Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-14 to 2016-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and because there is ample experience and background data on this species and strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD)
- Females (if applicable) nulliparous and non-pregnant: yes (virgin)
- Age at study initiation: (P) 6-7 wks; (F1) x wks
- Weight at study initiation: (P) Males: 171-198 g; Females: 153-167 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: Not specified
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in clear solid bottomed polysulfone cages measuring 59 x 38.5 x 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). 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 polysulfone cages measuring 42.5 x 26.6 x 18 cm with a stainless steel mesh lid and floor (Tecniplast – Gazzada S.a.r.l.). 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 measuring 42.5 x 26.6 x 18 cm (Tecniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Suitable nesting material was provided and changed as necessary.
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles.
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light - rooms lit by artificial light

IN-LIFE DATES: From: 2016-06-03 To: 2016-09-25

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of the test material was suspended in the vehicle. The formulation was prepared daily (concentrations of 10, 60, and 120 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is unstable in water, so corn oil was used as vehicle
- Concentration in vehicle: concentrations of 10, 60, and 120 mg/mL
- Amount of vehicle (if gavage): dose volume of 5 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug in situ or copulation plugs found in the cage tray referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): transferred to individual solid bottomed cages measuring 42.5 x 26.6 x 18 cm (Tecniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation.
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method for formulation analysis was validated in RTC Study No. A2115 in the range from 5 to 150 mg/mL. Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure, in the range from 10 to 120 mg/mL, was acceptable (concentration and homogeneity). Final results for all levels were within the acceptability limits. Samples of the formulations prepared on Day 1 and last week (males and females) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits. Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A2115). The software used for this activity was Analyst 1.5.2.
Duration of treatment / exposure:
Males: dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Dose volumes were adjusted once per week for each animal according to the last recorded body weight).
Females: dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice (Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum).
Frequency of treatment:
Males and females: Once a day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day
Remarks:
Low Dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
Medium Dose
Dose / conc.:
600 mg/kg bw/day
Remarks:
High Dose
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 50, 300 and 600 mg/kg/day were selected by the Sponsor, based on a previous, preliminary non GLP compliant study (RTC Study No.: Y0030).
- Rationale for animal assignment (if not random): rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Working days: twice a day, all parental animals were checked early in each working day in the morning and in the afternoon. Weekends and Public Holidays: a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during treatment, each animal was observed and any clinical signs were recorded. Observations were performed approximately 0.5-1h after dosing each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. 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 Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

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

OTHER:

Clinical Observations (Functional Observation Battery Tests): Once before commencement of treatment and 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, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).

Grip strength and sensory reactivity to stimuli: 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) and for assessment of grip strength. Measurements were performed using a computer generated random order.

Motor activity assessment (MA): Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order.
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:

1. anomalies of the oestrous cycle
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
Parameters examined in all P male parental generations:
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 in control and treated animals.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Not specified

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving parental males were sacrificed after completion of the mating period
- Maternal animals: All surviving parenteral females with live pups were sacrificed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating were killed shortly after.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

All females were examined also for the following:
– number of visible implantation sites (pregnant animals)
– number of corpora lutea (pregnant animals)

Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [4] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The live F1 offspring were sacrificed at termination (Day 4 post partum)
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 4 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection

GROSS NECROPSY
- Gross necropsy consisted of external examinations and sex confirmation by gondal inspection
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 non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The mean values, standard deviations and statistically analysis were calculated from the actual values in the computer without rounding off.
Reproductive indices:
1) Copulatory Index:

Males and females: Copulatory Index (%): (no. of animals mated / no. of animals paired) x 100

2) Fertility Index:

a) Males: Fertility Index (%) = (no. of males which induced pregnancy / no. of males paired) x 100
b) Females: Fertility Index (%) = (no. of pregnant females / no. of females paired) x 100

3) Pre-coital Interval:

Males and females: Pre-coital interval = Mean number of days between pairing and mating

4) Pre-birth loss (females):

Pre-birth loss = ((no. of visible implantations – total litter size at birth) / no. of visible implantations) x 100

