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Toxicological information

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:
2012-02-14 to 2012-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
N° 2011/40
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Triethyl phosphonoacetate (TEPA)
- Storage condition : at room temperature.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France Crl CD® (SD) IGS BR, Caesarian Obtained,Barrier Sustained-Virus Antibody Free (COBS-VAF®)
- Age/Weight: on the first day of treatment, the animals were approximately 8 weeks old. The males had a mean body weight of 301 g (range: 276 g to 323 g) and the females had a mean body weight of 204 g (range: 176 g to 238 g).
- Fasting period before study:
- Housing: The animals were housed by five from the same sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained an enrichment (rat hut). The cages were placed in numerical order on the racks
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 5776558 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filteredwith a 0.22 μm filter)
- Acclimation period: the animals were acclimatized to the study conditions for a period of 7 days before the beginning of the treatment period. A larger number of animals than necessary were allocated to the study and acclimatized to permit selection and/or replacement of individuals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From 2012-02-21: To:2012-03-29

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water, treated by reverse osmosis using Elix 5 (Millipore SA, Saint-Quentin en Yvelines, France).
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 100, 300 and 1000 mg/kg bw
- Amount of vehicle (if gavage): 5mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle and validation of the method
The Gas Chromatography with FID detection (GC-FID) analytical method for the determination of Triethyl phosphonoacetate in dose formulation samples was provided by the Sponsor and this method was validated at CIT prior to dose formulation analysis.

The validation of the analytical method was conducted in CIT/Study No. 38534 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.

Composition of an analytical sequence
Each analytical sequence consisted of at least:
. a blank sample (diluent only),
. ten standard samples at nominal concentration, prepared from two independent standard solutions,
. study samples prepared from aliquots of the dose formulations.
The standard samples bracketed the dose formulation samples.
The blank sample was checked for the absence of chromatographic interference

Analytical sequence suitability rules
The acceptance of the analytical sequence depended on the precision of the standard samples and on the agreement of the standard sample results.
Acceptance criteria are defined in CIT Standard Operating Procedures (SOPs).
The acceptance criteria include:
. precision of the standard solutions [% RSD (Relative Standard Deviation) must pass defined acceptance criteria],
. agreement of the standard samples (the mean response factor of standard samples prepared
from standard solution #1 must agree with the mean response factor of standard samples prepared from standard solution #2)

The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1 and 4 was determined. These analyses were performed prior to administration of the dose formulations to the animals
Duration of treatment / exposure:
The dose formulations were administered daily for a period of 28 to 31 days.
Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once a day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
other: test material
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
other: test material
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
other: test material
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, based on the results of a 7-day toxicity study (CIT/Study No. 38532 TSR; Appendix 2). In this preliminary study, the test item was given under the same experimental conditions at 100, 300 or 1000 mg/kg/day to Sprague-Dawley rats. At 1000 mg/kg/day, ptyalism was noted in all animals from day 5. There were no clear effects on body weight or food consumption and there were no relevant necropsy findings.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.
Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then at least once a week until the end of week 4.
Observations included (but were not limited to) changes in the 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 (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once before group allocation, on the first day of treatment and then at least once a week until the end of week 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The quantity of food consumed by the animals in each cage was recorded once a week until the end of week 4. Food consumption was calculated per animal and per day. When one of the animals in the same cage died, the number of days for which that animal had been present in the cage was taken into consideration for the calculation of the food consumption.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:The following parameters were determined at the end of the treatment period. Males were sampled on the same nominal day of dosing at the end of the treatment period. Females were sampled on or after day 29 after a metestrus or after a first day of diestrus when there was no detectable metestrus before
- Anaesthetic used for blood collection: Yes (identity) light isoflurane anesthesia
- Animals fasted: Yes for at least 14 hours
- How many animals: All animals
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: The following parameters were determined at the end of the treatment period. Males were sampled on the same nominal day of dosing at the end of the treatment period. Females were sampled on or after day 29 after a metestrus or after a first day of diestrus when there was no detectable metestrus before
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes for at least 14 hours.
- Parameters checked in table 7.5.1/1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once towards the end of the treatment period.
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete macroscopic post-mortem examination was performed on all study animals. This includes examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
HISTOPATHOLOGY: Yes (see table 7.5.1/2)
Other examinations:
THYROID HORMONES:
An additional blood sample (approximately 0.8 mL) was taken from each animal at the end of the treatment period into lithium heparin tubes. The blood was centrifuged (approximately 3000 g for 10 minutes at +4°C) and the plasma was kept at -20°C pending possible analysis. The levels of the thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) will not be determined as there was no indication for an effect on the pituitary-thyroid axis.

