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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:
09 September 2015 - 01 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Version 22 March 1996
Deviations:
yes
Remarks:
no Functional Observation Battery performed
GLP compliance:
yes
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
EC Number:
212-985-6
EC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Cas Number:
901-44-0
Molecular formula:
C19H24O4
IUPAC Name:
2-(4-{2-[4-(2-hydroxyethoxy)phenyl]propan-2-yl}phenoxy)ethan-1-ol
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age/Weight: at the beginning of the treatment, the males were 10 weeks old and had a mean body weight of 435 g (range: 382 g to 483 g) and the females were 9 weeks old had a mean body weight of 237 g (range: 193 g to 272 g).
- Housing: polycarbonate cages- Diet: SSNIFF R/M-H pelleted diet (free access)- Water: tap water filtered with a 0.22 µm filter (free access)- Acclimation period: for a period of 7 days before the beginning of the treatment period. ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.
- IN-LIFE DATES: 13 October 2015 to 01 December 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) methylcellulose 4000 cps aqueous solution
Details on oral exposure:
PREPARATION OF DOSING FORMULATIONS:Type of formulation (visual observation): solution in the vehiclePreparation:
- weigh the required quantity of test item,
- ground the test item using a mortar and pestle,
- add a few drops of vehicle and mix with the test item to obtain a homogeneous mixture,
- progressively add the vehicle and transfer the mixture into a gauged flask,
- rinse the mortar and pestle and add the rinsing liquid into the gauged flask,
- homogenize the suspension by manual stirring before completing to final volume with vehicle,
- homogenize the suspension by magnetic stirring for at least 10 minutes,
- homogenize the suspension by ultraturrax at 11000 rpm for 10 minutes,
- stir the formulations by magnetic stirring at room temperature and protected from light for at least 15 minutes

