Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
06 Jun 2013 - 20 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Jun 2013 - 20 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 22 Mar 1996
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
no endocrine disruptor endpoints adressed
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 191 to 204 g (males) and 172 to 179 g (females)
- Housing: From arrival to pairing: animals were housed 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating: animals were housed one male to one female in clear polysulphone cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating; the females were transferred to individual solid bottomed cages (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy) for the gestation period and parturition.
- Diet: laboratory rodent diet, 4 RF 21 (Mucedola S.r.l., Settimo Milanese (MI), Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous carboxymethylcellulose (0.5% CMC)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in the vehicle. Formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL for dose levels of 100, 300 and 1000 mg/kg bw/day, respectively
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1 male to 1 female (monogamous).
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged singly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test substance in the vehicle were verified by gas chromatopgraphy with a flame ionisation detection (FID). Concentration verification was conducted on a weekly basis.
Duration of treatment / exposure:
Males: The daily administration of the test item was started two weeks before mating and lasted until test day 28 to 29, which was one day before sacrifice.
Females: The daily administration of the test item was started two weeks before mating and continued until day 3 post-partum.
Maximum: 54 days of treatment.
Frequency of treatment:
once daily; 7 days/week
Details on study schedule:
not applicable for OECD 422 study
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 14-day range-finding study (Rossiello, 2013. RTC Study No.: 93730EXT)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during the study, each animal was observed and any clinical signs recorded.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).

BODY WEIGHT: Yes
- Time schedule for examinations: females: weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

For further observations and examinations (water intake, haematology, clinical chemistry, neurobehaviour), see "Repeated dose toxicity: oral" (chapter 7.5.1)

OTHER:
Reproduction paramters: number of pregnant females, pre-coital time, gestation length
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following: anomalies of the oestrous cycle and pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, and qualitative sperm staging.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter weight, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [males were sacrificed on day 29 or 30]
- Maternal animals: All surviving animals [females were sacrifices on day 4 post-partum or shorty thereafter]

GROSS PATHOLOGY: Yes
-Organ weights: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, heart, kidneys, liver, ovaries with oviducts, parathyroid glands, prostate gland, seminal vesicles with coagulating glands, spleen, testes, thymus (where present), thyroid and uterus-cervix,
-Fixation: adrenal glands, bone marrow (from sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, clitoral gland, colon, duodenum, epididymides, heart, ileum (including Peyer’s patches), jejunum, kidneys, liver, lungs (including mainstem bronchi), lymph nodes (mesenteric and cervical), ovaries with oviducts, parathyroid glands, pituitary gland, penis, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating glands, spinal column, spinal cord (cervical, thoracic, lumber), spleen, stomach, testes, thymus (where present), thyroid, trachea, urinary bladder, uterus-cervix and vagina.

HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)
Postmortem examinations (offspring):
SACRIFICE
- The surviving F1 offspring were sacrificed at 4 days of age.


