[No public or meaningful name is available]

Endpoint summary

Description of key information

Under the conditions of an OECD 407 study, the test substance did not produce adverse effects, the No Adverse Effect Level (NOAEL) could be established at 1000 mg/kg/day.

The test substance did produce treatment related effects consisting of hepatocellular hypertrophy, thyroid activation (increased synthesis/ reabsorption phase) and tubular swelling of the kidney resembling osmonephrosis. These findings were not accompanied by increased incidence or severity of inflammatory or degenerative / regenerative lesions and were deemed to represent adaptive changes (hepatocellular hypertrophy, renal tubular changes) or secondary effects (thyroid activation due to increased hepatic turnover/metabolization of thyroid hormones). The histopathological changes in livers and kidneys correlated well with the organ weights and weight ratios recorded in the 4-week necropsy group and resolved after the recovery period.

 

In an OECD 422 study the oral administration of the test substance to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, was well tolerated.

The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

28 April 2015 to 01 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Official notice of MHLW, METI and MOE (31 March 2011) YAKUSHOKUHATSU 0331 No 7 SEIKYOKU No 5 KANPOKIHATSU No 110331009

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

With recovery groups

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST

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 system
For the purpose of this study the test item was prepared at the appropriate concentrations as a
suspension in Polyethylene glycol 400 (PEG 400). The stability and homogeneity of the test
item formulations were determined. Results show the formulations to be homogenously prepared and stable for at least fifteen days when stored at approximately 4 °C, in the dark. Formulations were therefore prepared approximately weekly, aliquoted and stored as above before use.

Environmental conditions
Male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. The animals were examined upon receipt for signs of ill-health or injury. The animals were acclimatized for seven days before the start of dosing during which time their health status was assessed. A total of one hundred and twenty six animals (fifty eight males and sixty eight nulliparous, non-pregnant females) were accepted into the study. At the start of treatment the males weighed 300 to 342g, the females weighed 175 to 219g, and were approximately twelve weeks old and weight variation did not exceed ±20% of the mean weight for either sex.

Initially, all main study animals were housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, main study animals were transferred to polypropylene grid floor cages suspended over polypropylene trays lined with absorbent paper on a one male:one female basis (where applicable) within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation, birth and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery animals and satellite females were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding throughout the study. Appropriate color coded cage cards were prepared with details of test item, study number, dose level, sex, number of animals, route of administration and Study Director responsible for the study.

The animals were allowed free access to food and water. A pelleted diet was used. Tap drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals during mating and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered once daily by gavage using a stainless steel cannula attached to a
graduated, disposable plastic syringe.

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test item formulation were taken and analyzed for concentration of the test item on five occasions. The results indicate that the prepared formulations were within 91 to 102% of the nominal concentration.

Duration of treatment / exposure:

Males were dosed for approximately six weeks (42 or 43 days) whilst females were
dosed for up to eight weeks including a two week pre-pairing phase, pairing, gestation and early
lactation.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Twelve males and twelve females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day. Males were dosed for approximately six weeks (42 or 43 days) whilst females were dosed for up to eight weeks including a two week pre-pairing phase, pairing, gestation and early lactation. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400). These animals were paired for mating and dosed until the day before necropsy and are referred to as main study animals. Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for 42 consecutive days and then maintained without treatment for a further 14 days. In addition, two satellite groups, each of five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for 42 consecutive days. All surviving animals were killed on the day following their last dose or in the case of the recovery groups, on the day following the 14-day recovery period.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of main study animals within each dose group was undertaken on a one male:one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. Female 68 treated with 300 mg/kg bw/day was found dead on Day 12 of dosing, and therefore, Male 56 from this dose group was not paired with any female.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected main study males from each dose group during the final week of dosing, and for five selected main study parental females from each dose group on Day 4 post partum. These evaluations were also performed on all satellite females during the final week of dosing and on all recovery animals during the final week of the treatment-free period. Urinalysis was performed on five main study males per dose group and all satellite females during the final week of treatment and on all recovery animals during the final week of the treatment-free period. Five main study males and females from each dose group were selected for hematology and blood chemistry assessments prior to termination. These investigations were also performed on all satellite females and recovery animals prior to termination.