ESTROUS CYCLE MONITORING:
The stage of the estrous cycle of each female was determined from a fresh vaginal lavage (stained with methylene blue) every morning from day 15 of dosing, including the day of sacrifice.

SEMINOLOGY:
Before scheduled sacrifice, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.

Epididymal sperm
The left epididymis was removed, weighed and sperm from the cauda was sampled for motility and morphology investigations. Animals were then sacrificed. The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at -20°C pending further investigation.

Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results are expressed as the percentage of motile and non-motile spermatozoa.

Epididymal sperm morphology
Sperm morphology was determined from a smear, after eosin staining, counting 100 spermatozoa per slide. Results are expressed as the percentage of spermatozoa in each of the following categories:
. normal,
. normally shaped head separated from flagellum,
. misshapen head separated from flagellum,
. misshapen head with normal flagellum,
. misshapen head with abnormal flagellum,
. degenerative flagellar defect(s) with normal head,
. other flagellar defect(s) with normal head.

Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron. An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber. Results are expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm
The left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber. Results are expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10).
Statistics:
Citox software (version D.05) was used to perform the statistical analyses of body weight, hematology, blood chemistry and urinalysis.
PathData software (version 6.2d2) was used to perfrom the statistical analysis of organ weight data (level of significance 0.05 or 0.01).
Reprotox software (version B.1) was used to perform the statistical analysis of estrous cycle data by one-way variance analysis and Dunnett's test (mean values being considered as normally distributed, variance being considered as homogeneous) or by Fisher exact probability test (proportions).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One animal found dead in control group. Ptyalism in all animal treated with 1000 mg/kg bw/d from day 7.
Mortality:
mortality observed, treatment-related
Description (incidence):
One animal found dead in control group. Ptyalism in all animal treated with 1000 mg/kg bw/d from day 7.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below and Table 7.5.1/3
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see table 7.5.1/4
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below and table 7.5.1/6
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver and kidney at 1000 mg/kg bw/d. See table 7.5.1/8.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyaline droplets in kidney in males treated at all doses and minimal centrilobular hypertrophy in 3/5 males at 1000 mg/kg bw/d
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test item-related deaths.
One control male (X28604) was found dead on day 25 just after lateral recumbency and dyspnea had been suddenly observed. Minor changes seen at necropsy consisted of red discoloration on the thymus and white deposit in stomach. None of these changes had histological correlation. Microscopic examination did not show any relevant changes and the cause of death could not be determined. The slight congestion seen in the lungs was considered to be non specific, probably agonal.
All animals treated at 1000 mg/kg/day had ptyalism from day 7. This was considered to be related to treatment with the test item.
Incidental findings included alopecia and chromodacryorrhea were observed in isolated animals.

BODY WEIGHT AND WEIGHT GAIN:
In the second week of treatment, there were lower mean body weight gains in females treated at 300 or 1000 mg/kg/day (up to -71% vs. controls, p<0.01, due at least in part to one high individual control body weight gain during this period following low bodyweight gain in week 1) and in the males treated at 300 mg/kg/day (-20%). However, there was no dose-relationship, this was transient and had no impact on terminal mean body weights or on mean body weight gains over the whole treatment period. Therefore, these findings were considered of no toxicological significance. See table 7.5.1/3.

FOOD CONSUMPTION:
There were no effects of treatment with the test item on mean food consumption. See table 7.5.1/4.

