Registration Dossier

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:
2013-04-03 - 2013-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Name: N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)
(abbreviated as MPA)

Batch no. P70/05/S-6

Characteristics Cream white, solid, powder

Content 96.2%

Test animals

Species:
rat
Strain:
other: CD / Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany

- Age at study initiation: Males: 42 days, Females: 42 days

- Weight at study initiation: Males: 183.5 – 207.8 g, Females: 129.9 – 155.6 g

- Housing: singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm

- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ssniff® R-Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum
- Water (e.g. ad libitum): Tap water offered ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C - 21°C
- Humidity (%): approx. 44% - 55%

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Methocel
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle 0.5% Methocel to concentrations of 20, 60 and 200 mg test item/ml. The test item formulations were freshly prepared and adjusted to the animal's current body weight on each administration day.
In addition, the stability, concentration and homogeneity of the test item mixture were monitored

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg test item/ml
- Amount of vehicle (if gavage) :5 mL/kg b.w./day
Details on mating procedure:
Sexually mature male and female rats were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. If findings were negative, mating was repeated. The day of conception (day 0 of gestation) was considered to be the day on which sperm was found. In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered. This procedure was repeated until at least 8 pregnant dams were available for each group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test item that was mixed with vehicle, the concentration, stability and homogeneity of the test item in the mixture was determined, using an HPLC method.

At study initiation:
Analysis of stability and concentration
Immediately after preparation of the suspension as well as after 8 and 24 hours storage of the test item preparations at room temperature.
(3 samples/dose level group).
Number of samples: 3 x 3 = 9

Homogeneity
At start of administration, during (middle) administration and before administration to the last animal of each dose level group:
(3 samples/dose level group).
Number of samples: 3 x 3 = 9

At termination of the administration period at a time point when the majority of animals is dosed:
Analysis of concentration:
During treatment with the test item always before administration to the last animal/dose level group:
(1 sample per dose level group).
Number of samples: 1 x 3 = 3

Sum of all samples: 21
Duration of treatment / exposure:
Males
Once daily. Beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed.

Females
Once daily. Beginning 2 weeks prior to mating continuing up to, and including, day 3 post partum or the day before sacrifice.
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg test item/kg b.w./day
Basis:
actual ingested
No. of animals per sex per dose:
12 males, 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor based on the results of a 14-day dose-range-finding study. No test item related influence was noted in the dose range finding study at any dose level (100, 300 and 1000 mg test item/kg b.w./day) concerning the occurrence of premature deaths, signs of behavioural toxicity, body weight and body weight gain, drinking water consumption and macroscopic post mortem findings.
A reduction in food consumption was noted for the female rats of the high dose group (1000 mg test item/kg b.w./day), but not for the male rats.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, mortality twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum) and day 4 post-partum. Body weights were recorded individually for each adult animal. The pups were weighed within 24 hours of parturition (day 1 post-partum) and on day 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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily by visual appraisal throughout the study

OTHER:
Oestrous cyclicity (parental animals):
Gestation length calculated from day 0 of pregnancy
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

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

GROSS EXAMINATION OF DEAD PUPS:
yes
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, on test day 36
- Maternal animals: All surviving animals, on day 4 post-partum, or shortly thereafter

GROSS NECROPSY
At the time of sacrifice the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI .
The numbers of corpora lutea and implantation sites were recorded in the female adult animals and reported
The left and right testis and epididymis of all adult male animals were weighed in-dividually before fixation and identified as left or right.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs or parts of organs of all adult animals were fixed in 7% buffered formalin: Ovaries, coagulating gland, preputial gland, prostate, seminal vesicle, uterus (including cervix and oviducts) and vagina. Additionally all organs of all adult animals showing macroscopic lesions were preserved. Detailed histopathologic examinations were performed on the left and right ovary, testis and epididymis of the animals of the highest dose group and the control group.
Postmortem examinations (offspring):
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
Statistics:
For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p≤0.05, p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p≤0.05 and p ≤ 0.01.
For body weight, body weight gain and food consumption, statistical calculation was performed by Provantis. Homogeneity of variances and normality of distribution was tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality distribution, transformation of the values into logarithmic or rank values was performed before performing the DUNNETT test.
For the comparison of classification measurements (for example the Fertility Index) the
FISHER's exact test, n < 100
or
chi2-test with Yates' correction for continuity, n ≥ 100
(p ≤ 0.05 and p ≤ 0.01)
were employed.
These statistical procedures were used for all data.
Reproductive indices:
The following indices were calculated for each group:

Fertility index female [%] = (Number of pregnant rats/ Number of rats used)x 100

The female fertility index reflects the total number of dams that had achieved pregnancy, including those that delivered at term, aborted or had fully resorbed litters.