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations].
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
The criterion for statistical significance was p<0.05
Reproductive indices:
Male Copulatory Index (%) = No. of animals mated/No. of animals paired x 100
Male Fertility Index (%) = No. of males which induced pregnancy/ No. of males paired x 100
Female Copulatory Index (%) = No. of animals mated/No. of animals paired x 100
Female Fertility Index (%) = No. of pregnant females/No. of females paired x 100
Males and females:
Precoital interval = Mean number of days between pairing and mating
Offspring viability indices:
Pre-implantation loss [%] = (No. of corpora lutea - No. of implantations/ No. of corpora lutea) x 100
Pre-birth loss [%] = (No. of visible implantations - total litter size at birth/ No. of visible implantations
) x 100
Pup loss at birth [%] = (Total litter size - live litter size/ Total litter size) x 100
Cumulative pup loss on Day 4 post-partum [%] = (Total litter size at birth - live litter size at Day 4/ Total litter size at birth) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were observed in males and females throughout the study.
Mortality:
no mortality observed
Description (incidence):
No mortality were observed in males and females throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No difference of toxicological significance were seen in body weight or body weight gain. No relevant differences in terminal body weight were seen between the controls and treated animals of both sexes.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No changes of toxicological significance were observed. The statistically significant differences be tween control and treated animals (erythrocytes, mean corpuscular haemoglobin and leucocytes in males, haemoglobin, haematocrit and platelets in females) were of low magnitude and/or not dose related, therefore considered incidental. No changes were recorded at the coagulation tests.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant fluctuations of some biochemical parameters (mean group values) were recorded such as: increase of glucose in males dosed with 100 and 1000 mg/kg/day (48% for both dosages), increase in urea in those receiving 100 mg/kg/day (19%), increase of aspartate aminotrans ferases in females dosed with 300 mg/kg/day (35%), decrease of bilirubin (81%) and increase of potassium (10%) in those treated with 1000 mg/kg/day when compared to controls. Due to the lack of dose- and sex-consistency and to the absence of other relevant findings, the above alterations were considered unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. No significant differences were observed between groups, in motor activity, grip strength and sensory reactivity to stimuli, evaluated at the end of treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No relevant difference in oestrous cycle was observed in treated females when compared to controls.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
The number of corpora lutea, implantations, total litter size, pre-implantation loss and pre-birth loss did not differ significantly between groups. Gestation length was also comparable between groups. No dif
ferences were observed in the pre-coital interval, copulatory and fertility indices between control and treated groups.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Similar signs such as pallor, cold to touch and/or bruise muzzle were recorded in pups of both control and high dose groups. Small foetuses were seen in mid- and high dose groups as well as in control. No toxicological relevance was attributed to the signs since they were present in treated as well as in control animals.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
All pregnant females gave birth to live pups with the exception of one high dose female which had a total litter loss on day 3 post-partum. This female and two control females had unilateral implantation but was not treatment related. This finding was considered incidental since it was observed also in two control females.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Litter data including mean litter and pup weights were comparable between groups.
Food efficiency:
not examined
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 signs were seen in pups sacrificed on Day 4 post partum.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio of pups showed a slight increased number of males in high dose group. No toxicological relevance was attributed to this observation.
VIABILITY (OFFSPRING)

All pregnant females gave birth to live pups with the exception of one high dose female which had a total litter loss on day 3 post-partum. This female and two control females had unilateral implantation. This finding was considered incidental since it was observed also in two control females.

CLINICAL SIGNS (OFFSPRING)

Clinical signs of pups such as pallor, cold to touch, small and/or bruise muzzle, were observed in control, mid- and high dose groups. No toxicological relevance was attributed to these signs since they were seen in treated as well as in control groups.

BODY WEIGHT (OFFSPRING)

Litter data including mean litter and pup weights were comparable between groups. Sex ratio of pups showed a slight increased number of males in high dose group respect to control. No toxicological relevance was attribute to the statistical significant increase observed on Day 4.

GROSS PATHOLOGY (OFFSPRING)

Decedent pups were generally autolysed. No signs were seen in pups sacrificed on Day 4 post partum.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed in the study
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1: Fate of females: Group incidence

 

Treatment (mg/kg/bw/d)

0

100

300

1000

Initial group site

10

10

10

10

Unilateral Implantation

2

0

0

1

Total litter loss

0

0

0

1

With live pups on day 4 post-partum

10

10

10

9

Table 2: Implantation, pre-implantation loss data, pre-birth loss data and gestation length of females – Group mean data

Treatment (mg/kg/bw/d)

 

Corpora Lutea

Implantations

Total Litter size at birth

Pre-implantation loss %

Pre-birth loss %

Gestation length (days)

0

Mean

18.40

18.10

15.60

1.44

12.70

22.10

 

SD

3.37

3.18

5.27

3.17

25.82

0.32

 

n

10

10

10

10

10

10

 