5) Pre-implantation loss (females):

Pre-implantation loss = ((no. of corpora lutea – no. of visible implantations) / no. of corpora lutea) x 100

Offspring viability indices:
1) Pup loss at birth:

Pup loss at birth = ((Total litter size – live litter size) / Total litter size) x 100

2) Cumulative pup loss on Day 4 post-partum:

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

3) Sex Ratios: Sex ratios were calculated at birth and on Day 4 post-partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs were seen, on occasions, in animals treated at 600 mg/kg/day: salivation in males, rales and/or piloerection in females. A single male treated at 300 mg/kg/day showed rales on occasion. No compound related effects were seen in animals treated at 50 mg/kg/day. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Mortality:
mortality observed, treatment-related
Description (incidence):
There were three unscheduled deaths at the high dose level (600 mg/kg/day): one male and one female were humanely killed on Day 4 and 6 of treatment (premating phase), respectively and one male was found dead on Day 22 of the mating phase.

Macroscopic changes such as red colour, pale firm areas or distended red/dark depressed areas in the stomach and reduced size of the thymus were confirmed at the histopathological evaluation that revealed marked mucosal ulceration in the non glandular region of the stomach and atrophy of thymus. The factors contributory to the death of these animals could be attributed to gastric lesions and thymus atrophy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the post coitum and post partum periods, slight but statistically significant reductions in body weight were seen in females receiving 300 and 600 mg/kg/day (up to approximately 10%).

Reductions, significant at the statistical analysis, were seen in the body weight gain of males receiving 600 mg/kg/day on Day 8 before pairing, in males receiving 50 mg/kg/day at the end of the study, and in females receiving 300 and 600 mg/kg/day on Day 20 post coitum period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males receiving 600 mg/kg/day showed decreases in food consumption on Day 8 (-16%) statistically significant, and on Day 15 (-12%) of the premating period; a decrease (-20%) was also seen in females of the same group on Day 8 of premating period, compared to controls.
Statistically significant decreases in food consumption were seen in females receiving 300 and 600 mg/kg/day during the gestation period (up to -16% and -19%, respectively) and on Day 4 post partum (-19%, not statistically significant, and -28%, respectively).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Changes as slight reticulocytosis and related changes in erythrocytes/haemoglobin or leucocytosis, observed in animals receiving 600 mg/kg/day, were considered of no toxicological relevance due to the absence of dose-relation and/or the minimal severity and/or incidence
and/or presence of the same changes in control animals. No changes were seen in the coagulation parameter.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in alanine and aspartate aminotransferases, urea, creatinine, phosphorus and bile observed in single animals in all treated groups, not dose-related, were not considered to be suggestive of organ/tissue injury, due to the mild severity and the absence of histopathological related changes.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No relevant differences were noted in all parameters investigated between control and treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative thymus weights were decreased in males and females receiving 600 and 300 mg/kg/day statistically significant in the high dose group. This change was confirmed by the histopathological diagnosis of atrophy.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The most relevant changes observed at post mortem examination were dark/red colour or areas and/or thickened glandular and/or non glandular region of the stomach in high dose males and females and in low dose males, as well as an increased incidence of small thymus
in most treated females, when compared with controls.
Neuropathological 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.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were reported in the gastric tract (hyperplasia of the squamous non glandular epithelium, in some instances associated with ulceration and acute inflammation) and thymus (atrophy) of most treated animals of both sexes dosed at 300 and
600 mg/kg/day. No relevant changes were observed in the gastric tract and thymus of treated animals dosed at 50 mg/kg/day, when compared with controls.