VEHICLE:
- Justification for use and choice of vehicle: suitable formulation in the vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC/UVTest item concentrations: remained within an acceptable range of variation compared to nominal values when analysed in Weeks 1, 3 and 6
Duration of treatment / exposure:
Males: 5 weeks of treatment in total
Females: 6 to 7 weeks of treatment
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:The dose-levels were selected in agreement with the Sponsor, and are based on the results of a previous study. In this study, five male and five female Sprague Dawley rats were given the test item at 100, 300 or 1000 mg/kg/day for 2 weeks.There were no unscheduled deaths and the only test item treatment-related clinical sign was ptyalism in all animals at 1000 mg/kg/day. There were no toxicologically relevant test item-related changes in mean body weight. When compared with controls, mean body weight gains were transiently lower in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day during the first week of treatment which was considered as non-adverse. Minor toxicologically significantly lower mean food consumption was observed in males at 1000 mg/kg/day in the first week of treatment. The test item administration induced minimal to slight increases in mean liver weights at 1000 mg/kg/day but there were no macroscopic changes related to the test item administration at any doses.Therefore, the same dose-levels were used in the present study
- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.
CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.
DETAILED CLINICAL SIGNS
- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study.
BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.
FOOD CONSUMPTION:
- Time schedule: Males: on the first day of treatment, then once a week until the start of the mating period. Females: on the first day of treatment, then once a week until mating, on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.
HEMATOLOGY
- Time schedule: on the day of scheduled sacrifice.
CLINICAL CHEMISTRY
- Time schedule: on the day of scheduled sacrifice.
Sacrifice and pathology:
SACRIFICE
- Male animals: all surviving animals after 5 weeks of treatment,
- Female animals: all surviving animals = on Day 6 post-partum (p.p).
ORGAN WEIGHTS
- See table below.
GROSS PATHOLOGY
- A complete macroscopic post-mortem examination was performed on all animals.
HISTOPATHOLOGY
- All tissues listed in the Tissue Procedure Table from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. from the control and high-dose groups (groups 1 and 4),
- thymus (females), spleen (males), liver (both sexes), kidneys (females) and adrenals (females) from the first five males sacrificed as scheduled and the first five females sacrificed on Day 6 p.p., from the low- and mid-dose groups (groups 2 and 3),
- all macroscopic lesions from all groups,
- all tissues listed in the tissue Procedure Table from all animals that died or were sacrificed prematurely.
Statistics:
Statistical analyses were performed on body weight, food consumption, hematology, blood biochemistry and organ weight data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see Table 1 : Ptyalism was noted at 300 and 1000 mg/kg/day in a dose-related manner (incidence and duration) and was ascribed to the test item treatment.At 1000 mg/kg/day, piloerection and round back were observed in both sexes in several animals. One female was pale and cold to the touch at the same time on the first days of lactation (low food consumption was concomitantly recorded in this female) and reminded clinical signs noted in the prematurely sacrificed female in lactation. All these clinical signs were considered to be test item treatment related.At 300 mg/kg/day, there was 1/20 animals with piloerection and round back in premating and gestation period, and one other female with piloerection on Days 5 and 6 p.p.. There were no test item treatment-related clinical signs at 100 mg/kg/day. The other clinical signs recorded in the study (chromorhinorrhea, cutaneous lesion, area of hair loss, soft feces, necrosed tail, scab, short tail, reflux at dosing) were not considered to be test item treatment-related: there were in limited incidences and/or are of common background of laboratory rats.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no premature deaths in males.
At 1000 mg/kg/day:
-one female was prematurely sacrificed on Day 6 due to poor clinical condition (piloerection, round back, emaciated appearance, dehydration, hypoactivity and dyspnea). Reduced size of thymus and spleen and distended colon/rectum were observed. Lymphoid atrophy was noted in the thymus (cortex and medulla), in the spleen (all compartments) and in lymph nodes (mesenteric and mandibular). Slight hepatocellular hypertrophy and slight tubular vacuolation, dilated tubules along with hyaline casts in the kidney were also recorded. The cause of death was not evident but the lesions in kidney may have been a contributing factor,
- one other female was prematurely sacrificed during lactation on Day 2 p.p. due to poor clinical condition (piloerection, emaciated appearance, pallor of extremities, cold to the touch, hypoactivity, bent head, locomotory difficulties and dyspnea; loss of 9% of body weight from Days 1 to 2 p.p.). 67% of its pups did not have milk in the stomach on the day of sacrifice. Brown contents were observed in the female stomach, which was associated with multifocal erosion of the mucosa on microscopic examination. Likewise female, lymphoid atrophy was observed in several immune tissues, including thymus (cortex and medulla), spleen (all compartments), and lymph nodes (mesenteric and mandibular). Minimal tubular vacuolation in the kidney were also recorded. The cause of death was not evident.Both deaths were likely related to the test item treatment (both at the high-dose).
At 300 mg/kg/day:
- one female was prematurely sacrificed on Day 22 p.c. due to difficulties to deliver. The female had on that day piloerection, pallor of extremities and what was thought by the animal technician to be a fetus blocked in the vagina but which was in fact a vaginal septum transverse (seen at necropsy) preventing the delivery. Brown translucent contents were observed in both uterine horns, along with one implantation scar, three live and one dead fetuses in the right horn and two live and one dead fetuses in the left one. Vagina was enlarged and contained brown, translucent materials. As the vaginal septum transverse was a congenital anomaly, these difficulties to deliver and death were not considered to be test item treatment-related.There were no other premature deaths of females in the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Table 2At 1000 mg/kg/day and when compared with controls:- in males, statistically significantly lower mean body weight was noted as soon as in the first week of treatment (down to -10% vs. controls at termination). Mean body weight gain was statistically significantlylower in the first week of treatment and over the entire treatment period,- in females, mean body weight was statistically significantly lower during gestation and lactation (down to -12% vs. controls at the beginning of lactation). Mean body weight gain was particularly low in the firstweek of treatment as in males. These effects were not considered to be adverse as they were of slight amplitude.At 100 and 300 mg/kg/day, when compared with controls, mean body weight gains of males were statistically significantly lower in the first week of treatment and/or over the entire treatment period. These effects were considered to be of limited toxicological significance; mean body weights were not toxicologically affected. There were no toxicologically significant effects in