ESTROUS CYCLES:
Evaluation of the estrous cycle showed that females given the test item at 1000 mg/kg/day were either in late diestrus or early proestrus when females given the vehicle were all in diestrus. This was considered not to be test item-related. See Table 7.5.1/5.

SEMINOLOGY
There were no effects of treatment with the test item on sperm parameters: see Table 7.5.1/7.

HAEMATOLOGY
There were no toxicologically relevant effects on hematology parameters.
The statistically lower mean monocyte count noted in the low-dose females (0.14 G/L, vs. 0.29 G/L in controls, p<0.05) was considered incidental

CLINICAL CHEMISTRY
There was a non dose-related, but statistically significant, reduction in inorganic phosphorus blood level at all dose-levels in females when compared with controls. This was not observed in males.
At 1000 mg/kg/day, there was a high and statistically significant mean bile acid blood concentration in females and this level was also high in one male of the same group (66.8 µmol/L), hence the higher mean male group level when compared with controls.
There was also a trend to minimally higher mean cholesterol (both sexes) and triglyceride (all male results above the control range) levels at this high dose-level.
In the absence of correlation with other laboratory results or with microscopic findings (except maybe the minimal centrilobular hypertrophy of the liver in males) and in view of their slight amplitude, all these findings were considered to be of limited toxicological relevance and non adverse. See Table 7.5.1/6.

URINALYSIS
There were no effects of treatment with the test item on urinalysis parameters.

NEUROBEHAVIOUR
There were no test item-related effects at Functional Observation Battery and motor activity examinations.
Hypoactivity was observed in one animal of each test item group versus none in the controls. As there were no dose-relationship and no consistency with motor activity results, a relation with the test item treatment was considered unlikely. Moreover, this clinical sign was not observed over the study.
Increases in forelimb grip strength observed in one animal of each test item group and in three control females was considered to be not treatment-related.

ORGAN WEIGHTS
When compared with controls, the mean absolute and relative liver weights were higher in males given 1000 mg/kg/day, reaching statistical significance value for the relative weight (p<0.05).
The mean absolute and relative kidney weights were higher in both sexes at 1000 mg/kg/day reaching statistical significance for the relative weight in females (p<0.05). A similar not dose related and not statistically significant trend was seen in females given 100 or 300 mg/kg/day.
The mean absolute and relative ovaries weight were higher in females given 300 mg/kg/d (p<0.05 or p<0.01, respectively). However, in the absence of similar trend at higher dose-levels this was considered to be fortuitous.
The mean absolute and relative spleen weights were higher in males given 1000 mg/kg/day but without statistical significance. In the absence of a similar trend in females and relevant histopathological changes, and as spleen weights are generally highly variable in rats, any relationship with treatment was considered to be unlikely.
The mean absolute and relative thymus weights were lower in females given 1000 mg/kg/day without statistical significance. In the absence of a similar trend in males and histopathological correlates and as this was mainly due to individual values, any relationship with the test item was considered to be unlikely. See Table 7.5.1/8.


GROSS PATHOLOGY
No treatment-related changes were seen at necropsy. Changes observed were considered to be part of the normal background commonly seen in the rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
Kidneys
Hyaline droplets were observed in the epithelial cells of proximal tubules of males given the test item at 100, 300 or 1000 mg/kg/day but not in controls with the following incidence and severity. At 1000 mg/kg/day, this was associated with minor increased severity of tubular basophilia.
In females, there were no histopathological changes which correlated with the higher kidney weight at necropsy.
Liver
In the liver, minimal centrilobular hypertrophy was seen in 3/5 males given 1000 mg/kg/day but not in females and correlated with the higher mean liver weight in this sex at this dose-level.
Genital tract
In the female genital tract, no differences were seen between controls and treated animals.
Careful examination of testes did not show any treatment-related changes