100

Mean

16.30

15.80

14.20

3.26

10.09

22.10

 

SD

3.23

3.61

3.49

8.44

8.48

0.32

 

n

10

10

10

10

10

10

 

300

Mean

16.90

16.90

15.60

0.00

7.65

22.0

 

SD

1.97

1.97

2.17

0.00

7.35

0.00

 

n

10

10

10

10

10

10

 

1000

Mean

18.10

17.40

16.40

6.16

8.21

22.10

 

SD

5.17

5.32

5.44

12.73

9.93

0.32

 

n

10

10

10

10

10

10

Table 3: Litter data at birth, on day 1 and on day 4 post-partum of pregnant females – Group mean data

Treatment (mg/kg/bw/d)

 

At birth

On day 1 post-partum

On day 4 post-partum

Total litter size

Live litter size

Pup loss (%)

Litter weight (g)

Mean pup weight (g)

Live litter size

Cumulative loss (%)

Litter weight (g)

Mean pup weight (g)

0

Mean

15.60

15.40

1.13

101.62

7.00

14.40

6.37

132.38

9.61

SD

5.27

5.19

2.40

30.0

1.09

4.60

7.58

36.25

1.58

n

10

10

10

10

10

10

10

10

10

100

Mean

14.20

14.20

0.00

96.87

7.01

14.10

0.56

145.70

10.51

SD

3.49

3.49

0.00

19.08

0.81

3.38

1.77

29.27

1.08

n

10

10

10

10

10

10

10

10

10

300

Mean

15.60

15.60

0.00

106.70

6.94

14.80

5.51

143.13

9.76

SD

2.17

2.17

0.00

13.11

0.44

2.66

5.60

22.54

0.97

n

10

10

10

10

10

10

10

10

10

1000

Mean

16.40

16.20

5.50

115.50

7.10

15.60

13.30

163.63

9.46

SD

5.44

5.67

15.71

41.59

0.55

5.82

30.67

22.38

0.74

n

10

10

10

10

10

10

10

9

9

Table 4: Sex ratio of pups – Group mean data

Treatment (mg/kg/bw/d)

 

At birth

On day 4 post-partum

Males

Females

Total

% Males

Males

Females

Total

% Males

0

Mean

6.90

8.70

15.60

49.67

6.40

8.00

14.40

49.66

SD

2.02

4.03

5.27

19.96

1.71

3.59

4.60

19.93

n

10

10

10

10

10

10

10

10

100

Mean

6.90

7.30

14.20

50.50

6.80

7.30

14.10

50.14

SD

1.79

2.95

3.49

15.38

1.81

2.95

3.38

15.72

n

10

10

10

10

10

10

10

10

300

Mean

6.20

9.40

15.60

39.23

5.90

8.90

14.80

39.36

SD

2.20

1.65

2.17

11.28

2.08

1.66

2.66

10.29

n

10

10

10

10

10

10

10

10

1000

Mean

9.10

7.30

16.40

55.58

9.56*

7.78

17.33

55.66

SD

3.57

3.62

5.44

12.47

2.24

2.86

2.06

12.97

n

10

10

10

10

9

9

9

9

*= mean value of group is significantly different from control 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1996
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
yes
Remarks:
no endocrine disruptor endpoints addressed
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetradecyl oleate
EC Number:
244-949-0
EC Name:
Tetradecyl oleate
Cas Number:
22393-85-7
Molecular formula:
C32H62O2
IUPAC Name:
tetradecyl octadec-9-enoate