mean body weight or mean bodyweight gain in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see Table 3At 1000 mg/kg/day and when compared with controls, mean food consumption was statistically significantly lower during the first week of treatment in both sexes. These variations were considered to be non-adverse as there were not severe and transient.There were no test item treatment-related effects on mean food consumption at 100 and 300 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 4 In males at 1000 mg/kg/day and when compared with controls, there were statistically significantly lower mean reticulocyte count indicating decreased red blood cells turnover and correlating with decreased extramedullary hematopoïesis seen microscopically in the spleen. This finding was considered of minor toxicological significantly since the change was mild in amplitude and not associated with changes in other mean red blood cell parameters.In females at 1000 mg/kg/day and when compared with controls, lower mean hemoglobin concentration was observed along with red blood cell count and hematocrit and higher reticulocyte count. These differences suggested an accelerated red blood cells turnover and correlated with increase of hematopoietic foci seen microscopically. The cause for these changes remained unknown since no evidence of blood loss or hemolysis could be found.There were no toxicologically significant effects at 100 and 300 mg/kg/day.At 1000 mg/kg/day and when compared with controls, there was a tendency towards a shortened APTT in both sexes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 5 When compared with controls, there were dose-related higher mean cholesterol levels in test item groups. Triglycerides level was also higher in females treated at 1000 mg/kg/day (same tendency at 300 mg/kg/day). The higher mean ALAT activity and mean bile acids level in females at this dose, as well as higher mean bile acids level in females at 300 mg/kg/day, were considered to be of low toxicological significance (low amplitude of variation or no statistical significance).At 1000 mg/kg/day, higher mean urea and calcium levels were also noted, in both sexes. Mean creatinine concentration tended to be higher in males but the contrary was observed in females, therefore a relationship with the test item treatment was unclear.The statistically significant differences in potassium, phosphorus and albumin concentrations detailed in the above table were considered to be incidental (no dose-relationship).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see Table 6 Increased absolute and/or relative-to-body liver weights were recorded in males and females treated at 300 or 1000 mg/kg/day. This finding correlated with hepatocellular hypertrophy and therefore was attributed to test item treatment. Lower absolute and/or relative-to-body adrenal weights were noted in females treated at 1000 mg/kg/day. This correlated with a trend toward decreased size of cortical cells and therefore, this was considered to be test item treatment-related.Lower absolute and/or relative-to-body spleen weights were observed in males treated at 300 or 1000 mg/kg/day, possibly as a results of decreased hematopoïsesis in the red pulp. This was considered to be test item treatment-related.There was a trend toward lower thymus weights in females at 1000 mg/kg/day. As this correlated with a trend toward lymphoid atrophy in this tissue, this was attributed to treatment with the test item, possiblyas a result of decreased terminal body weights and the associated stress.The higher relative-to-body mean brain weight observed in males treated at 1000 mg/kg/day was due to lower terminal body weights and therefore it was not considered to be toxicologically relevant.Lower absolute heart weights were recorded in males treated at 300 and 1000 mg/kg/day. As these weight changes did not correlate with any macroscopic or microscopic finding, a relationship to test item treatment was considered to be unlikely.As other mean weight changes were minimal, or of opposing trend between males and females, a relationship to test item treatment was considered to be unlikely.
Gross pathological findings:
no effects observed
Description (incidence and severity):
see Table 7
Prematurely dead rats : Two females treated at 1000 mg/kg/day were euthanized prematurely on ethical grounds. Brown contents were observed in the stomach of one female, which was associated with multifocal erosion of the mucosa on microscopic examination. Reduced size of thymus and spleen and distended colon/rectum were observed in one female. The cause of death was not evident. One female treated at 300 mg/kg/day was euthanized prematurely on ethical grounds. Brown translucent contents were observed in both uterine horns, along with one implementation scar, three live and one dead fetuses in the right horn and two live and one dead fetuses in the left one. A transverse septum was observed in vagina, which was enlarged and contained brown, translucent materials. Terminal sacrifice No gross findings were attributed to treatment with the test item. The macroscopic findings were of those commonly reported in the rat without any indication of relationship to test item treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Table 1 Prematurely dead rats Two females treated at 1000 mg/kg/day were euthanized prematurely on ethical grounds. Multifocal foci of mucosal erosion were noted in one female, which correlated well with brown contents on gross examination. Minimal tubular vacuolation in the kidney were also recorded. Lymphoid atrophy was observed in several immune tissues, including thymus (cortex and medulla), spleen (all compartments),and lymph nodes (mesenteric and mandibular).One likewise female, lymphoid atrophy was noted in the thymus (cortex and medulla), in the spleen (all compartments) and in lymph nodes (mesenteric and mandibular) of one female. Slight hepatocellular hypertrophy and slight tubular vacuolation, dilated tubules and hyaline casts in the kidney were also recorded.The findings in the liver (hypertrophy correlated with higher liver weights) and kidney (at least the vacuoles) were attributed to treatment with the test item.The lymphoid atrophy was considered to be possibly related to poor health condition and the associated stress.One female treated at 300 mg/kg/day was euthanized prematurely on ethical grounds.Periportal liver hypertrophy was observed, along with hematopoietic foci.
Extramedullary hematopoiesis was marked in the spleen. These findings were also noted in the surviving rats and were considered to be test item treatment-related.Terminal sacrificeLiverMinimal to slight hepatocellular hypertrophy (centrilobular to diffuse) was observed in all males and females at 1000 mg/kg/day and in 1/5 male at 300 mg/kg/day. This microscopic finding was not considered to be adverse, but rather adaptive.
Hematopoietic system Extramedullary hematopoïesis was decreased in the spleen from males at 300 and 1000 mg/kg/day and there was an increase of hematopoietic foci in the liver from females treated at 1000 mg/kg/day, which correlated well with a trend toward decrease in reticulocyte counts in males and an increase in females.
KidneyMinimal tubular vacuolation was observed in the kidneys from females from 100 mg/kg/day onwards, in a dose-related manner.Thymus There was a trend toward lymphoid atrophy in the thymus (decreased cortex and medulla) in females treated at 1000 mg/kg/day.
Adrenals There was a trend toward decreased cortical cell size in adrenals from females treated at 1000 mg/kg/day, which correlated with lower adrenal weights. This was better observed in the zona reticularis.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1. Clinical signs noted in surviving animals