DISCUSSION
Presence of hyaline droplets in tubular epithelial cells of proximal tubules in kidneys of treated males is suggestive of treatment-related enhancement of production of the specific male rat alpha 2 microglobulin or of binding of the test item to this protein, thus impairing its catabolism. This change is known to occur following administration of a wide range of xenobiotics (Greaves, 2007) and is considered to be not relevant to human and therefore non adverse. The marginal increased tubular basophilia observed at 1000 mg/kg/day may be part of the same process.
Minimal centrilobular hypertrophy in the liver was considered to represent an adaptative response following administration of the test item and correlated with the higher liver weight in this sex. Due to the low severity of this change and the absence of associated degenerative changes this observation was considered to be non adverse.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No mortality, No change considered to be adverse

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 7.5.1/3: Mean body weight change/Mean body weight (g)

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Body weight change

Days 1 to 8

+51

+49

+48

+50

+12

+20

+19

+24

Days 8 to 15

+35

+38

+28

+36

+31

+20

+9**

+17*

Days 15 to 28

+43

+42

+47

+47

+12

+10

+29

+13

Days 1 to 28

+123

+128

+122

+132

+55

+50

+57

+55

Body weight

Day 1

299

300

299

304

204

199

210

202

Day 28

418

428

421

436

259

250

267

256

Statistical relevance: *: p<0.05, **: p<0.01.

 

Table 7.5.1/4: Mean food consumption (g/animal/day)

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

. Days 1 to 7

29

29

28

30

20

18

20

20

. Days 8 to 14

30

31

29

32

23

21

20

21

. Days 15 to 21

30

31

28

32

21

21

20

20

. Days 22 to 27

28

29

28

31

21

20

21

20

 

Table 7.5.1/5: Estrous cyclicity data

Dose-level (mg/kg/day)

0

100

300

1000

Mean number of cycles per female

3.0

2.6

3.0

3.0

Mean cycle length (days)

4.0

4.4

4.1

4.0

Number of normally cycling females

5/5

4/5

5/5

5/5

An abnormally cycling female is considered to have a mean average cycle of less than 4 days or more than 5 days.

Table 7.5.1/6: Blood biochemistry data

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Inorganic phosphorus (mmol/L)

2.57

2.51

2.65

2.48

2.56

2.13**

2.17*

2.08**

Bile acids (µmol/L)

17.6

14.2

11.5

26.4

13.6

17.4

15.5

23.6**

Cholesterol (mmol/L)

1.4

1.3

1.3

1.7

1.6

1.8

1.9

2.2

Triglycerides (mmol/L)

0.46

0.55

0.54

0.74

0.32

0.44

0.35

0.46

 

Table 7.5.1/7: Sperm analysis data

Dose-level (µg/kg/day)

0

100

300

1000

% of motile sperm

92.9

95.6

95.6

92.4

% of morphologically normal sperm

94.3

94.2

93.4

93.8

Mean number of epididymal sperm(106/cauda)

145

125

140

136

Mean number of sperm heads(106/g testis)

147

136

138

137

 

Table 7.5.1/8: Organ weight data

Sex.

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

100

300

1000

100

300

1000

Exam. animals / Num. of animals

5/5

5/5

5/5

5/5

5/5

5/5

Body weight

+3

0

+3

-1

+3

-1

- Kidneys

  . absolute

-1

+1

+10

+11

+11

+14

  . relative

-4

0

+6

+13

+7

+15*

- Liver

  . absolute

+5

+3

+16

+1

+11

+5

  . relative

+2

+3

+12*

+2

+8

+6

- Ovaries

  . absolute

 

 

 

+8

+19*

-2

  . relative

 

 

 

+9

+16**

-1

- Spleen

  . absolute

+22

+18

+33

-12

+3

-10

  . relative

+19

+18

+30

-11

0

-8

- Thymus

  . absolute

+18

-7

+13

-9

-6

-17

  . relative

+14

-6

+9

-10

-9

-17

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this subacute study, the No Observed Adverse Effect Level (NOAEL) was considered to be of 1000 mg/kg bw/day.
Executive summary:

The test substance triethyl phosphonoacetate (purity of 99%) was administered in drinking water to 5 males and 5 females Sprague Dawley rats by oral gavage at 0, 100, 300 and 1000 mg/kg bw/day for 28 days according to OECD Guideline 407 and in compliance with the GLP. Animals were observed twice daily for mortality and clinical signs. Body weights were recorded before treatment and once weekly during the study, food consumption was also determined once weekly. Haematological examination, clinical chemistry and urinalysis were performed at study termination. The rats were sacrificed between day 29 to32. A microscopic examination was performed on selected tissues from the control- and high-dose animals sacrificed at the end of the treatment period and on liver, kidneys and macroscopic lesions from low- and intermediate-dose animals sacrificed on completion of the treatment period. Sperm analysis was also performed on all males sacrificed at the end of the treatment period and estrous cycle were analysed in females every morning from day 15 of dosing, including the day of sacrifice. Towards the end of the dosing period, a Functional Observation Battery (FOB) including motor activity measurement was performed.

One male animal of the vehicle control group died on day 25. All other animals survived.

All animals treated at 1000 mg/kg bw/day had ptyalism from day 7. This was considered to be related to treatment with the test item.

In the second week of treatment, there were lower mean body weight gains in females treated at 300 or 1000 mg/kg bw/day, these findings were considered of no toxicological significance. There were no effects upon the mean daily food.

There were no test item-related effects at Functional Observation Battery and motor activity examinations.

There was a non dose-related, but statistically significant, reduction in inorganic phosphorus blood level at all dose-levels in females when compared with controls. This was not observed in males. At 1000 mg/kg bw/day, there was a high and statistically significant mean bile acid blood concentration in females and this level was also high in one male of the same group (66.8 µmol/L), hence the higher mean male group level when compared with controls. There was also a trend to minimally higher mean cholesterol (both sexes) and triglyceride (all male results above the control range) levels at this high dose-level.

There were no effects of treatment with the test item on urinalysis parameters. There were no effects of treatment with the test item on sperm parameters nor on the estrous cycle.

The mean absolute and relative kidney weights were higher in both sexes at 1000 mg/kg bw/day reaching statistical significance for the relative weight in females (p<0.05). The histopathological examination, revealed the presence of hyaline droplets in the epithelial cells of proximal tubules of kidney of the males given the test item at 100, 300 or 1000 mg/kg bw/day but not in controls with the following incidence and severity. At 1000 mg/kg bw/day, this was associated with minor increased severity of tubular basophilia. Presence of hyaline droplets in tubular epithelial cells of proximal tubules in kidneys of treated males is suggestive of treatment-related enhancement of production of the specific male rat alpha 2 microglobulin or of binding of the test item to this protein, thus impairing its catabolism. This change is known to occur following administration of a wide range of xenobiotics (Greaves, 2007) and is considered to be not relevant to human and therefore non adverse. The marginal increased tubular basophilia observed at 1000 mg/kg bw/day may be part of the same process.

The mean absolute and relative liver weights were higher in males given 1000 mg/kg bw/day, reaching statistical significance value for the relative weight (p<0.05). Minimal centrilobular hypertrophy was seen in 3/5 males given 1000 mg/kg bw/day but not in females and correlated with the higher mean liver weight in this sex at this dose-level. Minimal centrilobular hypertrophy in the liver was considered to represent an adaptative response following administration of the test item and correlated with the higher liver weight in this sex. Due to the low severity of this change and the absence of associated degenerative changes this observation was considered to be non adverse.

The mean absolute and relative ovaries weight were higher in females given 300 mg/kg bw/d (p<0.05 or p<0.01, respectively). However, in the absence of similar trend at higher dose-levels this was considered to be fortuitous. No difference in the genital tract for the females was revealed by the histopathological examination.

The mean absolute and relative thymus weights were lower in females given 1000 mg/kg bw/day without statistical significance. No treatment-related changes were seen at necropsy. Changes observed were considered to be part of the normal background commonly seen in the rat.

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be of 1000 mg/kg bw/day. Therefore, the submitted substance is not classified for repeated dose toxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 407.