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 191 to 204 g (males) and 172 to 179 g (females)
- Housing: From arrival to pairing: animals were housed 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating: animals were housed one male to one female in clear polysulphone cages measuring approximately 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating; the females were transferred to individual solid bottomed cages (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy) for the gestation period and parturition.
- Diet: laboratory rodent diet, 4 RF 21 (Mucedola S.r.l., Settimo Milanese (MI), Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous carboxymethylcellulose (0.5% CMC)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in the vehicle. Formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL for dose levels of 100, 300 and 1000 mg/kg bw/day, respectively
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test substance in the vehicle were verified by gas chromatopgraphy with a flame ionisation detection (FID). Concentration verification was conducted on a weekly basis.
Duration of treatment / exposure:
Males: The daily administration of the test item was started two weeks before mating and lasted until test day 28 to 29, which was one day before sacrifice.
Females: The daily administration of the test item was started two weeks before mating and continued until day 3 post-partum.
Maximum: 54 days of treatment.
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 14-day range-finding study (Rossiello, 2013. RTC Study No.: 93730EXT)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during the study, each animal was observed and any clinical signs recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).

BODY WEIGHT: Yes
- Time schedule for examinations: females: weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: the weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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 : Yes
- Time schedule for examinations: daily by visual appraisal

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment
- Anaesthetic used for blood collection: Yes with isofluorane
- Animals fasted: Yes
- How many animals: 5/sex/dose
- Parameters examined: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count and differential leucocyte count

Coagulation tests: Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment
- Animals fasted: Yes
- How many animals :5/sex/dose
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, total bilirubin, total cholesterol, creatinine, glucose, urea, total protein, calcium, chloride, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT), gamma-glutamyltransferase, triglycerides, inorganic phosphorus

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the study, towards the end of treatment, 5 males and 5 females were randomly
selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli), for assessment of grip strength and for the motor activity was measured (for approximately 5 min).
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad), as well as the assessment of grip strength (Meyer) and motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
-Organ weights: adrenal glands, brain (cerebrum, cerebellum, medulla/pons), epididymides, heart, kidneys, liver, ovaries with oviducts, parathyroid glands, prostate gland, seminal vesicles with coagulating glands, spleen, testes, thymus (where present), thyroid and uterus-cervix,
-Fixation: adrenal glands, bone marrow (from sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, clitoral gland, colon, duodenum, epididymides, heart, ileum (including Peyer’s patches), jejunum, kidneys, liver, lungs (including mainstem bronchi), lymph nodes (mesenteric and cervical), ovaries with oviducts, parathyroid glands, pituitary gland, penis, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating glands, spinal column, spinal cord (cervical, thoracic, lumber), spleen, stomach, testes, thymus (where present), thyroid, trachea, urinary bladder, uterus-cervix and vagina.

HISTOPATHOLOGY: Yes, all organs that were included for fixation (5/sex of control and high dose group)

In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Other examinations:
Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation was made.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test.
The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were observed in males and females throughout the study.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed in males and females throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences of toxicological significance were seen in terminal body weight or body weight gain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No intergroup differences were seen in food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No changes of toxicological significance were observed. The statistically significant differences between control and treated animals (erythrocytes, mean corpuscular haemoglobin and leucocytes in males, haemoglobin, haematocrit and platelets in females) were of low magnitude and/or not dose-related, therefore considered incidental.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No changes of toxicological significance were observed. Statistically significant fluctuations of some biochemical parameters (mean group values) were recorded such as: increase of glucose in males dosed with 100 and 1000 mg/kg bw/day (48% for both dosages), increase in urea in those receiving 100 mg/kg bw/day (19%), increase of aspartate aminotransferases in females dosed with 300 mg/kg bw/day (35%), decrease of bilirubin (81%) and increase of potassium (10%) in those treated with 1000 mg/kg/day when compared to controls.
Due to the lack of dose- and sex-consistency and to the absence of other relevant findings, the above alterations were considered unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No significant differences were observed between groups, in motor activity, grip strength and sensory reactivity to stimuli, evaluated at the end of treatment.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No relevant differences in organ weights were seen between the controls and treated animals of both sexes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No remarkable changes were noted at post mortem examination in treated animals when compared with controls.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed. The lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. No relevant difference in estrous cycle was observed in treated females when compared to controls.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Categories Display