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Number of animalsa

10

10

10

10

10

10(P,G), 9(L)

10(P), 9(G),

8(L)

9(P,G), 8(L)

Ptyalism

 

 

6

10

 

 

4(P), 3(G), 1(L)

9(P,G), 6(L)

Pallor of extremities

 

 

 

 

 

 

 

1(L)

Cold to the touch

 

 

 

 

 

 

 

1(L)

Piloerection

 

 

 

4

 

 

1(P,G), 1(L)

1(P),3(G;L)

Round back

 

 

 

3

 

 

1(P,G)

2(P,L)

P: premating and mating periods, G: gestation period, L: lactation period.

a: for females: No. of femalesin the different phases, excluding theprematurely sacrificed females.

Table 2 Mean body weights and mean body weight changes

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Pre-mating (females) or whole study (males)

 

 

 

 

Day 1

436

436

429

437

237

238

237

236

 

 

(0%)

(-2%)

(0%)

 

(0%)

(0%)

(0%)

Day 8

483

468

462

442*

250

253

250

239

 

 

(-3%)

(-4%)

(-8%)

 

(+1%)

(0%)

(-4%)

Day 15

509

488

479

463**

261

263

258

254

 

 

(-4%)

(-6%)

(-9%)

 

(+1%)

(-1%)

(-3%)

Day 22

520

499

489

470**

/

/

/

/

 

 

(-4%)

(-6%)

(-10%)

 

 

 

 

p.c.: post-coitum, p.p.: post-partum, /: not applicable, in brackets: differences from control values.

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

(continued)

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Day 29

536

521

502

480**

/

/

/

/

 

 

(-3%)

(-6%)

(-10%)

 

 

 

 

Day 36

559

537

520

504**

/

/

/

/

 

 

(-4%)

(-7%)

(-10%)

 

 

 

 

Days 1-8

46

32*

32*

5#

13

15

13

4*

Days 8-15

26

20

18

20

10

9

9

15

Days 1-15

73

52*

50**

26#

24

24

21

19

Days 15-36

50

49

41

41

/

/

/

/

Days 1-36

122

101

91*

67#

/

/

/

/

Gestation

 

 

 

 

 

 

 

 

Day 0 p.c.

/

/

/

/

268

271

262

249

 

 

 

 

 

 

(+1%)

(-2%)

(-7%)

Day 7 p.c.

/

/

/

/

312

308

308

284*

 

 

 

 

 

 

(-1%)

(-1%)

(-9%)

Day 14 p.c.

