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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-09-29 to 2009-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 422 and in compliance with GLP
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-09-29 to 2009-08-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 422 and in compliance with GLP.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
OECD GLP (inspected on 05th to 09th and 26th to 30th November 2007, Signed on 12th November 2008)
Limit test:
no
Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 44414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 284 to 318 g; Females: 178 to 214 g
- Fasting period before study: none
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular oestrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 31/08).
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with music during the light period

IN-LIFE DATES: From: 2008-09-28 To: 2009-08-27
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker on a tarred precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added.
Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 24897436
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day, one sample (middle) from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose
formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analysed by GC coupled to an FI detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.

The identity of the test material was confirmed by its retention time, which was similar to that measured in the working standards. The application formulations investigated during the study were found to comprise the test material in the range of 91.7% to 108.9% and thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of the test material in the preparations was approved because single results did not deviate more than 5.9% (<15%) from the corresponding mean. The application formulations were considered to be stable for at least 7 days when kept at room temperature.
Duration of treatment / exposure:
Males: minimum 4 weeks. Females: approximately 7 weeks.
Frequency of treatment:
Daily
Details on study schedule:
See Table 7.8.1/1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C05595, using dose levels of 100, 300, and 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Positive control:
None
Parental animals: Observations and examinations:
VIABILITY / MORTALITYS: Yes
- Time schedule: Twice daily

CLINICAL SIGNS: Yes
- Time schedule: Daily cage-side clinical observations (once daily during acclimatization and up to day of necropsy).
Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter, performed outside the home cage. Animals were observed
- Parameters examined: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION:
Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1-8, 8-14 and 14-16; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.
No food consumption was recorded during the pairing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 animals/sex/dose
- Parameters checked in table 5.8.1/2.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals:5 animals/sex/dose
- Parameters checked in table 5.8.1/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males shortly before the scheduled sacrifice and females on day 3 or 4 post partum
- Dose groups that were examined: five P generation males and five P generation females from each group (erroneously six females were investigated in group 4)
- Battery of functions tested:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and faecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or faeces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in male parental animals: testis weight, epididymis weight. The testes were stained by PAS hematoxylin for qualitative sperm staging.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS:
No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects.
- Maternal animals: All surviving animals were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

GROSS PATHOLOGY: Yes (see table 7.8.1/3)

HISTOPATHOLOGY: Yes (see table 7.8.1/3)

The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.
Postmortem examinations (offspring):
SACRIFICE
Pups were sacrificed on day 4 post partum

GROSS NECROPSY
- All pups were examined macroscopically for any structural changes at the scheduled necropsy.
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied to the macroscopical findings.
Reproductive indices:
not applicable
Offspring viability indices:
not applicable
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All males and females treated with 400 or 800 mg/kg bw/d pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. This was considered to be a sign of discomfort following treatment rather than a toxic effect of the test item. One male and one female treated with 800 mg/kg bw/d had salivation for isolated days. In addition, one female had salivation for most of the prepairing, pairing and gestation periods. This was considered to be a sign of discomfort following the treatment.
Other clinical signs noted were a wound on the shoulder of one male in low-dose group and hair loss in one dam in the control group. These findings were considered to be incidental.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Normal responses to the taste of fragrances.

- Males: In high-dose group, body weight gain was statistically significantly reduced on days 3, 6 and 10 of the pre-pairing period and on the last day of the after pairing period. However, mean body weight was similar in all dose groups throughout the study. No test item-related effects were noted in the other groups.
- Females: In high-dose group 4, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight on days 3, 4 and 6 during the pre-pairing period.This was considered to be a result of treatment with the test item. No other test item-related effects were noted during the study at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Normal responses to the taste of fragrances.