Adult main study males were terminated on Day 43 or 44 relative to the start of dosing, followed by the termination of all surviving females and offspring on Day 5 post partum. Any main study female which did not produce a pregnancy was terminated on Day 26 post coitum. All satellite females were terminated on Day 43 whilst all recovery animals were terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Observations and examinations performed and frequency:

All animals were examined for overt signs of toxicity, ill-health and behavioral change
immediately before dosing, up to thirty minutes after dosing, and one hour after dosing, (except
for females during parturition where applicable). During the treatment-free period, recovery
animals were observed daily. All observations were recorded.

Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for
signs of functional/behavioral toxicity. Functional performance tests were also performed on
five selected males and females from each main study dose level and all satellite females during
the final week of dosing, together with an assessment of sensory reactivity to various stimuli.
Functional performance tests together with with an assessment of sensory reactivity to various
stimuli were also performed on all recovery animals during the final week of the treatment-free
period.

Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for main
study males until termination and weekly for main study females until pairing. During the
pairing phase main study females were weighed daily until mating was confirmed. Body
weights were then recorded for these females on Days 0, 7, 14 and 20 post coitum, and on Days
0 and 4 post partum (see deviations from Study Plan). Satellite females and recovery animals of
either sex were weighed on Day 1 (prior to dosing) and then weekly until termination.

During the pre-pairing period, weekly food consumption was recorded for each cage of main
study animals. This was continued for males after the completion of mating phase on the study.
For main study females showing evidence of mating, food consumption was recorded for the
periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food
consumption was recorded for the period covering post partum Days 0-4 (see deviations from
Study Plan). Weekly food consumptions were performed for each cage of satellite females and
recovery group animals throughout the study period.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively
for main study males (except during the mating phase), satellite and recovery group animals
throughout the study period and for main study females during the pre-pairing phase. Due to
offspring growth and milk production, food efficiency could not be accurately calculated during
gestation and lactation.

Water intake was observed daily by visual inspection of water bottles for any overt changes.

Hematology and blood chemistry investigations were performed on five males and five females
selected from each main study test and control group prior to termination (Day 42 for males and
Day 4 post partum for females). These evaluations were also performed on all satellite females
on Day 42. In addition hematology and blood chemistry investigations were performed on all
recovery group animals after the fourteen day treatment-free period (Day 56).

were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac
puncture at termination. Animals were not fasted prior to sampling.

Urinalyses were performed on five main study males from the control and each test group during
the final week of treatment and on all recovery animals during the final week of the recovery
period. These investigations were also performed on all satellite females during the final week
of treatment. Urine samples were collected overnight by housing the rats in metabolisms cages.
Animals were maintained under conditions of normal hydration during collection but without
access to food.

Sacrifice and pathology:

Adult main study males were killed by intravenous overdose of pentobarbital sodium followed
by exsanguination on Day 43 or 44 relative to the start of dosing. Surviving adult main study
females were killed by intravenous overdose of pentobarbital sodium followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of pentobarbital sodium on Day 5 post partum. Any main study females which failed to achieve pregnancy were killed on Day 26 post coitum.

For all main study females, the uterus was examined for signs of implantation and the number of
uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by
staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora
lutea were also counted.

Satellite females and recovery group animals were killed by intravenous overdose of
pentobarbital sodium followed by exsanguination on Days 43 or 57, respectively.
All adult animals and offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded. A vaginal smear was taken at necropsy from all surviving females and the stage of estrus was recorded.

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-related effects on
pituitary-thyroid axis were identified, therefore, these samples will be discarded following report
finalization.

The following organs were dissected free from fat and weighed before fixation from five
selected males and five selected females from each main study dose group and from all satellite
and recovery group animals:

- Adrenals
- Brain
- Epididymides*
- Heart
- Kidneys
- Liver
- Ovaries*
- Pituitary (post fixation)*
- Prostate*
- Seminal vesicles*
- Spleen
- Testes*
- Thymus
- Thyroid (weighed post-fixation with Parathyroid)
- Uterus (weighed with Cervix)*

* Tissues weighed from all remaining main study animals.