The remaining lesions reported in control and/or treated animals such as atrophy of testes and epididymides were considered to be an expression of spontaneous and/or incidental pathology changes, commonly seen in this species and age under our experimental conditions.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Measurement of oestrous cycle and the outcome of mating performance in terms of pre-coital intervals did not show important differences between groups. Reductions in the copulatory and fertility indices were seen in mid- (300 mg/kg/day) and high dose (600 mg/kg/day) males; a slight reduction in the fertility index was also seen in 300 mg/kg/day females.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No alterations were noted in the seminiferous tubules when evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages, in all treated males respect to controls
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reductions in the number of corpora lutea and implantations (up to-21%) were seen in females receiving 600 and 300 mg/kg/day, when compared with controls. Decreases in the total litter size at birth were seen in females receiving 300 (-18%) and 600 (-7%) mg/kg/day respect to controls. Gestation periods were similar between treated animals and controls. All dams gave birth between Days 21 and 23 post coitum.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Sytemic Toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of small pups/litter was seen in the mid- and high dose groups, compared to controls. Pups with apparently no food intake were seen in two litters in the high dose group (600 mg/kg/day).

Pups cold to touch, pale, with the tip of tail missing or black were generally observed in all groups including controls, without substantial differences.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Slight increases in pup loss at birth and in the cumulative loss on Day 4 post partum, significant at the statistical analysis, were seen in high dose females (600 mg/kg/day). The percentage of the pup loss on Day 4 post partum in high dose females (8 fold higher than that of controls) was due to three females (contributing with 25-54% of loss).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Litter weight was significantly reduced at the statistical analysis (up to -34%), in females receiving 300 and 600 mg/kg/day on both Days 1 and 4 post partum, due to a reduction in the number of pups/litter.
Sexual maturation:
no effects observed
Description (incidence and severity):
No toxicologically relevant differences were seen in sex ratio of pups on birth and Day 4 post partum.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased incidence of decedent/missing pups was seen in the mid- and high dose groups (300 and 600 mg/kg/day). Autolysis of all organs was observed for the majority of the decedents pups, in all groups.

No abnormalities were detected in pups of all groups, with the exception of few lesions of no toxicological significance, such as abnormal area of the skin (abrasion/scab) or tip of tail missing or dark recorded in single pups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive/Developmental Toxicity

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 5. BODY WEIGHT (g) OF PREGNANT FEMALES – POST COITUM AND POST PARTUM PERIODS – GROUP MEAN DATA

Group(s)

 

Day of Phase

0!

7

14

20

1’’

4

1

(Control)

(n)

9

9

9

9

9

9

Mean

253.45

282.49

317.29

415.07

315.95

307.43

SD

10.89

7.05

9.51

11.34

12.21

10.09

 

2

(50 mg/Kg bw/day)

(n)

10

10

10

10

10

10

Mean

253.46

280.17

311.23

407.79

311.48

305.88

SD

10.14

8.17

9.65

23.98

9.48

17.18

 

3

(300 mg/Kg bw/day)

(n)

8

8

8

8

8

8

Mean

238.09*

261.83* ($)

294.37**

370.52**

297.11*

283.48

SD

17.62

18.42

17.58

22.25

20.89

29.79

 

4

(600 mg/Kg bw/day)

(n)

8

8

8

8

8

8

Mean

238.41*

260.24**

($)

295.42**

375.44**

283.22**

275.52* ($)

SD

9.71

6.27

6.38

17.52

14.47

24.57

! = Gestation phase; " = Post-partum phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

 

Table 6. BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES POST COITUM AND POST PARTUM PERIODS – GROUP MEAN DATA

Group(s)

 

Day of Phase

7!

14

20

4’’

1

(Control)

(n)

9

9

9

9

Mean

4.149

4.971

16.296

-2.840

SD

0.952

0.676

1.228

6.198

 

2

(50 mg/Kg bw/day)

(n)

10

10

10

10

Mean

3.815

4.438

16.092

-1.865

SD

0.789

0.929

3.260

5.930

 

3

(300 mg/Kg bw/day)

(n)

8

8

8

8

Mean

3.391

4.649

12.691*

-4.544

SD

1.416

1.488

1.807

4.758

 

4

(600 mg/Kg bw/day)

(n)

8

8

8

8

Mean

3.118

5.025

13.338*

-2.567

SD

1.407

0.925

2.461

6.559

! = Gestation phase; " = Post-partum phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = mean daily body weight gain over the previous period starting from Day 0 post coitum and Day 1 post-partum

Table 7. FOOD CONSUMPTION° (g/animal/day) OF PREGNANT FEMALES - POST COITUM AND POST PARTUM PERIODS – GROUP MEAN DATA

Group(s)

 

Day of Phase

7!