- Males: Mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period (-5.6% and -6.1%, respectively). Thereafter, food consumption was not affected by treatment with the test item in any dose group.
- Females: In high-dose group, mean food consumption was statistically significantly reduced over the first week of the pre-pairing period (-18.4% compared to the control group). This reduction was considered to be a test item-related effect. The statistically significant increase in low-dose group in the first week of the pre-pairing period was considered to be incidental due to the lack of a dose-dependent pattern.
No other test item-related effects were noted in the food consumption during the study at any dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Males The level of platelets was statistically significantly increased in medium-dose groups (+23.2%, compared to the control group) and 4 (+33.8%) and was outside the historical control data in high-dose group.
- Females: In the females in high-dose group, although the prothrombin time was not statistically significantly increased (+15.3%), the level was outside the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Males The level of cholesterol was statistically significantly increased in medium- and high-dose dose groups (+28.5% and +44.0%, respectively). Both these values were outside the range of the historical control data. The level of protein (+5.3% in medium-dose group and +8.6% in high-dose group) as well as globulin (+9.6% in medium-dose group and +16.5% in high-dose group) were increased and although the values were not statistically significant, they were outside the range of the historical control data.
- Females: The level of cholesterol in medium- and high-dose groups (+59.0% and +125.5%, respectively) was outside the range of the historical control data and was statistically significantly increased in high-dose group. The level of globulin was statistically significantly increased in medium- and high-dose groups (+17.3% and +16.1%, respectively) and both levels were outside the range of the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In high-dose group, there was an increased incidence in males of a reduced number of rearings (80%) compared to the control group (20%). In addition, body temperature was statistically significantly decreased in males (37.9 °C compared to the control group 38.5 °C) and females (38.3 °C compared to 39.0 °C in the control group).
In medium-dose group, the body temperature of the females was statistically significantly reduced (38.5 °C compared to 39.0 °C in the control group).
Although the body temperatures were within the range of the historical control data, since they decreased in a dose-dependent manner, it may be a slight effect of the test item.
No other test item-related effects were noted.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The weights and ratios of the liver were statistically significantly increased in males and females in the groups receiving the test item. In medium- and high-dose groups, this corresponded to histopathological findings. In low-dose group, since no test item-related histopathological findings were noted and the mean absolute weight was within the historical control data, the increase was considered to be incidental.
The weights and organ/body weight ratios of the kidney were statistically significantly increased in high-dose group in the females. Since this did not correspond to any histopathological findings and the mean absolute weight was within the historical control data, it was therefore considered to be incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
2 females in high-dose group had an enlarged liver. No other test item-related findings were noted for males or females in any group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the liver, there was centrilobular hepatocellular hypertrophy in males and females in medium- and high-dose groups. There was no further lesion along with this hypertrophy and therefore, an adaptive change in liver metabolism was concluded.
In the thyroid glands, there was diffuse follicular hypertrophy in some high dose males and females at minor degrees of severity. This was considered to be the result of increased metabolism in the liver. The changes in the liver and in the thyroid glands were not considered to be adverse.
In the kidneys of males in medium- and high-dose groups, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent alpha-2-microglobulin and the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in male rats due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.
In low-dose group, no test item-related findings were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Mating Performance and Fertility: The mean precoital time was unaffected by treatment with the test item. Mean precoital time was 4.3, 3.0, 2.7 and 2.7 days, in order of increasing dose level. Two females in control group, and one female each in low- and high-dose groups were not pregnant.
- Duration of Gestation: The mean duration of gestation was unaffected by exposure to the test item and was similar in all dose groups.
- Corpora Lutea Count: Mean number of corpora lutea per dam (determined at necropsy) gave no indication of a test item-related effect.
- Implantation Rate and Post-implantation Loss: The mean number of implantations per dam (13.9, 14.9, 14.4, 12.7, in order of ascending dose
level) was not affected by treatment with the test item. Post-implantation loss was also unaffected by the test item (1.3, 1.1, 1.5 and 0.4, as a mean per dam and in order of increasing dose level).
- There were no effects on the completeness of stages or cell populations. There was no indication for maturation arrest, re-absorption of sperm or any other degenerative type.
Key result
Dose descriptor:
NOAEL
Remarks:
Screening for Fertility
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related findings were noted at the first litter check or during the lactation period.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No dead pups were found at the first litter check at any dose level. Thereafter, three pups were lost from 2 litters in high-dose group. Since only 2 out of 9 litters were affected, this was considered to be incidental. No other pups were lost from any other litter.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Reduced mean body weights of the pups at 800 mg/kg bw/d, but not statistically significant and in the range of historical control data.