Samples of the following tissues were removed from five selected males and five selected
females from all main study dose groups and all satellite and recovery animals and preserved in
buffered 10% formalin, except where stated:

- Adrenals
- Aorta (thoracic)
- Bone & bone marrow (femur including stifle joint)
- Bone & bone marrow (sternum)
- Brain (including cerebrum, cerebellum and pons)
- Caecum
- Coagulating gland**
- Colon
- Duodenum
- Epididymides**
- Esophagus
- Eyes
- Gross lesions**
- Heart
- Ileum (including peyer’s patches)
- Jejunum
- Kidneys
- Liver
- Lungs (with bronchi)
- Lymph nodes (mandibular and mesenteric)
- Mammary gland**
- Muscle (skeletal)
- Ovaries**
- Pancreas
- Pituitary**
- Prostate**
- Rectum
- Salivary glands (submaxillary)
- Sciatic nerve
- Seminal vesicles**
- Skin
- Spinal cord (cervical, mid-thoracic and lumbar)
- Spleen
- Stomach
- Thyroid/parathyroid
- Trachea
- Testes
- Thymus
- Urinary bladder
- Uterus/Cervix**
- Vagina**

Tissues preserved from all remaining main study animals **

The tissues from five selected main study control and 1000 mg/kg bw/day dose group animals, all satellite females, any animals dying during the study, and any animals which failed to achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues marked with ** from the remaining control and 1000 mg/kg bw/day main study animals were also processed. In addition, sections of testes from all main study control and 1000 mg/kg bw/day males were also stained with Periodic AcidSchiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular
stages of the spermatogenic cycle. The examination was conducted in order to identify
treatment-related effects such as missing germ cell layers or types, retained spermatids,
multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell-or stage-specificity of testicular findings was noted.

Microscopic examination was conducted by the Test Facility. A peer review of the histopathology results for the study were conducted by the Test Facility.

Statistics:

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, Body Weight Change, Food Consumption during
gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex
Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body
Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood
Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights, Urine Volume and
Specific Gravity.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as
detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The
homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup
variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate
covariates. Any transformed data were analyzed to find the lowest treatment level that showed a
significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means,
the data were analyzed 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 MannWhitney U test (non-parametric).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical observations considered to be related to the toxicity of the test item at any
dose level.

At 1000 mg/kg bw/day, animals of either sex showed sporadic instances of increased post-dose
salivation from Day 11 of dosing. A small number of males and females treated with 300 mg/kg
bw/day also showed similar clinical signs towards the end of the dosing period. Although
increased post-dose salivation was also recorded for three control females on Day 12 relative to
the start of dosing, such observations are commonly observed following the oral administration
of an unpalatable or slightly irritating test item formulation and are considered to be of no
toxicological importance. During the treatment-free period, none of the recovery animals
previously treated with 1000 mg/kg bw/day showed such observations.

One male from the recovery control group showed generalized fur loss during the treatment- and
treatment-free periods whilst another male from this dose group was observed with a scab during
the last week of dosing. These clinical observations were not related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A female given the test item at 300 mg/kg bw/day was found dead on Day 12 of dosing. There
were no clinical signs for this animal prior to death and macroscopic findings at necropsy
included red or dark discoloration of liver, mesenteric lymph nodes, uterus/cervix and lungs.
Microscopic examination showed agonal congestion which was considered to be due to the
animal being found dead. Although the cause of the death for this female could not be
established, there were no further unscheduled deaths in this or any other group, and this death
was considered unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Throughout the study, there was considered to be no adverse effect of treatment with the test
item on body weight development in animals of either sex.

At 1000 mg/kg bw/day, group mean body weight gain in main study males over the second week
of dosing was slightly but statistically significantly lower than controls (p<0.05). Overall body
weight gain in these males was similar to controls with recovery males treated with 1000 mg/kg
bw/day also showing comparable overall body weight gain relative to their respective controls
during the treatment period. During the fourteen day treatment-free phase of the study, group
mean body weight gains in recovery males previously treated with 1000 mg/kg bw/day remained
similar to controls and, as such, any intergroup differences during the dosing period were
considered to be of no toxicological significance. In spite of minor fluctuations, body weight development in males receiving 100 or 300 mg/kg bw/day was generally similar to controls
throughout the treatment period.

Over the first week of dosing, group mean body weight gain in main study females receiving
1000 mg/kg bw/day was statistically significantly higher than controls (p<0.01) (Table 9), which
resulted in slightly higher overall body weight gain for this group during the pre-pairing phase of
the study. Satellite females treated with 1000 mg/kg bw/day also showed statistically
significantly higher group mean body weight gain over the first week of treatment (p<0.01). In
contrast, group mean body weight gain in recovery females given 1000 mg/kg bw/day over the
first week of dosing remained similar to controls. It remains unclear whether the statistically
significant increase in body weight gain in main study and satellite females over the first week of
dosing was treatment-related, but an increase in body weight gains is generally considered not to
be adverse and as such this observation was considered to be of no toxicological significance.