/

/

/

/

350

346

343

316**

 

 

 

 

 

 

(-1%)

(-2%)

(-10%)

Day 20 p.c.

/

/

/

/

438

428

423

398*

 

 

 

 

 

 

(-2%)

(-3%)

(-9%)

Days 0-20 p.c.

/

/

/

/

170

157

162

149

Lactation

 

 

 

 

 

 

 

 

Day 1 p.p.

/

/

/

/

328

328

325

288**

 

 

 

 

 

 

(0%)

(-1%)

(-12%)

Day 5 p.p.

/

/

/

/

346

337

341

313*

 

 

 

 

 

 

(-3%)

(-1%)

(-10%)

Days 1-5 p.p.

 

 

 

 

18

9

17

27

 

p.c.: post-coitum, p.p.: post-partum, /: not applicable, in brackets: differences from control values.

*: p<0.05, **: p<0.01, #: p<0.001.

Table 3 Mean food consumption

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Pre-mating

 

 

 

 

 

 

 

 

Days 1 - 8

  36

34

(-6%)

36

(0%)

28**

(-22%)

21

21

(0%)

20

(-5%)

16#

(-24%)

Days 8 -15

36

33

34

33

21

22

21

21

Gestation

 

 

 

 

 

 

 

 

Days 0 - 7 p.c.

/

/

/

/

27

25

27

25

Days 7 - 14 p.c.

/

/

/

/

31

33

30

28

Days 14 - 20 p.c.

/

/

/

/

32

32

29

30

Lactation

 

 

 

 

 

 

 

 

Days 1 - 5 p.p.

/

/

/

/

47

41

44

42

p.c.: post-coitum, p.p.: post-partum, /: not applicable, in brackets: differences from control values.

**: p<0.01, #: p<0.001.

Table 4 Relevant mean hematology data

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Red blood cells (T/L)

9.01

8.79

9.26

8.94

7.28

7.34

7.31

6.72

Hemoglobin (g/dl)

15.9

15.5

16.1

15.8

14.1

14.1

13.7

12.6*

Hematocrit (PCV; L/L)

0.49

0.47

0.49

0.48

0.42

0.42

0.41

0.39

Reticulocytes (%)

(T/L)

2.58

0.23

2.32

0.20

1.96

0.18

1.69

0.15*

5.13

0.38

5.12

0.37

4.66

0.34

7.28

0.49

*: p<0.05. The significance concerned the organ weights values and not the percentages.

 

 (continued) 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

APTT (s)

14.4

15.4

14.3

11.5

14.0

11.5

12.5

9.6

 

Table 5 Relevant mean blood biochemistry data

   

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Cholesterol (mmol/L)

1.89

2.23

2.80*

4.31**

1.36

2.28*

2.74**

4.27**

Triglycerides (mmol/L)

0.60

0.86

1.08

0.76

0.74

0.87

1.33

1.92**

ALAT (U/L)

43

41

45

56

80

71

87

108*

Bile acids (µmol/L)

37.3

28.0

44.6

59.1

56.3

50.4

106

118

Calcium (mmol/L)

2.61

2.55

2.64

2.82*

2.70

2.68

2.73

2.86*

Urea (mmol/L)

5.5

5.0

5.6

7.1*

7.3

7.2

8.1

9.2*

Creatinine (µmol/L)

31.8

31.4

30.9

34.4

35.3

37.3

34.8

30.2

Potassium (mmo/L)

4.22

3.82*

3.89

3.80*

3.90

3.89

4.11

4.05

Phosphorus (mmol/L)

2.21

2.00

1.95

2.15

2.81

2.67

2.41*

2.46

Albumin (g/L)

39

36**

39

37

38

37

36

39

*: p<0.05, **: p<0.01. The significance concerned the organ weights values and not the percentages.

Table 6 Percentage of differences in mean organ weights vs. control means

 

Sex

Male

Female

Dose-level (mg/kg/day)

100

300

1000

100

300

1000

- Final body weight

-2

-7

-11**

-1

0

-9*

- Adrenal glands

. absolute

-15

-32*

-18

-19

-21

-23*

. relative

-13

-27

-8

-18

-27*

-22*

- Liver

. absolute

0

+7

+10

+3

+27*

+41**

. relative

+4

+15*

+24**

+4

+17*

+44**

- Spleen

. absolute

-12

-23**

-23**

+5

+2

+12

. relative

-8

-16**

-12

+5

-6

+13

- Thymus

. absolute

+6

+2

-25

0

+4

-24

. relative

+10

+11

-14

0

-5

-23

*: p<0.05, **: p<0.01. The significance concerned the organ weights values and not the percentages.