In high-dose group, the mean body weight of the pups was reduced on day 1 post partum (5.9 g compared to 6.4 g in the control group). In addition, the pups did not increase in weight as much as in the control group (+39.0% compared to +46.9% in the control group), resulting in reduced mean body weights of pups on day 4 post partum (8.2 g compared to 9.4 g in the control group). Since these effects were not statistically significant and in the range of the historical control data (5.7 - 6.3 g on day 1 post partum and 8.1 - 9.4 g on day 4 post partum - even though borderline to the lower limit), they were considered to be test item-related but not adverse.
The mean body weights and body weight gain of the pups in low- and medium-dose groups were not affected by treatment with the test item
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related abnormal findings were noted at necropsy.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratios at first litter check and on day 4 post partum were not considered to have been affected by the test item. The statistically significant increase in the proportion of females in high-dose group (59%) was considered to be due to biological variability.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Screening for development (foetal and pup growth survival until day 4)
Generation:
F1
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Discussion:

At 800 mg/kg bw/day, salivation was noted in isolated individuals. There was an increased incidence of a decreased number of rearings in the males in group 4 compared to the control group. In addition, body temperature was statistically significantly decreased in males and females in group 4. Although this was within the range of the historical control data, since body temperature decreased in a dose-dependent manner, it may be a slight effect of the test item. Mean food consumption and body weight gain were reduced slightly and transiently in the males and females in the first week of the pre-pairing period. Clinical laboratory investigations revealed that the level of platelets was statistically significantly increased in the males. In the females, the prothrombin time was outside the range of the historical control data. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. From the macroscopical examination, two females had an enlarged liver. Microscopically, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity. These findings were not considered to be adverse. In the kidneys of males, there was an increased degree of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent α2-microglobulin and that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

At 400 mg/kg bw/day, mean food consumption was very slightly reduced in the males during the first week of the pre-pairing period. This had no effect on the mean absolute body weights. The level of platelets was statistically significantly increased in the males. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. Microscopical examination revealed centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. The changes that were observed for platelets may be related to changed renal function and increased excretion. Biochemical data reveal also an affection of the female kidneys, by changes in protein and ion parameters. The cause of increased globulin levels remains unclear. Other biochemical changes recorded, including cholesterol and bilirubin are likely due to changes in the liver metabolism.

At 100 mg/kg bw/d, no test item-related effects were noted in the in-life phase. No effects were noted in the clinical laboratory investigations. At necropsy, no test item-related macroscopical or microscopical findings were noted.

Conclusions:
The NOAEL for fertility and development was considered to be 800 mg/kg bw/day.
Executive summary:

In a combined repeated dose toxicity study with the reproduction/development toxicity screening test performed in accordance with OECD test guideline No. 422 and in compliance with GLP, ST 10 C 08 diluted in corn oil was administered to 10 HanRcc: WIST(SPF) rats/sex/dose by gavage at dose levels of 0, 100, 400 and 800 mg/kg bw/day. Control rats were given the vehicle alone. The test material was administered to male rats for at least 28 days and to female rats for 16 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following results were obtained for the Parent animals:

- Mortality and General tolerability

All males and females in medium- and high dose groups pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. One male and one female in high-dose group had salivation for isolated days. In addition, one female had salivation for most of the pre-pairing, pairing and gestation periods. These findings were considered to be a sign of discomfort following the treatment.

- Functional Observational Battery

Body temperature was statistically significantly decreased in high-dose group in males and females as well as in the females in medium-dose group. Body temperature decreased in a dose-dependent manner. However, since these changes were within the range of the historical control data, they were considered of no toxicological importance.

