Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 November 2010 - 15 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study. No gross examination of pups was performed after sacrifice.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
No gross examination of pups was performed after sacrifice
GLP compliance:
yes (incl. certificate)
Remarks:
Slovak National Accreditation Service
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SP-334LM
- Physical state: solid (powder)
- Analytical purity: 99.7%
- Purity test date: 9 Jul 2010
- Lot/batch No.: 34692
- Expiration date of the lot/batch: 01 January 2012
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Velaz Praha, Czech Republic
- Age at study initiation: adult males/females
- Weight at study initiation: mean body weight: males 205.3 g, females 197.8 g
- Housing: All animals were kept in cages with bedding (wood shavings) under standard conditions. During the pre-mating period 4 rats/sex/cage; duringthe mating period 1 manle and 1 female per cage; during the gestation period pregnant females were caged individually; during the lactation period females were caged individually together with the offspring; after the mating procedure males were returned to the original cages, 4 males per cage
- Diet: conventional laboratory diet (MP-OŠ-06, BIOFER, 06901, Snina, Slovak Republic)
- Water: drinking water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 October 2010 To: 12 November 2010 (males) and 11 or 12 November 2010 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The solution with the test article was prepared daily before application and administered within 2 hours. SP-334 at concentrations of 10 mg/mL, 40 mg/mL and 200 mg/mL in olive oil were prepared by mixing an accurately weighed amount of SP-334 with olive oil. The solution of SP-334 is stable up to 24 hours at laboratory temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): SP-334 is not soluble in the water; therefore the olive oil was used as the vehicle.
- Concentration in vehicle: 1, 4 and 10% (w/v)
- Amount of vehicle (if gavage): 0.5 mL/100 g body weight
- Lot/batch no. (if required): 100
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum 14 days, until proof of pregnancy
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- After 6 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The precision, homogeneity and stability of formulation was analysed against standard samples by HPLC. The precision and homogeneity met the requirements of 90-110% of the nominal concentration (relative SD ≤3%). The stability after 24 hours at room temperature was acceptable; as the mean values measured at 24 hours were shown to be within ±5% of the values measured at 0 hours.
Duration of treatment / exposure:
males: 28 days
females: up to 56 days (14 days before the mating procedure, up to 14 days during the mating process, 22–24 days during gestation, and 4 days during the lactation)
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: adults
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 200 and 1000 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a subacute 28-day oral toxicity study, 12 rats/sex/group were administered 50, 200 and 1000 mg/kg bw/d of the test substance by gavage (Pels Rijken, 1998). There was no treatment-related mortality. One female administered 50 mg/kg bw/d died on day 27, most likely due to mis-dosing. Red staining of the fur was observed in females in the highest dose group. A discharge from the nose and eyes that is spread over the fur during grooming is normal in rats. The increased discharge is probably a consequence of the stress rats are subject to during the repeated dosing by gavage, and is therefore related to treatment but not to the test substance. The NOAEL was considered to be 1000 mg/kg bw/d, based on the lack of effects at the highest dose level. The same dose levels as those used in the 28-day repeated dose study were therefore selected for the reproduction/developmental screening toxicity study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVAIONS: Yes
- Time schedule: daily
- Cage side observations included: mortality, clinical signs (piloerection, alopecia, dyspnoea, salivation, smooth stool, diarrhoea, tremors, spasms, hyperactivity, lethargy, cannibalism, aborts, moribund state)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed on the first day of dosing, then weekly thereafter, and at study termination. During the gestation period, females were weighed on day 0, 7, 14, 20; within 24 hours of parturition (day 1 post-partum); and on day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured weekly
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

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, physical or behavioural abnormalities.
The litters were weighed 24 hours after the birth and on day 4 post-partum.

GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed after 28 days of treatment
- Maternal animals: All surviving females were sacrificed on day 5 post-partum

GROSS NECROPSY
- A gross necropsy, consisted of external and internal macroscopic examination of for abnormalities or pathological changes, was performed on all animals that died during the study period and the animals sacrificed at the end of the study

HISTOPATHOLOGY / ORGAN WEIGHTS
A histopathologic examination was performed on the animals in the control and highest dose group. The liver, kidneys (target organs), testes, epididymes, uterus, and ovaries were weighed. The samples from the above-mentioned organs including the prostate (including coagulating glands) and mammary gland were processed for microscopic examination. All tissue samples were fixed in formaldehyde.
Postmortem examinations (offspring):
SACRIFICE
- The surviving offspring were sacrificed on day 5 post-partum
Statistics:
The results (body weight of animals, litters, and organs, food consumption, number of corpora lutea and implantation sites) were statistically evaluated by the statistical programme Statgraphics. The statistical evaluated was made separately for the males, females, and litters. For the identification of homogeneity of the groups Bartlett’s test was used. In the case of homogeneitya One-Way Analysis of variance with consecutive Multiple-Ranges tests was used. In the case of non-homogeneity and for the length of pregnancy Kruskal-Wallis One Way Analysis by Ranks was applied.
Reproductive indices:
The following reproductive indices were calculated: fertility index and gestation (pregnancy) index
Offspring viability indices:
The following indices were calculated: live birth index and viability (survival) index

Live birth index = (number of live offspring / number of offspring delivered) x 100
Viability (survival) index = (number of live offspring at lactation day 4/number of live offspring delivered) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
alopecia
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
gestation period

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality: 0, 2, 1 and 0 in the female control, 50, 200 and 1000 mg/kg bw/d groups, respectively. The animals had inflammation of the endometrium, which is not considered to be test substance-related. No males died during the study period.

Alopecia was noted in several females on one or several days in all groups during day 27-44; in total in 2, 1, 3 and 1 females in the control, 50, 200 and 1000 mg/kg bw/d groups, respectively. This was not considered to be a treatment-related occurrence.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no statistically significant difference in body weight or food consumption between the control group and treament groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
A statistically significant increase in gestation period (≥23 days) was noted in the 200 and 1000 mg/kg bw/d groups (see table 1). However, the number was highest in the 200 mg/kg bw/d group (8 females) and the number in the 1000 mg/kg bw/d group was the same as for the control group (4 females). As the results are not dose-related and there are no other effects on reproductive parameters, this is not considered to be a treatment-related effect, despite a statistical significance. There was no statistically significant difference in reproductive performance, measured as pregnancy rate (fertility index), gestation index, live birth index and viability index, between the control group and treament groups (see table 1). There was a reduction in the number of implants and number of live pups (not statistically significant), showing a seemingly dose-related trend. However, historical data (see table 2) indicate that the results in this study fall within the expected range and are therefore not related to treatment with the test substance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The relative liver weight of males administered 1000 mg/kg bw/d was statistically significantly increased, compared to the male control group. An increase in liver weight is frequently observed in repeated dose studies, as the liver is enlarged due to the increased metabolising activity caused by the high intake the test substance. This effect is considered to be an adaptation, rather than an adverse effect (see table 3).

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related effects on gross pathology.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related effect on histopathology.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to the highest dose level
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to the highest dose level
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to the highest dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
A slight reduction in the viability on day 4 was observed (not statistically significant), showing a seemingly dose-related trend. However, historical data (see table 2) indicate that the results in this study fall within the expected range and are therefore not related to treatment with the test substance.

CLINICAL SIGNS (OFFSPRING)
No clinical signs of toxicity were observed.

BODY WEIGHT (OFFSPRING)
There were no treatment-related effects on body weight.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: results of mating procedure, pregnancy parameters and offspring viability:  

Observations

Values

Dosage

Control

group

Low dose

group

Medium dose

group

High dose

group

Pairs started (N)

12

12

12

12

Females showing evidence of copulation (N)

12

12

12

11

Females achieving pregnancy (N)

10

11

12

9

Conceiving days 1 – 5 (N)

12

12

11

11

Conceiving days 6 – 14(N)

0

0

1

0

Pregnancy ≤ 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

6

6

3

4

Pregnancy ≥ 23 days (N)

4

5

8*

4*

Dams with live young born (N)

10

9

10

7

Dams with live young at day 4 pp (N)

8

8

8

7

Corpora lutea / dam (mean)

15

14

14

11

Implants / dam (mean)

13

11

10

7

Live pups / dam at birth (mean)

11

8

9

7

Live pups / dam at day 4 (mean)

12

9

8

7

Sex ratio (m / f) at day 4 (mean)

5/7

4/5

4/5

4/4

Litter weight at birth (mean)

56.50

51.67

50.00

46.43

Litter weight at day 4 (mean)