 

Table 7 Microscopic examination

Incidences and severities of the test item-related microscopic findings in the liver

 

Finding

Male

Dose-level (mg/kg/day)

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Number examined

6

5

5

5

5

5

5

5

Liver

 

 

 

 

 

 

 

 

Hepatocellular hypertrophy

0

0

1

5

0

0

0

5

- grade 1

- grade 2

-

-

-

-

1

-

-

5

-

-

-

-

-

-

3

2

-: not examined or observed.

Grade 1: minimal. Grade 2: slight.

(continued)

Incidences and severities of the test item-related microscopic findings in the liver and spleen

 

Finding

Male

Dose-level (mg/kg/day)

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Number examined

6

5

5

5

5

5

5

5

Hematopoiesis in the liver

- grade 1

 

0

-

 

0

-

 

0

-

 

0

-

 

0

-

 

1

1

 

1

1

 

4

4

 

Hematopoiesis in the spleen

- grade 1

- grade 2

- grade 3

- grade 4

 

4

1

3

-

-

 

4

2

2

-

-

 

5

4

1

-

-

 

1

-

1

-

-

 

5

-

1

4

-

 

-

-

-

-

-

 

-

-

-

-

-

 

5

-

1

3

1

 

-: not examined or observed.In bold: considered to be test item treatment-related.

Grade 1: minimal; grade 2: slight, grade 3: moderate, grade 4: marked.

(continued)

Incidences and severities of the test item-related microscopic findings in the kidneys

 

Finding

Male

Dose-level (mg/kg/day)

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Number examined

5

0

0

5

5

5

5

5

Vacuolation, tubule

- grade 1

 

0

-

 

-

-

 

-

-

 

0

-

 

0

-

 

1

1

 

2

2

 

3

3

 

-: not examined or observed.

Grade 1: minimal.

 

Incidences and severities of the test item- related microscopic findings in the thymus

 

Finding

Male

Dose-level (mg/kg/day)

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Number examined

5

0

0

5

5

5

5

5

Lymphoid atrophy

- grade 2

- grade 3

0

-

-

-

-

-

-

-

-

0

-

-

0

-

-

0

-

-

0

-

-

3

2

1

-: not examined or observed.

Grade 2: slight, grade 3: moderate.

(continued)

Incidences and severities of the test item-related microscopic findings in the adrenals

 

Finding

Male

Dose-level (mg/kg/day)

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Number examined

5

0

0

5

5

5

5

5

Decreased cortical cell size

- grade 1

 

0

-

 

-

-

 

-

-

 

0

-

 

0

-

 

0

-

 

0

-

 

5

5

 

-: not examined or observed.

Grade 1: minimal.

Applicant's summary and conclusion

Conclusions:
The test item, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 5 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day. Based on the experimental conditions and results of this study:- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the deaths and the accumulation of effects (clinical signs, effects on mean body weight, food consumption, hematology and blood biochemistry data, pathology findings) at 1000 mg/kg/day.
Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item, following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 5 post-partum (p.p.).

This study provides initial information on possible effects:

- on basic indication on immunological effects likely to arise from repeated exposure over a relatively limited period of time.

- on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus, and parturition (not discussed in this part).

 

Methods

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item, daily by oral (gavage) administration before mating, through mating and, for the males, until sacrifice, and for the females, through gestation until Day 5 p.p.. The test item was administered at dose-levels of 100, 300 and 1000 mg/kg/day in the vehicle [0.5% (w/v) methylcellulose4000 cpsaqueous solution]. Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

The concentration of the dose formulations was checked in study Weeks 1, 3 and 6.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating, mating (food consumption not during mating) and gestation (0, 7, 14 and 20 p.c.) periods and during lactation on Days 1 and 5 p.p..

Prior to sacrifice, blood samples were taken from five males and five females per group for analysis of hematology and blood biochemistry parameters.