There was an increased incidence of a reduced number of rearings in high-dose group compared to the control group. However, in general, when only one FOB measurement is affected, the results are not considered evidence of a neurotoxic effect, mostly when the effects occurred at the high dose, in the presence of sign of toxicity such as body weigh or body temperature descreases (Guidelines for Neurotoxicity Risk Assessment, EPA, April 1998).

- Food consumption

In the males, mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period. In the females, it was statistically significantly reduced in high-dose group over the first week of the pre-pairing period. These variations are normal responses to the taste of fragrances, like ST 10 C 08.

- Body weights

In high-dose group in the males, body weight gain was statistically significantly reduced on isolated days during the pre-pairing period and on the last day of the after pairing period. In the females, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight was occasionally statistically significantly reduced. These findings were correlated to lower mean food consumption and were fully reversible; therefore they are not considered as toxicologically significant.

- Clinical Laboratory Investigations

Haematology: The level of platelets was statistically significantly increased in the males in medium- and high-dose groups (+23.2 and +33.8%, respectively) and was outside the historical control data in high-dose group. In the females in high-dose group, although the prothrombin time was not statistically significantly increased, the level was outside the range of the historical control data.

Clinical biochemistry: In both males and females, the levels of cholesterol and globulin in medium- and high-dose groups were outside the range of the historical control data. The level of protein in the males in the same groups was outside the range of the historical control data.

These changes were considered as adaptive in nature, and of no toxicological concern.

- Organ Weights

The weights and ratios of the liver were statistically significantly increased in males and females in medium- and high-dose groups.

- Macroscopical Findings and Histopathological Examinations

At macroscopical examination, two females in high-dose group were noted to have an enlarged liver.

In this dose group, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity.

These findings were considered to be adaptive responses to the test material and not to be adverse. In the kidneys of males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions representα2-microglobulin. It may be considered that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

In medium-dose group, there was centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia.

 

The following results were obtained for the Pups:

-      Findings at First Litter Check and during Lactation

No test item-related findings were noted at the first litter check or during the lactation period. Sex ratios at first litter check and on day 4 post partum were not considered to have been affected by the test item.

-      Pup Weights to Day 4 Post Partum

In high-dose group, the mean body weight of the pups was reduced on day 1 post partum. In addition, the pups did not increase in weight as much as in the control group, resulting in reduced mean body weights of pups on day 4 post partum. Since these effects were not statistically significant and in the range of the historical control data, they were considered to be test item-related but not adverse.

-      Macroscopical Findings

No test item-related abnormal findings were noted at necropsy.

 

Based on the results, the NOAEL for fertility and development (foetal and pup growth until Day 4) was considered to be 800 mg/kg bw/d.

 

Under the test conditions, ST 10 C 08 is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
OECD GLP (inspected on 05th to 09th and 26th to 30th November 2007, Signed on 12th November 2008)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state: solid
- Stability under test conditions: at least 7 days when kept at room temperature.
- Storage condition of test material: In the refrigerator (2 - 8 °C), protected from light
- Molecular formula : C16 H28 O
- Molecular weight : 236.4 g/mol

Test animals

Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Details on species / strain selection:
Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 44414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 284 to 318 g; Females: 178 to 214 g
- Fasting period before study: none
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular oestrus cycles.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 31/08).
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with music during the light period

IN-LIFE DATES: From: 2008-09-28 To: 2009-08-27

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was weighed into a glass beaker on a tarred precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added.
Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 24897436
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day, one sample (middle) from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose
formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analysed by GC coupled to an FI detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.