Throughout the remainder of the treatment period, minor fluctuations in body weight
development were apparent in satellite and recovery females in relation to their respective
controls with some intergroup differences achieving statistical significance; as these differences
were only evident in one or the other dose group, they were deemed likely to be due to biological
variation. During the treatment-free period, body weight fluctuations were still evident for
recovery females but none of the intergroup differences achieved statistical significance. As
there was no adverse effect of treatment with the test item at any dose level on body weight
performance in these females during the dosing period, any intergroup differences during the
recovery period were considered to be of no toxicological relevance.

At the start of the gestation phase of the study, group mean body weight in 1000 mg/kg bw/day
main study females was slightly but statistically significantly higher than controls (p<0.01)
(Table 9). This was deemed likely due to a combination of slightly higher group mean body
weight for these females at the start of dosing and slightly higher overall body weight gain
during the pre-pairing phase of the study. Over Days 7 to 14 and 14 to 20 of gestation, group
mean body weight gains for main study females treated with the test item at all dose levels were
generally slightly higher than controls but statistical significance was only achieved for
1000 mg/kg bw/day females over Days 7 to 14 of gestation (p<0.05). With this exception,
overall group mean weight gains in main study females during gestation at all dose levels were
slightly higher than controls but without attaining statistical significance. Group mean body
weights in main study females from all test item-treated dose groups on Days 7, 14 and 20 of
gestation were slightly higher than controls, but statistical significance was only achieved for the
1000 mg/kg bw/day dose group on Days 7 and 14 of gestation (p<0.05 and p<0.01, respectively).
Intergroup differences were not dose related and these findings were considered to be due to a
slightly higher number of fetuses for test item-treated main study females relative to the
corresponding control females. This is further supported by the fact that group mean body
weights for all main study females on Day 0 of lactation were comparable to controls.

During the lactation phase of the study, group mean body weight gains in main study females
treated with 100 or 1000 mg/kg bw/day were slightly higher than controls over Days 0 to 4 post
partum, which resulted in slightly higher cumulative body weight gains for these females. There
was, however, no dose relationship and statistical significance was not achieved. As such, these
differences were considered to be incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Throughout the study, there was no detrimental effect of treatment with the test item at any dose
level on food consumption for main study males, satellite females or recovery animals of either
sex. Any differences in food conversion efficiency were considered to be reflective of minor
intergroup differences in body weight gains.

Food efficiency:

no effects observed

Description (incidence and severity):

At all dose levels, food intake and food conversion efficiency values for main study females
were comparable to controls during the pre-pairing phase of the study. During gestation and
lactation, dietary intake for the test item-treated females from all dose groups was generally
comparable with controls.

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

no effects observed

Description (incidence and severity):

Visual inspection of water bottles did not indicate any differences for the animals given the test
item in relation to controls.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant effects were detected in treated animals of either sex at any dose
level.

At all dose levels, group mean hemoglobin (Hb) values in main study males at the end of the
dosing period were statistically significantly lower than controls in a dose related manner
(p<0.01 at 1000 mg/kg bw/day and p<0.05 at 300 or 100 mg/kg bw/day). Group mean red blood
cell counts (RBC) and hematocrit (Hct) values in these males were also generally slightly lower
than controls in a dose-dependent fashion, achieving statistical significance at 1000 mg/kg
bw/day (p<0.05 for RBC and p<0.01 for Hct) and 300 mg/kg bw/day (Hct only; p<0.05). The
mean corpuscular haemoglobin (MCH) and the mean corpuscular volume (MCV) in main study
males treated with 1000 mg/kg bw/day were also slightly lower than controls but there was no
dose relationship and statistical significance was not attained for these differences. It is worth
noting that whilst the majority of the individual values for the test item-treated males were within
the historical control data ranges, the corresponding values in some main study control males
were close to or marginally above the higher end of these ranges. At the end of the fourteen day
treatment-free period, group mean Hb, Hct, MCH and MCV values in recovery males previously
treated with 1000 mg/kg bw/day remained slightly lower than controls with the intergroup
differences for Hb and Hct attaining statistical significance (p<0.01 and p<0.05, respectively).
At the end of the treatment period, satellite females receiving 1000 mg/kg bw/day showed
statistically significantly lower Hb, MCH and reticulocyte counts in relation to the respective
controls (p<0.05). Group mean Hct and MCV values in these females were also slightly lower
than controls but the differences were not statistically significant. It is worth noting that whilst
recovery females previously given 1000 mg/kg bw/day also showed slightly lower group mean
Hct, MCH and MCV values in relation to controls at the end of the treatment-free period,
achieving statistical significance for the latter two parameters (p<0.05 and p<0.01, respectively),
the corresponding values in main study females at the end of the dosing period were comparable
with controls.