The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p.. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on several organs from five males and five females in the control and high-dose groups, on thymus, spleen, liver, kidneys and adrenals from five males and/or females in the low- and mid-dose groups, on all macroscopic lesions and reproductive organs from animals that did not conceive.

Results

 

The test item concentrations in dose formulations analyzed were within an acceptable range of variations when compared with the nominal values (nominal concentration ± 15%). There was no test item observed in the control dose formulations.

 

At 1000 mg/kg/day, the test item treatment-related effects are described below:

- there were two females prematurely sacrificed on Day 2 or 6 p.p. following poor clinical condition (piloerection, emaciated appearance, hypoactivity, dyspnea, etc) which were likely related to the test item treatment,

- ptyalism was noted in all animals at several occasions; piloerection and round back were observed in both sexes, and one female was also pale and cold,

- mean body weight was lower than in controls from the first week of treatment in males (down to -10%, p<0.01) and during gestation and lactation in females (down to -12%, p<0.01),

- mean food consumption was lower than in controls during the first week of treatment in both sexes (-22 to -24%, p<0.01 or 0.001),

- in males there were lower mean reticulocyte count (0.15 T/L vs. 0.23, p<0.05). In females there were lower mean hemoglobin concentration (12.6 g/dL vs. 14.1 in controls, p<0.05) along with lower mean red blood cell count and mean hematocrit (6.72 G/L and 0.39 L/L vs. 7.28 and 0.42), while mean reticulocyte count was higher (0.49 T/L vs. 0.38),

- there was a tendency towards a shortened APTT (11.5 s in males and 9.6 s in females,vs.14.4 and 14.0, respectively, in controls),

- there were higher mean cholesterol (both sexes: 4.31 and 4.27 mmol/L vs. 1.89 and 1.36 in controls, p<0.01) and triglycerides (females: 1.92 mmol/L vs. 0.74, p<0.01) concentrations. Mean ALAT activity (108 U/L vs. 80, p<0.05) and mean bile acids level (118 µmol/L vs. 56.3) were also higher in females but considered of low toxicological significance. Higher mean urea (7.1 and 9.2 mmol/L vs. 5.5 and 7.3, p<0.05) and calcium (2.82 and 2.86 mmol/L vs. 2.61 and 2.70, p<0.05) levels were also noted, in both sexes,

- there were higher mean liver weight in both sexes and lower mean adrenals (females) and spleen (males) weights, as well as a tendency towards lower mean thymus weights in females,

- minimal to slight hepatocellular hypertrophy was observed in both sexes, andminimal renal tubular vacuolation and a trend towards lymphoid atrophy in the thymus or towards decreased cortical cell size in adrenals in females. Extramedullary hematopoïesis was decreased in the spleen from males while increase of hematopoietic foci was noted in the females and correlated with reticulocyte count.

At 300 mg/kg/day, ptyalism was also noted in about half of the animals and two females had piloerection plus round back for one of them. Mean body weight gains of males were lower in the first week of treatment (+32 g vs. +46, p<0.05) and over the entire treatment period when compared with controls. Mean cholesterol level was also higher in both sexes than in controls (2.80 and 2.74 mmol/L, p<0.05 or 0.01) and there was a trend towards higher mean bile acids level in females (106 µmol/L).

Increased absolute and/or relative-to-body mean liver weights were recorded. This finding correlated with hepatocellular hypertrophy only in one male. Lower absolute and/or relative-to-body mean spleen weights were observed in males, which was associated with a trend toward decreased hematopoïsesis in the red pulp.


Minimal tubular vacuolation was observed in the kidneys from 2/5 females.

 

At 100 mg/kg/day and when compared with controls, mean body weight gains of males were lower in the first week of treatment (+32 g vs. +46, p<0.05) and mean cholesterol level was higher in both sexes (2.23 and 2.28 mmol/L, p<0.05 in females).

Minimal tubular vacuolation was observed in the kidneys from 1/5 females.

  

Conclusion

 

The test item, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 5 p.p.for females, at dose-levels of 100, 300 and 1000 mg/kg/day.

 

Based on the experimental conditions and results of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the deaths and the accumulation of effects (clinical signs, effects on mean body weight, food consumption, hematology and blood biochemistry data, pathology findings) at 1000 mg/kg/day,