14

20

4’’

1

(Control)

(n)

9

9

9

9

Mean

18.36

21.82

26.82

29.57

SD

1.11

1.25

1.73

5.56

 

2

(50 mg/Kg bw/day)

(n)

10

10

10

10

Mean

18.29

20.30

25.37

30.61

SD

1.19

1.49

2.00

6.77

 

3

(300 mg/Kg bw/day)

(n)

8

8

8

8

Mean

15.68**

18.79**

22.58**

23.90

SD

2.19

2.03

2.53

4.26

 

4

(600 mg/Kg bw/day)

(n)

8

8

8

8

Mean

14.81**

18.77**

24.21*

21.36*

SD

1.19

0.99

2.46

8.05

! = Gestation phase; " = Post-partum phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = food consumed over the previous period starting from gestation Day 0 or post-partum Day 1

Table 8. HAEMATOLOGY - GROUP MEAN DATA

Parameter/units

 

Group(s)

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Reticulocytes (%)

Mean

2.124

2.208

2.380

3.095+

SD

0.273

0.268

0.456

0.393

n

5

5

5

4

 

Reticulocytes

(10^9/L)

Mean

167.56

174.44

188.92

231.98+

SD

22.23

18.80

30.80

22.26

n

5

5

5

4

Females

Mean Red Blood Cell Volume (fl)

Mean

61.30

60.34

59.76

65.56*

SD

1.72

2.65

1.53

2.99

n

5

5

5

5

 

Mean Corpuscular HB (pg)

Mean

20.12

19.92

19.80

21.82*

SD

0.87

0.79

0.97

1.26

n

5

5

5

5

 

White Blood Cell Count

(10^3/uL)

Mean

4.500

4.492

3.230

7.084+

SD

0.672

1.270

0.441

1.783

n

5

5

5

5

 

Lymphocytes (10^3/uL)

Mean

3.440

3.384

2.430

4.794*

SD

0.397

0.953

0.464

0.748

n

5

5

5

5

* = mean value of group is significantly different from control at p < 0.05

+ = mean value of group is significantly different from control at p < 0.01

 

Table 9. CLINICAL CHEMISTRY - GROUP MEAN DATA

Parameter/units

 

Group(s)

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Alkaline Phosphatase (U/L)

Mean

218.58

288.90

264.12

311.16*

SD

55.34

45.97

57.02

33.07

n

5

5

5

5

 

Alanine Amino Transferase

(U/L)

Mean

46.30

43.20

87.34

77.86*

SD

9.26

8.29

35.42

15.96

n

5

5

5

5

 

Bile Acids (µmol/L)

 

Mean

12.94

25.12

23.70

30.12*

SD

7.11

10.43

11.34

9.01

n

5

5

5

5

 

Globulin (g/dL)

Mean

2.44

2.46

2.26*

2.26*

SD

0.05

0.11

0.09

0.15

n

5

5

5

5

 

Inorganic Phosphorous (mg/dL)

Mean

6.192

5.946

7.424

9.088+

SD

1.171

0.488

0.984

1.980

n

5

5

5

5

Females

Alkaline Phosphatase (U/L)

Mean

219.36

200.46

251.46

377.68*

SD

57.95

86.72

18.56

97.73

n

5

5

5

5

 

Alanine Amino Transferase

(U/L)

Mean

47.96

62.78

58.12

83.90*

SD

4.15

44.17

10.01

18.75

n

5

5

5

5

 

Urea (mg/dL)

Mean

39.54

47.62

47.04

65.42+

SD

8.94

4.38

7.37

12.86

n

5

5

5

5

 

Creatinine ((mg/dL)

Mean

0.408

0.436

0.478*

0.536+

SD

0.38

0.025

0.031

0.038

n

5

5

5

5

 

Sodium (mmol/L)

