of an unpleasant tasting or slightly irritant formulation rather than systemic toxicity. A local irritation study (Harlan Laboratories Study No. 41200600) confirmed the test item to cause an irritant dermal response.

Further indication of formulation palatability issues identified was a probable compensatory increase in water consumption identified in the males tested at 750 mg/kg bw/day. Such findings can give rise to a form of dehydration which may cause associated slightly impaired body weight and minor haematological blood changes (haemo-concentration) similar to those seen in males at 750 mg/kg bw/day. However, it is reasonable to assume in the absence of any convincing affect on dietary intake, food utilisation and absence of supporting signs of systemic toxicity that these treatmentrelated changes were of minor importance. Perhaps the most noteworthy findings were associated with the organ weights, in that males treated with 750 mg/kg bw/day showed elevated liver and kidney weights. The individual liver weights were generally above the anticipated historical ranges and although there was no supporting microscopic findings detected to suggest treatment-related hepatic changes a relationship with treatment should not on this occasion be wholly discounted. In relation to the kidneys, while individual values in these animals fell within the historical ranges; histopathological findings identified renal lesions in the collecting tubules of both sexes treated at 300 and 750 mg/kg bw/day with severities ranging between minimal and mild although there was one instance of tubular necrosis (one male at 750 mg/kg bw/day). It is possible that the necrosis identified in one high dose male may be a result of biological variability however, in view this was a degenerative tissue change it cannot be wholly excluded from animals from this test group. However, in view that a number of animals showed mitotic changes which are indicative of renewal and repair it is possible certainly at 300 mg/kg bw/day that the renal changes may have been an adaptive response to a slightly irritant test item. For the reasons previously expressed a No Observed Adverse Effect Level (NOAEL) could not be established for animals treated at 750 mg/kg bw/day. However, given the frequency and severity of renal findings identified in either sex that received 300 mg/kg bw/day; this dose level can be considered under the conditions of this study to be the NOAEL. The absence of convincing evidence of treatment-related findings in either sex at 30 mg/kg bw/day would indicate this dose level to be the 'No Observed Effect Level'.

Applicant's summary and conclusion

Conclusions:

The oral administration of ST 01 C 11 to rats by gavage for a period of twenty-eight consecutive days, at dose levels of 30, 300 and 750 mg/kg bw/day resulted in microscopic renal changes in either sex of animals treated at 300 and 750 mg/kg bw/day. There were no convincing changes detected in either sex treated at 30 mg/kg bw/day. Based on results from this study, the NOAEL in males and females is considered to be 300 mg/kg bw/day. The NOEL in either sex was indicated to be 30 mg/kg bw/day.

Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 750 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP).

 

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Clinical findings were confined to transient episodes of increased salivation which developed during the first week of treatment in animals treated at 300 and 750 mg/kg bw/day and persisted (sporadically) through to Day 28 (the final day of treatment). Such observations are commonly observed following gavage administration of a test item formulation, and in the absence of associated evidence of toxicity they are considered to be related to the oral administration of an unpleasant tasting or slightly irritant formulation rather than systemic toxicity. A local irritation study (Harlan Laboratories Study No. 41200600) confirmed the test item to cause an irritant dermal response.

Further indication of formulation palatability issues identified was a probable compensatory increase in water consumption identified in the males tested at 750 mg/kg bw/day. Such findings can give rise to a form of dehydration which may cause associated slightly impaired body weight and minor haematological blood changes (haemo-concentration) similar to those seen in males at 750 mg/kg bw/day. However, it is reasonable to assume in the absence of any convincing affect on dietary intake, food utilisation and absence of supporting signs of systemic toxicity that these treatmentrelated changes were of minor importance. Perhaps the most noteworthy findings were associated with the organ weights, in that males treated with 750 mg/kg bw/day showed elevated liver and kidney weights. The individual liver weights were generally above the anticipated historical ranges and although there was no supporting microscopic findings detected to suggest treatment-related hepatic changes a relationship with treatment should not on this occasion be wholly discounted. In relation to the kidneys, while individual values in these animals fell within the historical ranges; histopathological findings identified renal lesions in the collecting tubules of both sexes treated at 300 and 750 mg/kg bw/day with severities ranging between minimal and mild although there was one instance of tubular necrosis (one male at 750 mg/kg bw/day). It is possible that the necrosis identified in one high dose male may be a result of biological variability however, in view this was a degenerative tissue change it cannot be wholly excluded from animals from this test group. However, in view that a number of animals showed mitotic changes which are indicative of renewal and repair it is possible certainly at 300 mg/kg bw/day that the renal changes may have been an adaptive response to a slightly irritant test item. For the reasons previously expressed a No Observed Adverse Effect Level (NOAEL) could not be established for animals treated at 750 mg/kg bw/day. However, given the frequency and severity of renal findings identified in either sex that received 300 mg/kg bw/day; this dose level can be considered under the conditions of this study to be the NOAEL. The absence of convincing evidence of treatment-related findings in either sex at 30 mg/kg bw/day would indicate this dose level to be the 'No Observed Effect Level'.