The identity of the test material was confirmed by its retention time, which was similar to that measured in the working standards. The application formulations investigated during the study were found to comprise the test material in the range of 91.7% to 108.9% and thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of the test material in the preparations was approved because single results did not
deviate more than 5.9% (<15%) from the corresponding mean. The application formulations were considered to be stable for at least 7 days when kept at room temperature.
Duration of treatment / exposure:
Males: minimum 4 weeks. Females: approximately 7 weeks.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C05595, using dose levels of 100, 300, and 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
VIABILITY / MORTALITYS: Yes
- Time schedule: Twice daily

CLINICAL SIGNS: Yes
- Time schedule: Daily cage-side clinical observations (once daily during acclimatization and up to day of necropsy).
Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter, performed outside the home cage. Animals were observed
- Parameters examined: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION:
Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1-8, 8-14 and 14-16; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum.
No food consumption was recorded during the pairing period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 animals/sex/dose
- Parameters checked in table 5.7.1/2.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals:5 animals/sex/dose
- Parameters checked in table 5.7.1/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males shortly before the scheduled sacrifice and females on day 3 or 4 post partum
- Dose groups that were examined: five P generation males and five P generation females from each group (erroneously six females were investigated in group 4)
- Battery of functions tested:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes.
Sacrifice and pathology:
Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
GROSS PATHOLOGY: Yes (see table 7.5.1/3)
HISTOPATHOLOGY: Yes (see table 7.5.1/3)
Other examinations:
The testes were stained by PAS hematoxylin for qualitative sperm staging.
Statistics:
The following statistical methods were used to analyse food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied to the macroscopical findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All males and females treated with 400 or 800 mg/kg bw/d pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. This was considered to be a sign of discomfort following treatment rather than a toxic effect of the test item. One male and one female treated with 800 mg/kg bw/d had salivation for isolated days. In addition, one female had salivation for most of the pre-pairing, pairing and gestation periods. This was considered to be a sign of discomfort following the treatment.
Other clinical signs noted were a wound on the shoulder of one male in low-dose group and hair loss in one dam in the control group. These findings were considered to be incidental.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Males: In high-dose group, body weight gain was statistically significantly reduced on days 3, 6 and 10 of the pre-pairing period and on the last day of the after pairing period. However, mean body weight was similar in all dose groups throughout the study. No test item-related effects were noted in the other groups.
- Females: In high-dose group 4, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight on days 3, 4 and 6 during the pre-pairing period. This was considered to be a result of treatment with the test item. No other test item-related effects were noted during the study at any dose level.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Normal responses to the taste of fragrances

- Males: Mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period (-5.6% and -6.1%, respectively). Thereafter, food consumption was not affected by treatment with the test item in any dose group.
- Females: In high-dose group, mean food consumption was statistically significantly reduced over the first week of the pre-pairing period (-18.4% compared to the control group). This reduction was considered to be a test item-related effect. The statistically significant increase in low-dose group in the first week of the pre-pairing period was considered to be incidental due to the lack of a dose-dependent pattern.
No other test item-related effects were noted in the food consumption during the study at any dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increased platelets counts in medium- and high-dose males; prothrombin time outside the historical control data in high-dose females. Not adverse

- Males The level of platelets was statistically significantly increased in medium-dose groups (+23.2%, compared to the control group) and 4 (+33.8%) and was outside the historical control data in high-dose group.
- Females: In the females in high-dose group, although the prothrombin time was not statistically significantly increased (+15.3%), the level was outside the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increased cholesterol and globulin level in medium- and high-dose animals; increased globulin levels in medium- and high-dose males. Not adverse.

- Males The level of cholesterol was statistically significantly increased in medium- and high-dose dose groups (+28.5% and +44.0%, respectively). Both these values were outside the range of the historical control data. The level of protein (+5.3% in medium-dose group and +8.6% in high-dose group) as well as globulin (+9.6% in medium-dose group and +16.5% in high-dose group) were increased and although the values were not statistically significant, they were outside the range of the historical control data.
- Females: The level of cholesterol in medium- and high-dose groups (+59.0% and +125.5%, respectively) was outside the range of the historical control data and was statistically significantly increased in high-dose group. The level of globulin was statistically significantly increased in medium- and high-dose groups (+17.3% and +16.1%, respectively) and both levels were outside the range of the historical control data.
All other values noted for the males and females, including statistically significant changes, were within the range of the historical control data.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Reduced number of rearing in high-dose males. Dose-dependent decrease in body temperature.