Mean

140.74

139.32

139.84

139.02*

SD

0.85

3.68

0.97

0.87

n

5

5

5

5

* = mean value of group is significantly different from control at p < 0.05

+ = mean value of group is significantly different from control at p < 0.01

 

Table 10. REPRODUCTIVE PARAMETERS OF ANIMALS - SUMMARY

 

Group(s)

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Copulatory Index (%)

90.0

90.0

90.0

77.8

Fertility Index (%)

80.0

90.0

70.0

66.7

Females

Copulatory Index (%)

100.0

100.0

100.0

100.0

Fertility Index (%)

90.0

100.0

80.0

88.9

Table 11. IMPLANTATION, PRE-IMPLANTATION LOSS DATA, PRE-BIRTH LOSS DATA AND GESTATION LENGTH – GROUP MEAN DATA

Group(s)

 

Corpora

Lutea

Implantations

Pre-implantation Loss %

Total

Litter Size at Birth

Pre-Birth Loss %

Gestation

Length (Days)

1

(Control)

Mean

16.67

16.67

0.00

14.11

14.43

22.22

SD

1.73

1.73

0.00

2.37

15.94

0.44

n

9

9

9

9

9

9

 

2

(50 mg/Kg bw/day)

Mean

15.90

15.90

0.00

14.80

7.08

22.30

SD

3.54

3.54

0.00

3.29

5.22

0.48

n

10

10

10

10

10

10

 

3

(300 mg/Kg bw/day)

Mean

13.38*

13.13*

1.56

11.50*

11.75

22.25

SD

2.13

1.89

4.42

2.20

14.93

0.46

n

8

8

8

8

8

8

 

4

(600 mg/Kg bw/day)

Mean

13.63*

13.38*

1.79

13.13

1.93

22.25

SD

1.19

1.30

5.06

1.46

3.58

0.46

n

8

8

8

8

8

8

Statistical analysis: Kruskall Wallis test

William’s test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control at p < 0.05

Table 12. LITTER DATA AT BIRTH, ON DAY 1 AND ON DAY 4 POST PARTUM OF FEMALES - GROUP MEAN DATA

Group(s)

 

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)

1

(Control)

Mean

14.11

13.89

1.58

91.99

6.67

13.78

2.32

122.9

9.11

SD

2.37

2.42

3.13

11.96

0.48

2.39

3.49

13.50

1.48

n

9

9

9

9

9

9

9

9

9

 

2

(50 mg/Kg bw/day)

Mean

14.80

14.60

1.19

94.37

6.53

14.60

1.19

122.0

8.54

SD

3.29

3.17

2.51

18.59

0.51

3.17

2.51

23.15

1.27

n

10

10

10

10

10

10

10

10

10

 

3

(300 mg/Kg bw/day)

Mean

11.50*

11.38

1.56

71.41*

6.48

10.63

7.63

92.41*

8.85

SD

2.20

2.45

4.42

15.02

0.38

2.20

7.12

12.28

1.17

n

8

8

8

8

8

8

8

8

8

 

4

(600 mg/Kg bw/day)

Mean

13.13

12.38

6.08

72.88*

6.03

10.63

19.55*

81.59*

7.66

SD

1.46

2.00

7.43

11.39

0.42

2.77

16.33

22.01

1.22

n

8

8

8

8

8

8

8

8

8

Statistical analysis: Kruskall Wallis test

William’s test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control at p < 0.05

Table 13. ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA

Organ

Group:

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

Males

Adrenals

Number/Group

10

10

10

8

Mean

0.0528

0.0532

0.0566

0.0623

SD

0.0063

0.0040

0.0059

0.0082

Group diff atp<0.05

 

0.0068

0.0068

0.0072*

Group diff atp<0.01

 

0.0086

0.0086

0.0091*

Analysis of variance: F ratio = 4.36 Df = 3/ 34 F probability = 0.011

* indicates group mean is significantly different from control at level of significance shown

 

Heart

Number/Group

10

10

10

8

Mean

1.391

1.376

1.298

1.247

SD

0.099

0.060

0.114

0.126

Group diff atp<0.05

 

