Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 January to 25 April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD test guideline No. 407 and in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (Inspected on 2007-08-21)
Limit test:
no

Test material

Constituent 1
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): ST 22 C 07
- Substance type: pure active substance
- Physical state: clear colourless liquid
- Storage condition of test material: Approximately 4 °C under nitrogen in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Sprague-Dawley Crl:CD®(SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent
- Age at study initiation: ca. five to seven weeks old.
- Weight at study initiation: males 151 to 184g, females 129 to 162g
- Fasting period before study: animal were not fasted before blood sampling
- Housing: Groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels or suitable alternatives.
- Diet (e.g. ad libitum): ad libitum (pelleted diet Rodent 5LF2 (Certified) Diet, BCM IPS Limited, London, UK), routinely analysed
- Water (e.g. ad libitum): ad libitum (tap water, routinely analysed)
- Acclimation period: 8 days (during which time their health status was assessed).

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

IN-LIFE DATES: From: 10 January 2008 To: 20 March 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.
Formulations (showed to be stable for at least 14 days) were prepared weekly and stored at +4°C in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not sufficiently soluble in water
- Concentration in vehicle: 0, 3.75, 37.5 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity results show the formulations to be stable for at least 14 days. Formulations were therefore prepared weekly and stored at 4°C in the dark.
The concentration of ST 22 C 07 in the test material formulations was determined by gas chromatography (GC) using an external standard technique. Actual concentrations measured in test material formulations were between 101 and 107 % of nominal dosage, except on one occasion when the lowest dose was 19% above the nominal concentrations. The mean low dose level administered throughout the study was 9% above nominal concentrations, therefore the results indicate that the prepared formulations were within acceptable limits (± 10%) for the purpose of this study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily, 7 days each week
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 150 & 1000 mg/kg bw/day (m/f)
Basis:
other: nominal in vehicle (Arachis oil)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A range-finder (range finding study) was performed to establish the maximum tolerated dose level (up to 1000 mg/kg bw/day) of the test material following repeated oral administration to rats of the same strain and source, and to provide information for selection of dose levels for use in the 28 day oral toxicity phase. Three groups, each of six rats (three males and three females) were dosed with 0, 500 & 1000 mg/kg bw of ST 22 C 07. The test material was administered daily, for 14 consecutive days, by gavage. Control animals were treated in an identical manner with Arachis oil BP. The observations preformed were the following:
1. No death during the 14 days
2. Clinical observations: increased salivation ifor either sex especially with 1000 mg/kg bw/day
3. Bodyweight: slight increase in bodyweight gain for male with 500 mg/kg bw/day and clear increase for both sexes with 1000 mg/kg bw/day
4. Water consumption: increase for both sexes with 1000 mg/kg bw/day
5. Necropsy: One male treated with 1000 mg/kg bw/day displayed gaseous distension of the small intestines and sloughing of the gastric glandular mucosa. One male treated with 500 mg/kg bw/day displayed hydronephrosis of the right kidney and one female treated with 500 mg/kg bw/day showed sloughing of the glandular region of the stomach. One control female also displayed sloughing of the gastric glandular mucosa.

In function of these observations, the following doses were chosen for the 28 day study: 15, 150 & 1000 mg/kg bw/day corresponding to low, intermediate & high dose, respectively (plus a control group treated with vehicle alone)

- Rationale for animal assignment (if not random): Not Applicable
- Rationale for selecting satellite groups: Not performed
- Post-exposure recovery period in satellite groups: Not performed
- Section schedule rationale (if not random): Not Applicable
Positive control:
None

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule for examination: twice daily, early and late during the working period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, immediately before dosing, immediately 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to start of dosing) and at weekly intervals thereafter. Individual bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): NOT A FEEDING STUDY
(Food consumption was recorded for each cage group at weekly intervals throughout the study)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): NOT A DRINKING WATER STUDY
(Water intake was measured and recorded daily for each cages)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (Day 28). Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Animals fasted: No
- How many animals: all
- Parameters checked in table 7.5.1/1.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed individual clinical observations were performed for each animal using a purpose built arena. Prior to the start of treatment and on Days 5, 12, 19 and 26, all animals were observed for signs of functional/behavioural toxicity.
Behavioural assessments: The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormall Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.
Functional Performance tests: sensory reactivity (auditory, visual, proprioceptive stimuli), forelimb/hindlimb grip strength, motor activity