81.88

75.63

65.00

65.71

ABNORMAL PUPS

Dams with 0

0

0

0

0

Dams with 1

0

0

0

0

Dams with ≥ 2

0

0

0

0

LOSS OF OFFSPRING

Pre-implantation (corpora lutea minus implantations)

Females with 0

6

2

2

3

Females with 1

1

3

1

0

Females wits 2

1

1

2

1

Females with ≥ 3

2

5

7

5

Pre-natal / post-implantations (implantation minus live birth)

Females with 0

0

5

3

2

Females with 1

4

2

5

6

Females with 2

3

1

1

0

Females with ≥ 3

3

3

3

1

Post-natal (live births minus alive at post-natal day 4)

Females with 0

6

10

9

9

Females with 1

3

1

1

0

Females with 2

0

0

0

0

Females with ≥ 3

1

0

2

0

* No 28, 46 - exact day of birth unknown

Table 2: Historical data from control groups treated with olive oil (vehicle) only

Parameter

Range

Number of implants/dam

5-16

Live pups/dam at birth

3-15

Pup survival to day 4

3-15

Table 3: Relative liver weight

Dose (mg/kg bw/d)

Control

50

200

1000

Relative liver weight, males (%)

3.81±0.21

3.90±0.24

3.85±0.48

4.17±0.18*

Relative liver weight, females (%)

5.09±0.72

4.63±0.59

5.27±0.58

5.82±0.77

* statistically significant difference compared to control

Applicant's summary and conclusion

Conclusions:
CLP: not classified
DSD: not classified
Executive summary:

In a screening study performed according to OECD 421, the potential adverse effect of SP-334 on reproduction and developmental parameters in rats was assessed.

12 Wistar rats/dose/sex were administered 0, 50, 200 and 1000 mg/kg bw/d; males for 28 days during the pre-mating and mating periods, and females for up to 56 days during the pre-mating, mating, and gestation period and during lactation until day 4. The control group received the vehicle (olive oil). A fixed application volume of 0.5 mL/100 g body weight was administered by gavage.

There were 3 unscheduled cases of mortality in female dose groups; 2/12 in the 50 mg/kg bw/d group and 1/12 in the 1000 mg/kg bw/d group, respectively. The animals had inflammation of the endometrium, which is not considered to be test substance-related. No males died during the study period. Alopecia was noted in several females on one or several days in all groups during day 27-44; in total in 2, 1, 3 and 1 female in the control, 50, 200 and 1000 mg/kg bw/d groups, respectively. This is a normal occurrence in rats during repeated dose studies and is not considered to be treatment-related.The relative liver weight of males administered 1000 mg/kg bw/d was statistically significantly increased, compared to the male control group. The liver weight was 3.81, 3.90, 3.85 and, in the control, 50, 200 and 1000 mg/kg bw/d dose groups, respectively. An increase in liver weight is frequently observed in repeated dose studies, as the liver is enlarged due to the increased metabolising activity caused by the high intake the test substance. This effect is considered to be an adaptation, rather than an adverse effect. No treatment-related findings were noted during gross necropsy and histopathological examination.

A statistically significant increase in gestation period (≥23 days) was noted in the 200 and 1000 mg/kg bw/d groups. However, the number was highest in the 200 mg/kg bw/d group (8 females) and the number in the 1000 mg/kg bw/d group was the same as for the control group (4 females). As the results are not dose-related and there are no other effects on reproductive parameters, this is not considered to be a treatment-related effect, despite a statistical significance. There was no statistically significant difference in other reproductive parameters between the control group and treatment groups.

 

The NOAEL (No-Observed-Adverse-Effect Level) for reproduction in males and females is considered to be 1000 mg/kg bw/d, as no effects were observed up to and including the highest dose level.

 

The NOAEL for systemic toxicity is also considered to be 1000 mg/kg bw/d for males and females, as no effects were noted up to and including the highest dose level.

 

In the offspring (F1 -generation) a slight reduction in the viability on day 4 was observed (not statistically significant), showing a seemingly dose-related trend. However, historical data indicate that the results in this study fall within the expected range and are therefore not related to treatment with the test substance. No clinical signs of toxicity were observed in the offspring and there were no effects on the body weight.

No treatment-related effects were observed up to and including the highest dose level, therefore the NOAEL for developmental effects is considered to be 1000 mg/kg bw/d.