Gestation Index = (Number of litters with live pups/ Number pregnant)x 100


For each litter and group the following indices were determined:
Birth Index = (Total number of pups born (live +dead)/ Number of implantation scars)x 100


Live Birth Index = (Number of pups live on day 0/1 of lactation/ Total number born (live + dead))x 100


Viability Index = (Number of pups live on day 4 / Number of pups live on day 0/1)x 100


Pre-implantation loss [%] = (Corpora lutea - implantations/ Corpora lutea)x 100


Post-implantation loss [%] = (Implantations - living pups/ Implantations)x 100



Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No signs of behavioural toxicity were noted for the male animals of the control group and the test item-treated groups (100, 300 and 1000 mg MPA/kg b.w./day) during the whole study.
No test item related signs of behavioural toxicity were noted for the female animals of the low and the intermediate dose group (100 and 300 mg MPA/kg b.w./day).
In the high dose group (1000 mg MPA/kg b.w./day) pale faeces were noted for the pregnant animal no. 90 on lactation days 1 to 3 and for the non-pregnant animals nos. 87 and 95 from gestation day 12 or 19 onwards until sacrifice on gestation day 24.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test item-related differences were noted for the body weight of the male rats between the control group and the low and intermediate dose group (100 and 300 mg MPA/kg b.w./day) during the entire study.
In the high dose group (1000 mg MPA/kg b.w./day) a slight reduction in body weight was noted between test day 8 (by 4.3%) and test day 36 (by 7.4%), statistically significant (p ≤ 0.05) on test days 8, 15, 22 and 36.
No test item-related differences were noted on the body weight of the female rats between the control group and the low and intermediate dose group (100 and 300 mg MPA/kg b.w./day) during the pre-mating period.
A slight but test item-related reduction was noted in the high dose group (1000 mg MPA/kg b.w./day) on test day 8 by 5.1% (p ≤ 0.05) and on test day 15 by 6.5% (p ≤ 0.01).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test item-related difference was noted between the gestation length of the females of the control group and those of the low dose group (100 mg MPA/kg b.w./day).
At the intermediate and the high dose group (300 and 1000 mg MPA/kg b.w./day) a slight statistically significantly (p ≤ 0.01) increased gestation length of 22.9 and 23.0 days were noted in comparison to 22.1 days in the control group. This was considered as test item-related.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item-related differences were noted for the gestation index between the control and the test item-treated groups (100, 300 and 1000 mg test item/kg b.w./day).
No test item related differences were noted between the control and the low dose group (100 mg MPA/kg b.w./day).
In the intermediate dose group (300 mg MPA/kg b.w./day) statistically significant (p≤ 0.01) and test item-related reductions were noted in the number of implantation sites per dam, the number of pups (born alive and dead) per dam, the number of pups (born alive) per dam. A test item-related increase was noted for the index of pre-implantation loss (p ≤ 0.01).
In the high dose group (1000 mg MPA/kg b.w./day) the following test item-related changes were noted: a reduction in the number of implantation sites per dam (not statistically significant), a reduction in the number of pups (born alive and dead) per dam (p ≤ 0.01), a reduction in the number of pups (born alive) per dam (p ≤ 0.01), a reduction in the birth index (p ≤ 0.01), a reduction in the live birth index (p ≤ 0.01), an increase in pre-implantation loss (p ≤ 0.01), an increase in post-implantation loss (p ≤ 0.01) and an increase in the number of stillbirth per dam (p ≤ 0.01).

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related differences were noted on body weight at autopsy for the rats of the low and intermediate dose group (100 and 300 mg MPA/kg b.w./day).
At the high dose group (1000 mg MPA/kg b.w./day), a slight and statistically not sig-nificant reduction by 6.9% was noted. As the reduction in live body weight on test day 36 by 7.4% was considered as test item-related, the reduced body weight at autopsy was also considered as test item-related.
Autopsy for the female rats was performed between test days 41 and 53.
No test item-related differences were noted on body weight at autopsy for the rats of the low and intermediate dose group (100 and 300 mg MPA/kg b.w./day).
At the high dose group (1000 mg MPA/kg b.w./day), body weight at autopsy was statistically significantly (p ≤ 0.01) reduced by 14.2%. This was in accordance with the test item-related reduction in live body weight on lactation day 4 by 14.9%

GROSS PATHOLOGY (PARENTAL ANIMALS)
No macroscopic changes were noted in the control group and the test item-treated groups (100, 300 and 1000 mg MPA/kg b.w./day).
No macroscopic changes were noted in the control group and the low and intermediate dose group (100 and 300 mg MPA/kg b.w./day).A test item-related thickened cardia part was noted in the stomach from animal nos. 85, 86 and 92 of the high dose group (1000 mg MPA/kg b.w./day). These changes were confirmed by the finding of squamous cell hyperplasia and hyperkeratosis in the non-glandular stomach of these 3 animals during microscopic examination.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination performed on testicles and epididymides (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test item-related effects in the male rats of the high dose group (1000 mg MPA/kg b.w./day).
No test item-related microscopic changes were noted in the ovaries from the female rats of the high dose group (1000 mg MPA/kg b.w./day).
4/12 females of group 4 (nos. 85, 86, 92 and 94 (premature sacrificed animal)) showed microscopic changes in the forestomach (non-glandular) which consisted of squamous cell hyperplasia / hyperkeratosis and could be associated with macroscopy for 3/12 animals of which the cardia part was thickened at macroscopy.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: small reduction in body weight (1000 mg/kg, males and females), reduction in food consumption (1000 mg/kg, females), thickened cardia part, squamous cell hyperplasia and hyperkeratosis (1000 mg/kg, females)
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
No test item-related differences were noted for the survival index of the pups of the low and intermediate dose group (100 and 300 mg MPA/kg b.w./day).
In the high dose group (1000 mg MPA/kg b.w./day) the survival index of the pups was statistically significantly (p ≤ 0.01) reduced to 2.9%. In detail, from 35 live born pups, only 1 pup was still alive on lactation day 4.