Whilst the above findings may indicate some minor perturbations in red blood cells, it is worth
noting that these observations were not consistent between sexes or even amongst main study or satellite females. The majority of individual values for test item-treated main study male dose groups at the end of the treatment period were within the background data ranges whilst the
corresponding parameters in main study females were similar to controls. There were no
associated microscopic findings in the main study or satellite females at the end of dosing and
these observations were considered not to be of any toxicological importance.

At all dose levels, main study males showed statistically significantly higher neutrophil counts
when compared with controls (p<0.05). There was no dose relationship and all individual values
were within the historical background data ranges. The corresponding values in main study
females from all test item-treated dose groups were also slightly higher than controls but all
individual values were within the control data ranges. There were no corresponding intergroup
differences for satellite females at the end of treatment or for recovery animals of either sex at
the end of the treatment-free period and, in the absence of any associated histopathological
findings, these observations were considered to be of no toxicological relevance.

Females from the 300 or 1000 mg/kg bw/day dose groups also showed statistically significantly
lower group mean monocyte and eosinophil counts in relation to controls (p<0.05), but this was
considered to be due to atypically high counts in some control females. The corresponding
values in main study males, satellite females and recovery animals of either sex were comparable
with controls and these findings were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant effects were detected in animals of either sex receiving the test
item at any dose level.

At the end of the dosing period, main study females treated with 300 or 1000 mg/kg bw/day
showed statistically significant increases in albumin/globulin ratios when compared with
controls (p<0.05). There was no dose relationship and all individual values from these females
were within the historical background data ranges. Whilst the corresponding parameters in main
study males were similar to controls, satellite females treated with 1000 mg/kg bw/day showed
statistically significant higher plasma concentrations of total protein, albumin and
albumin/globulin ratio (p<0.01) in comparison with the respective controls. At the end of the
fourteen day dose-free period, the corresponding values in recovery females were similar to
controls. In the absence of any associated histopathology observations, these findings were
considered to be of no toxicological importance.

At the end of the treatment period, the group mean plasma level of triglycerides in main study
males receiving 1000 mg/kg bw/day was statistically significantly lower than controls (p<0.01),
albeit without any dose relationship. Whilst the corresponding values in main study females and
satellite females treated with 1000 mg/kg bw/day were similar to controls, the satellite females
showed a statistically significant decrease in plasma concentration of cholesterol when compared with controls (p<0.01). At the end of the recovery period, these parameters in recovery animals of either sex were similar to their controls and, due to the lack of any histopathology correlates, these observations were deemed to be of no toxicological significance.

At 1000 mg/kg bw/day, main study females showed statistically significantly higher bile acid
concentration relative to controls at the end of the treatment period (p<0.05). Although, there
was no dose relationship, most individual values for the 1000 mg/kg bw/day females were above
historical data ranges. The corresponding values in satellite and recovery females were similar
to controls and, as there were no associated microscopic observations, these findings were
deemed of no toxicological relevance.

At all dose levels, group mean plasma phosphorus levels in main study males were statistically
significantly lower than control levels at the end of the dosing period (p<0.05 at 100 or 300
mg/kg bw/day and p<0.01 at 1000 mg/kg bw/day). There was no dose dependence and all
individual values from the test item-treated males were within the historical controls data ranges.
Whilst mean phosphorus plasma levels in main study females were similar to control levels,
satellite females treated with 1000 mg/kg bw/day also showed statistically significantly lower
phosphorus levels (p<0.01) and a statistically significant rise in calcium levels (p<0.05) relative
to controls. Additionally, main study females from all dose groups showed statistically lower
mean sodium concentrations with respect to controls (p<0.05) but there was no dose relationship, and all individual values from the test item-treated animals were within the background data ranges. Main study females treated with 1000 mg/kg bw/day also showed statistically significantly lower chloride levels in relation to controls (p<0.05) but not in a dose related manner, and most individual values were within the historical data ranges. The corresponding parameters in recovery animals of either sex were comparable with the respective controls and in the absence of any treatment-related microscopic findings, these observations were considered to be of no toxicological relevance.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment with the test item on urine volume and specific gravity in main
study males and satellite females during the final week of the treatment period. The
corresponding parameters in the recovery animals of either of either sex previously treated with
1000 mg/kg bw/day were comparable with the respective controls during the final week of the
treatment-free period.