In high-dose group, there was an increased incidence in males of a reduced number of rearings (80%) compared to the control group (20%). In addition, body temperature was statistically significantly decreased in males (37.9 °C compared to the control group 38.5 °C) and females (38.3 °C compared to 39.0 °C in the control group).
In medium-dose group, the body temperature of the females was statistically significantly reduced (38.5 °C compared to 39.0 °C in the control group).
Although the body temperatures were within the range of the historical control data, since they decreased in a dose-dependent manner, it may be a slight effect of the test item.
No other test item-related effects were noted.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The weights and ratios of the liver were statistically significantly increased in males and females in the groups receiving the test item. In medium- and high-dose groups, this corresponded to histopathological findings. In low-dose group, since no test item-related histopathological findings were noted and the mean absolute weight was within the historical control data, the increase was considered to be incidental.
The weights and organ/body weight ratios of the kidney were statistically significantly increased in high-dose group in the females. Since this did not correspond to any histopathological findings and the mean absolute weight was within the historical control data, it was therefore considered to be incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
2 females in high-dose group had an enlarged liver. No other test item-related findings were noted for males or females in any group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the liver, there was centrilobular hepatocellular hypertrophy in males and females in medium- and high-dose groups. There was no further lesion along with this hypertrophy and therefore, an adaptive change in liver metabolism was concluded.
In the thyroid glands, there was diffuse follicular hypertrophy in some high dose males and females at minor degrees of severity. This was considered to be the result of increased metabolism in the liver. The changes in the liver and in the thyroid glands were not considered to be adverse.
In the kidneys of males in medium- and high-dose groups, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent alpha-2-microglobulin and the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in male rats due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.
In low-dose group, no test item-related findings were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The induced hyaline droplet nephropathy in male rats is known to be a rat-specific lesion and is not relevant for human

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Discussion:

At 800 mg/kg bw/day, salivation was noted in isolated individuals. There was an increased incidence of a decreased number of rearings in the males in group 4 compared to the control group. In addition, body temperature was statistically significantly decreased in males and females in group 4. Although this was within the range of the historical control data, since body temperature decreased in a dose-dependent manner, it may be a slight effect of the test item. Mean food consumption and body weight gain were reduced slightly and transiently in the males and females in the first week of the pre-pairing period. Clinical laboratory investigations revealed that the level of platelets was statistically significantly increased in the males. In the females, the prothrombin time was outside the range of the historical control data. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. From the macroscopical examination, two females had an enlarged liver. Microscopically, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity. These findings were not considered to be adverse. In the kidneys of males, there was an increased degree of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions represent α2-microglobulin and that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

At 400 mg/kg bw/day, mean food consumption was very slightly reduced in the males during the first week of the pre-pairing period. This had no effect on the mean absolute body weights. The level of platelets was statistically significantly increased in the males. In both males and females, the levels of cholesterol and globulin were outside the range of the historical control data. The level of protein in the males was also outside the range of the historical control data. At necropsy, the weights and ratios of the liver were statistically significantly increased in males and females. Microscopical examination revealed centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. The changes that were observed for platelets may be related to changed renal function and increased excretion. Biochemical data reveal also an affection of the female kidneys, by changes in protein and ion parameters. The cause of increased globulin levels remains unclear. Other biochemical changes recorded, including cholesterol and bilirubin are likely due to changes in the liver metabolism.

At 100 mg/kg bw/d, no test item-related effects were noted in the in-life phase. No effects were noted in the clinical laboratory investigations. At necropsy, no test item-related macroscopical or microscopical findings were noted.