Sacrifice and pathology:
GROSS PATHOLOGY: Yes (See Table 7.5.1/2)
HISTOPATHOLOGY: Yes (See Table 7.5.1/2)
Other examinations:
None
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
All data was summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The 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 showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.0l **
p < 0.05 *
P > 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Increased salivation linked to the unpleasant tasting and/or locally irritant test material formulation
Mortality:
no mortality observed
Description (incidence):
Increased salivation linked to the unpleasant tasting and/or locally irritant test material formulation
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Decreased body weight gain of males treated with 1000 mg/kg bw/day
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Slight increase in animals treated with 1000 mg/kg bw/day
Food efficiency:
no effects observed
Description (incidence and severity):
Reduced in animals treated with 1000 mg/kg bw/day
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Increased water consumption linked to the unpleasant tasting and/or locally irritant test material formulation
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Slight increase in plasma calcium and reduction in inorganic phosphate in males treated with 1000 mg/kg bw/day
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Adaptive change (increased liver weights)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Adaptive change (centrilobular hepatocyte enlargement) or non-adverse change (spleen)
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths.
1000 mg/kg bw/day: Increased salivation was evident soon after dosing for males from Day I and for 1000 mg/kg bw/day females from Day 2 until the end of the treatment period. Increased salivation was also evident one hour after dosing during the treatment period for animals of either sex and females were observed with increased salivation, 5 hours after dosing on occasion. Associated incidents of wet fur and staining around the mouth were also reported.
150 mg/kg bw/day: evident soon after dosing for individual males during the treatment period and one female on Day 4.
15/mg/kg bw/day: no clinically observable signs of toxicity were detected for animals of either sex.

BODY WEIGHT AND WEIGHT GAIN
1000 mg/kg/day: Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in bodyweight gain when compared to controls during Week 1 (p<0.01). Reduced bodyweight gains were also evident during the remainder of the treatment period for these animals; however statistical significance was not achieved.
No adverse effect on bodyweight change was detected for 1000 mg/kg bw/day females or for animals of either sex treated with 150 or 15 mg/kg bw/day.

FOOD CONSUMPTION AND FOOD EFFICIENCY
Females treated with 1000 mg/kg bw/day showed a slight increase in dietary intake when compared to controls throughout the treatment period. Males treated with 1000 mg/kg bw/day showed an increase in dietary intake from Week 2 onward. Food efficiency (the ratio of bodyweight to dietary intake) was reduced for males throughout the treatment period and for females treated with 1000 mg/kg bw/day from Week 2 onwards when compared to control values. No adverse effect on dietary intake or food efficiency was detected for animals of either sex treated with 150 or 15 mg/kg bw/day.

WATER CONSUMPTION
A 22% increase in water intake was noted for males and a 67% increase in water intake was evident for females treated with 1000 mg/kg bw/day throughout the treatment period when compared to controls.
No overt inter-group differences were detected for animals of either sex treated with 150 or 15 mg/kg bw/day.

HAEMATOLOGY
No toxicologically significant effects were detected.

CLINICAL CHEMISTRY
Males treated with 1000 mg/kg bw/day displayed a statistically significant increase in blood calcium (p<0.05) and a statistically significant reduction in inorganic phosphate (p<0.01). No such effects were evident for females treated with 1000 mg/kg bw/day or for animals of either sex treated with 150 or 15 mg/kg bw/day.
Males from all treatment groups showed a statistically significant increase in plasma creatinine levels when compared to controls. The significance achieved was minimal (p<0.05) and in the absence of any supporting data to suggest an effect of treatment, these increases were considered to have arisen incidentally.
Females treated with 150 mg/kg bw/day showed a statistically significant increase in plasma albumin levels when compared to controls. All individual values were within the normally expected ranges for animals of the age and strain employed. In the absence of a dose-related response, this minimal increase was considered to have arisen accidentally and was considered to be unrelated to treatment.

ORGAN WEIGHTS
Animals of either sex treated with 1000 mg/kg bw/day showed statistically significant increases in liver weights, both absolute and relative to terminal bodyweights, when compared to controls (p<0.01). Increased liver weights were also observed for males treated with 150 mg/kg bw/day in comparison to control values (p<0.01).
No treatment-related changes in organ weights were detected for females treated with 150 mg/kg bw/day or for animals of either sex treated with 15 mg/kg bw/day.
Females treated with 1000 mg/kg bw/day showed a statistically significant increase in kidney weights, both absolute and relative to terminal bodyweight. The significance achieved was minimal (p<0.05) and review of the individual data showed one lower than expected control value. This increase was therefore considered to have arisen incidentally.
Statistically significant reductions in absolute and relative thymus weights were detected for males from all treatment groups when compared to controls (p<0.05 - p<0.01I). There were no histopathological correlates or a convincing dose-related response detected. Review of the individual data revealed two higher than expected control values for absolute thymus weights and as such, these reductions were considered to be unrelated to treatment.