Towards the end of the dosing period, urine samples from 3/5 satellite females treated with
1000 mg/kg bw/day tested positive for the presence of protein. These females also showed
statistically significant increases in total protein and albumin concentrations in plasma at the end
of the treatment period when compared with controls. In the absence of any histopathology
correlates, this finding was considered to be of no toxicological significance. Urine samples from a small number of main study males and recovery animals of either sex given the test item
also tested positive for protein, but these incidences were comparable with controls.

Urine samples from individual animals of one or the other sex from the main study, satellite or
recovery groups tested positive for glucose, erythrocytes or hemoglobin. There was no dose
relationship and these observations were considered to be incidental.

Sediments analysis of urine samples from main study males, satellite females and recovery males and females did not reveal any treatment related differences.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral Assessments

There were no changes in the behavioral parameters considered to be related to treatment with
the test item at any dose level.

Functional Performance Tests

There were no intergroup differences considered to be related to treatment with the test item.

Motor activity evaluation during the final week of the treatment period revealed statistically
significantly lower activity (last 20%) in main study males given 1000 mg/kg bw/day when
compared with controls (p<0.05). A dose-relationship was evident. Main study females
receiving the test item at dose levels of 300 or 1000 mg/kg bw/day showed statistically
significantly lower overall activities in relation to controls (p<0.05) but without any dose
relationship. When compared with the respective controls, there were no statistically significant
intergroup differences for satellite females toward the end of treatment or for recovery animals
of either sex during the second week of the dose-free period and in the absence of any signs of
neurotoxicity on this study, these findings in the main study animals were deemed unlikely to be
related to treatment with the test item.

Sensory Reactivity Assessments

Sensory reactivity scores across all treated main study, satellite and recovery animals were
similar to their respective controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the treatment period, group mean absolute and body weight related kidney weights
in main study females receiving 1000 mg/kg bw/day were statistically significantly higher than
controls (p<0.05). A dose relationship was not evident, although some individual values from
test item-treated animals were slightly above the historical control data ranges. The
corresponding values in satellite females were also slightly higher than controls, albeit without
achieving statistical significance. At the end of the fourteen day recovery period, females
previously given 1000 mg/kg bw/day still showed statistically significantly higher group mean
absolute and body weight-related kidney weights in comparison with the respective controls
(p<0.01). In the absence of any histopathology correlates, these observations were considered to
be of no toxicological significance.

At the end of the treatment period, absolute and body weight-related thyroid/parathyroid weights
in main study animals of either sex treated with 300 or 1000 mg/kg bw/day and main study
males treated with 100 mg/kg bw/day, were statistically significantly higher than controls
(p<0.01 at 1000 mg/kg bw/day and p<0.05 in the remaining instances). A dose relationship was
only evident in females, but a number of individual values, in particular from the 1000 mg/kg
bw/day dose group, were slightly above the background data ranges. The corresponding values
from satellite females were similar to controls. At the end of the treatment-free period, absolute
and body weight-related thyroid/parathyroid weights in animals of both sexes previously
receiving 1000 mg/kg bw/day were comparable to controls. In the absence of any associated
histopathology observations, these findings were considered to be of no toxicological
importance.

At the end of the treatment-free period, group mean absolute and body weight-related brain
weights in males previously treated with 1000 mg/kg bw/day were slightly, but statistically
significantly, higher than those of controls (p<0.01). The corresponding values in recovery 1000
mg/kg bw/day females were similar to those of controls, and as there were no intergroup
differences for main study or satellite animals or any histopathology correlates, this finding was
considered to be incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At terminal necropsy, no macroscopic findings were observed at any dose level in main study
and recovery males and females or satellite females.

Macroscopic findings for the female, found dead on Day 12 of treatment, included red or dark
discoloration of liver, mesenteric lymph nodes, uterus/cervix and lungs. As there were no
similar findings in animals from other dose groups, these observations were considered not
related to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathology examination of the selected tissues from main study animals and satellite females
from the 1000 mg/kg bw/day dose groups did not reveal any treatment-related abnormalities.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

mortality

organ weights and organ / body weight ratios

serum/plasma biochemistry

urinalysis

water consumption and compound intake

Critical effects observed:

no

Conclusions:

In an OECD 422 study the oral administration of the test substance to rats by gavage, at dose levels of 100, 300 or 1000 mg/kg bw/day, was well tolerated.

The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restriction.

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

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study conducted according to OECD Guideline and under GLP conditions.