0.111

0.111

0.118*

Group diff atp<0.01

 

0.141

0.141

0.150

Analysis of variance: F ratio = 4.02 Df = 3/ 34 F probability = 0.015

* indicates group mean is significantly different from control at level of significance shown

 

Thymus

Number/Group

10

10

10

8

Mean

0.4056

3.645

0.3379

0.2645

SD

0.0763

0.0474

0.0478

0.0408

Group diff atp<0.05

 

0.0610

0.0610*

0.0647*

Group diff atp<0.01

 

0.0775

0.0775

0.0822*

Analysis of variance: F ratio = 10.03 Df = 3/ 34 F probability = 0.000

* indicates group mean is significantly different from control at level of significance shown

Females

Kidneys

Number/Group

10

10

10

9

Mean

1.770

1.706

1.593

1.654

SD

0.134

0.121

0.202

0.163

Group diff atp<0.05

 

0.173

0.173*

0.178

Group diff atp<0.01

 

0.220

0.220

0.226

Analysis of variance: F ratio = 2.27 Df = 3/ 35 F probability = 0.097

* indicates group mean is significantly different from control at level of significance shown

 

Thymus

Number/Group

10

10

10

9

Mean

0.2272

0.1933

0.1826

0.1442

SD

0.0568

0.0533

0.0564

0.0737

Group diff atp<0.05

 

0.0661

0.0661

0.0679*

Group diff atp<0.01

 

0.0838

0.0838

0.0861

Analysis of variance: F ratio = 3.06 Df = 3/ 35 F probability = 0.040

Note: a * indicates group mean is significantly different from control at level of significance shown

Data homogeneous by Bartlett's test (Dunnett's test)


Table 14. MICROSCOPIC OBSERVATIONS - GROUP INCIDENCE

Tissue with Diagnoses

 

Animals Affected

Animal Sex

Males

Females

Dosage Group

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

1

(Control)

2

(50 mg/Kg bw/day)

3

(300 mg/Kg bw/day)

4

(600 mg/Kg bw/day)

No. in group

10

10

10

8

10

10

10

9

Stomach

Number Examined

10

10

10

8

10

10

10

9

Mucosal Ulceration

 

0

0

1

2

0

0

0

1

Squamous Hyperplasia

 

0

0

4

-6

0

0

1

-9

Acute Inflammation

 

0

0

1

1

0

0

0

4

Mucosal Erosion

 

0

0

0

0

0

0

0

0

Necrosis

 

0

0

0

0

0

0

0

0

Oedema

 

0

0

0

0

0

0

0

0

Dilated Mucosal Gland

 

1

3

0

0

2

0

0

0

Chronic Inflammation

 

0

1

0

0

0

0

0

0

 

Thymus

Number Examined

10

10

10

8

10

10

10

8

Atrophy

 

0

0

1

-6

2

4

5

-7

Necrosis

 

0

0

0

0

0

0

0

0

Haemorrhage

 

0

0

0

2

1

0

0

0

Note: Entries flagged with a - (minus) are significantly higher than control at the 0.05 level using the Kolmogorov-Smirnov one-tailed test.

Applicant's summary and conclusion

Conclusions:
Based on the results observed, the NOAEL (No Observed Adverse Effect Level) for systemic and developmental toxicity was determined to be 50 mg/Kg bw/day.
Executive summary:

In a key Guideline (OECD 422), combined repeated dose / reproductive developmental toxicity screening study, the test material in corn oil was administered via oral gavage to Sprague-Dawley rats (10/sex/dose) once daily at doses of 0, 50, 300, or 600 mg/Kg bw/day. Male rats were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day prior to necropsy. Female rats were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post-partum periods until Day 3 post-partum or the day prior to sacrifice.

 

Mortality was observed in the study as follows: Three unscheduled deaths in the high dose group (600 mg/Kg bw/day) - one male and one female humanely killed on Day 4/6 of the premating phase, respectively and one male found dead on Day 22 of the mating phase. Macroscopic changes such as red colour, pale firm areas or distended red/dark depressed areas in the stomach and reduced size of the thymus were confirmed by the histopathological evaluation that revealed marked mucosal ulceration in the non glandular region of the stomach and atrophy of thymus. Death of these animals could be attributed to gastric lesions and thymus atrophy.