Applicant's summary and conclusion

Conclusions:
Based on the results of the study and since the induced hyaline droplet nephropathy in male rats is known to be a rat-specific lesion and not relevant for human, the general NOAEL was considered to be 800 mg/kg bw/day.
Executive summary:

In a combined repeated dose toxicity study with the reproduction/development toxicity screening test performed in accordance with OECD test guideline No. 422 and in compliance with GLP, the test substance diluted in corn oil was administered to 10 HanRcc: WIST(SPF) rats/sex/dose by gavage at dose levels of 0, 100, 400 and 800 mg/kg bw/day. Control rats were given the vehicle alone. The test substance was administered to male rats for at least 28 days and to female rats for 16 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following results were obtained:

- Mortality and General tolerability

All males and females in medium- and high dose groups pushed their head through the bedding after application from day 14 of the pre-pairing period onwards. One male and one female in high-dose group had salivation for isolated days. In addition, one female had salivation for most of the pre-pairing, pairing and gestation periods. These findings were considered to be a sign of discomfort following the treatment.

- Functional Observational Battery

Body temperature was statistically significantly decreased in high-dose group in males and females as well as in the females in medium-dose group. Body temperature decreased in a dose-dependent manner. However, since these changes were within the range of the historical control data, they were considered of no toxicological importance.

There was an increased incidence of a reduced number of rearings in high-dose group compared to the control group. However, in general, when only one FOB measurement is affected, the results are not considered evidence of a neurotoxic effect, mostly when the effects occurred at the high dose, in the presence of sign of toxicity such as body weigh or body temperature descreases (Guidelines for Neurotoxicity Risk Assessment, EPA, April 1998).

- Food consumption

In the males, mean food consumption was very slightly reduced in medium- and high-dose groups during the first week of the pre-pairing period. In the females, it was statistically significantly reduced in high-dose group over the first week of the pre-pairing period. These variations are normal responses to the taste of fragrances, like ST 10 C 08.

- Body weights

In high-dose group in the males, body weight gain was statistically significantly reduced on isolated days during the pre-pairing period and on the last day of the after pairing period. In the females, mean body weight gain was statistically significantly reduced over days 2 - 7 of the pre-pairing period and absolute body weight was occasionally statistically significantly reduced. These findings were correlated to lower mean food consumption and were fully reversible; therefore they are not considered as toxicologically significant.

- Clinical Laboratory Investigations

Haematology: The level of platelets was statistically significantly increased in the males in medium- and high-dose groups (+23.2 and +33.8%, respectively) and was outside the historical control data in high-dose group. In the females in high-dose group, although the prothrombin time was not statistically significantly increased, the level was outside the range of the historical control data.

Clinical biochemistry: In both males and females, the levels of cholesterol and globulin in medium- and high-dose groups were outside the range of the historical control data. The level of protein in the males in the same groups was outside the range of the historical control data.

These changes were considered as adaptive in nature, and of no toxicological concern.

- Organ Weights

The weights and ratios of the liver were statistically significantly increased in males and females in medium- and high-dose groups.

- Macroscopical Findings and Histopathological Examinations

At macroscopical examination, two females in high-dose group were noted to have an enlarged liver.

In this dose group, in the liver there was centrilobular hepatocellular hypertrophy and, as a result, diffuse follicular hypertrophy in the thyroid glands in males and females at minor degrees of severity.

These findings were considered to be adaptive responses to the test material and not to be adverse. In the kidneys of males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia. It is considered that the hyaline inclusions representα2-microglobulin. It may be considered that the increased deposition is a consequence of increased liver metabolism. The nature of this lesion may be considered to be adverse in rat males due to the increased tubular basophilia that represent regeneration from a previous tubulopathy.

In medium-dose group, there was centrilobular hepatocellular hypertrophy in males and females. In addition, in the kidneys of the males, there were increased degrees of severity of hyaline inclusions along with an increased incidence and severity of tubular basophilia.

Based on the results of the study and since the induced hyaline droplet nephropathy in male rats is known to be a rat-specific lesion and not relevant for human, the general NOAEL was considered to be 800 mg/kg bw/day.

Based on the results of this study, the test substance is not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.