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Toxicological information

Toxicity to reproduction

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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:
06 Jun - 04 Aug 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
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
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetradecyl-Oleate
- Physical state: clear yellowish oil
- Analytical purity: 100% (UVCB)
- Lot/batch No.: K120711A
- Expiration date of the lot/batch: 2014-01-24
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals 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 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
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 1000 mg/kg bw/day
Basis:
actual ingested
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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

No relevant clinical signs or mortality were observed in males and females throughout the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

No difference of toxicological significance were seen in body weight or body weight gain. No intergroup differences were seen in food consumption.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

No relevant difference in oestrous cycle was observed in treated females when compared to controls.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

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 (PARENTAL ANIMALS)

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 differences were observed in the pre-coital interval, copulatory and fertility indices between control and treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)

No relevant differences in terminal body weight or organ weights were seen between the controls and treated animals of both sexes

GROSS PATHOLOGY (PARENTAL ANIMALS)

No remarkable changes were noted at post mortem examination in treated animals when compared with controls.

HISTOPATHOLOGY (PARENTAL ANIMALS)

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.

Effect levels (P0)

open allclose all
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 obseved in the study
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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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.
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)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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 

Applicant's summary and conclusion