 

Clinical signs such as salivation, rales and/or piloerection were seen, on occasion, in animals treated at 600 mg/Kg bw/day and in one animal dosed at 300 mg/Kg bw/day, while no treatment-related effects were seen in animals treated at 50 mg/Kg bw/day. Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test material. No relevant differences were noted in motor activity and sensory reactivity to stimuli between control and treated groups.

 

Slight reductions in body weight (up to approximately 10%) were observed in females dosed at 300 and 600 mg/Kg bw/day during the post coitum and post-partum periods. Slight reductions in food consumption were seen in animals receiving 600 mg/Kg bw/day on Days 8 and/or 15 of the premating period, compared to controls. Decreases in food consumption were also seen in females receiving 300 and 600 mg/Kg bw/day during the gestation period and on Day 4 post-partum. No toxicological relevance was attributed to slight, not dose-related, changes observed in clinical pathology parameters.

 

One control female, two females receiving 300 mg/Kg bw/day, and 1 female receiving 600 mg/Kg bw/day, were found not pregnant at necrospy. Unilateral implantation was observed in 1 female each in the low (50 mg/Kg bw/day) and mid-dose (300 mg/Kg bw/day) dose groups. The number of females with live pups on Day 4 post-partum was 9 in the control, 10 in the low dose (50 mg/Kg bw/day), 8 in each of the mid-dose (300 mg/Kg bw/day) and high dose groups (600 mg/Kg bw/day). One male in each of the control, low and mid-dose groups and two males in the high dose group did not mate. Measurement of oestrous cycle and the outcome of mating performance in terms of precoital intervals did not show important differences between groups. The copulatory and fertility indices were slightly lower in the 300 and 600 mg/Kg bw/day males and a slight reduction in the fertility index was also seen in 300 mg/Kg bw/day females, when compared to controls. Gestation periods were similar between treated animals and controls, the mean value being 22 days post coitum. Statistically significant reductions in corpora lutea and implantations were seen in females dosed at 300 and 600 mg/Kg bw/day.

 

A slight increase in pup loss at birth and in the cumulative loss on Day 4 post coitum were seen in 600 mg/Kg bw/day females. Litter weight was reduced in females receiving 300 and 600 mg/Kg bw/day on both Days 1 and 4 post-partum, due to a reduction in the number of pups/litter, compared to controls. No toxicologically relevant differences were seen in sex ratio of pups on birth and Day 4 post-partum. An increased incidence of small pups/litter was seen in the mid- and high dose groups, compared to controls. An increased incidence of decedent/missing pups was seen in mid- and high dose groups (300 and 600 mg/Kg bw/day).

 

A decrease (up to -9%) was observed in the terminal body weight of all treated males and in females receiving 300 and 600 mg/Kg bw/day, when compared to the controls. Reduction of thymus weights were seen in males and females receiving 300 and/or 600 mg/Kg bw/day. The most relevant changes observed at post mortem examination were dark/red colour or areas and/or thickened glandular and/or non glandular region of the stomach in 600 mg/Kg bw/day males and females and in 50 mg/Kg bw/day males, as well as an increased incidence of small thymus in most treated females, when compared with controls. Treatment-related changes were reported in the gastric tract (hyperplasia of the squamous non glandular epithelium, in some instances associated with ulceration and acute inflammation) and thymus (atrophy) of most treated animals of both sexes dosed at 300 and 600 mg/Kg bw/day.

No relevant changes were observed in the gastric tract and thymus of treated animals dosed at 50 mg/kg/day, when compared with controls.

 

No alterations were noted in the seminiferous tubules when evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages, in all treated males respect to controls.

 

Based on the results obtained in this study, the dose level of 50 mg/Kg bw/day was determined to be the NOAEL (No Observed Adverse Effect Level) for systemic and developmental toxicity.