GROSS PATHOLOGY
Sloughing of the stomach was evident for one female treated with 1000 mg/kg bw/day.
No other macroscopic abnormalities were detected

HISTOPATHOLOGY:
LIVER: Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 1000 mg/kg/day and for males treated with 150 mg/kg/day.
SPLEEN: A lower incidence of higher grades of severity of extramedullary haemopoiesis was seen in relation to treatment for males treated with 1000 mg/kg/day but not at any other dose level.

NEUROBEHAVIOUR
Weekly open-field arena observations revealed increased salivation for one male and one female treated with 1000 mg/kg bw/day and one male treated with 150 mg/kg bw/day during the final week of treatment. No treatment-related effects were detected for females treated with 150 mg/kg bw/day or for animals of either sex treated with 15 mg/kg bw/day.
There were no treatment-related changes in the functional performance parameters measured.
There were no treatment-related changes in sensory reactivity.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The effects detected at 1000 and 150 mg/kg bw/day in this study, in the absence of any degenerative changes, were considered adaptive in nature and not to represent serious damage to health. Therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg bw/day.
Executive summary:

In a sub-acute study performed according to the OECD test guideline No. 407 and in compliance with GLP, the test material diluted in Arachis oil BP was administered by oral route to Sprague-Dawley Crl:CD®(SD)IGS BR strain rats (5/sex/group) at 15, 150 or 1000 mg/kg bw/day for four weeks. A control group received the vehicle alone at the same treatment volume (4 mL/kg bw/day).

Clinical signs, functional observations, bodyweight development and food 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.

 

There were no unscheduled deaths. Clinical signs were confined to increased salivation noted after dosing for animals of either sex treated with 1000 mg/kg bw/day, and to a lesser degree, for animals treated with 150 mg/kg bw/day. Associated signs of wet fur and staining around the mouth were also detected at the highest dose levels. Increased salivation was also noted during the final week of the open-field arena observations for two animals treated with 1000 mg/kg bw/day and one male treated with 150 mg/kg bw/day. These observations are often recorded following the oral administration of an unpleasant tasting and/or locally irritant test material formulation. Supporting evidence of irritancy was obtained from daily measurements of water bottles, which revealed an increase in water consumption for high dose animals when compared to controls. Furthermore, post-mortem findings revealed sloughing of the gastric glandular mucosa, although this was confined to only one animal treated at the high dose. In the absence of any histopathological gastric changes, the irritancy effect of the test material was considered to be minimal.

Males treated with 1000 mg/kg bw/day showed lower bodyweight gains when compared to controls although dietary intake was increased for either sex treated with 1000 mg/kg bw/day. This resulted in reduced food utilization at this dose level. Slight blood chemical changes were also evident for males treated with 1000 mg/kg bw/day, although these were confined to an increase in plasma calcium and an associated reduction in inorganic phosphate. Organ weight data revealed increases in absolute and relative organ weights for animals of either sex treated with 1000 mg/kg/day and also for males treated with 150 mg/kg bw/day. Furthermore, histopathological examinations of the liver revealed centrilobular hepatocyte enlargement in these animals. This observation is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any associated inflammatory or degenerative changes, is generally considered adaptive in nature.

Finally, Microscopic examination of the spleen revealed a lower incidence of higher grades of severity of extramedullary haemopoiesis for males treated with the highest dose level, albeit marginal and considered to be non-adverse.

The effects detected at 1000 and 150 mg/kg bw/day in this study, in the absence of any degenerative changes, were considered adaptive in nature and not to represent serious damage to health. Therefore the 'No Observed Adverse Effect Level' (NOAEL) was considered to be 1000 mg/kg bw/day. Under the test conditions, the test material does not need to be classified for damage to organs through prolonged oral dose repeated exposure according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and of the Directive 67/548/EEC.

This sub-acute toxicity study is acceptable and satisfies the requirement for a sub-acute oral study in rats.