BODY WEIGHT (OFFSPRING)
No test item-related differences were noted for the mean litter weight of the pups per dam in the low and intermediate dose group (100 and 300 mg MPA/kg b.w./day). A severe reduction on the mean litter weight of the male and female pups was noted in the high dose group (1000 mg MPA/kg b.w./day) on lactation day 1, statistically significant at p ≤ 0.01. On lactation day 4 no statistical calculation was performed, as only one male pup was left in the high dose group.A dose dependent reduction in total litter weight was noted in all test item treated groups, due to the reduced number of born pups noted in all test item-treated groups in comparison to the control group.
In the high dose group (1000 mg MPA/kg b.w./day) the total litter weight was additionally reduced due to the reduced body weight of the pups. Hence, only the reduction in total litter weight noted in the high dose group was considered as an adverse effect on pup development.


SEXUAL MATURATION (OFFSPRING)

ORGAN WEIGHTS (OFFSPRING)

GROSS PATHOLOGY (OFFSPRING)

HISTOPATHOLOGY (OFFSPRING)

OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced survival index and a reduced body weight noted for the pups of the high dose group (1000 mg MPA/kg b.w./day)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Fertility indices:

Group / Dose level

Fertility index in %

Non-pregnant rats/ rats used

Group 1

(control)

83.3

2 / 12

Group 2

(100 mg/kg)

66.7

4 / 12

Group 3

(300mg/kg)

83.3

2 / 12

Group 4

(1000 mg/kg)

75.0#

3 / 12

Gestation indices:

Group / Dose level

Gestation index in %

Pregnant rats without live pups (NVP) / Number of pregnant rats

Group 1

(control)

100

0 / 10

Group 2

(100 mg/kg)

87.5

1 / 8

Group 3

(300mg/kg)

80.0

2 / 10

Group 4

(1000 mg/kg)

87.5#

1 / 8

Reproductive parameters and indices:

 

Data

Reproductive parameters

 and indices#1

Control

Group 1

Group 2

Group 3

Group 4

Number of corpora lutea

per dam#2

15.3

12.0

9.5**

12.8

Number of implantation sites per dam

14.3

11.3

7.8**

10.5

Number of pups (born aliveand dead) per dam

13.9

10.0

6.9**

7.3**

Number of pups (born alive) per dam

13.8

9.9

6.9**

4.4**

Fertility index %

83.3

66.7

83.3

75.0

Gestation index %

100.0

87.5

80.0

87.5

Birth index %

97.2

88.9**#3

88.5**#3

69.1**

Live birth index %

99.3

98.8

100.0

60.3**

Pre-implantation loss %

6.5

6.3

17.9**

17.6**

Post-implantation loss %

3.5

12.2**#4

11.5*#4

58.3**

*:p≤0.05; **: p≤0.01, Fisher or Chi2 t-test

#1: The listed indices are calculated from the total number of corpora lutea, implantation sites and fetuses per group

#2: The number of corpora lutea per dam was below the background data (13.8 – 16.5 corpora lutea per dam) in all test item treated groups and no dose response relationship was noted. Hence, the observed low numbers were considered as not test item-related in all dose groups.

#3: The reduced birth index was still in range of background data (87 – 97%) and not considered as test item-related.

#4: The increased post-implantation loss was still in the range of background data (3.6 – 12.7%) and not considered as test item-related.

Test item-related changes aremarked in bold.

Applicant's summary and conclusion

Conclusions:
The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg MPA/kg b.w./day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 36 for the male rats and on lactation day 4 or shortly thereafter for the female rats.

The following no-observed-adverse-effect levels were established:

Parental toxicity
NOAEL (no-observed-adverse-effect level): 300 mg/kg b.w./day, p.o.

Reproductive toxicity
a) adverse effects on sexual function and fertility
NOAEL (no-observed-adverse-effect level): 100 mg/kg b.w./day, p.o.

b) adverse effects on development of the offspring
NOAEL (no-observed-adverse-effect level): 300 mg/kg b.w./day, p.o.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 421. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg MPA/kg b.w./day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 36 for the male rats and on lactation day 4 or shortly thereafter for the female rats.

Parental Toxicity

No test item-related premature deaths were noted in the test item treated groups

Pale faeces were noted for 3 of 12 animals of the high dose group (1000 mg MPA/kg b.w./day) (one pregnant and 2 non-pregnant) during the gestation or the lactation period.

A small reduction in body weight was noted for the male and female rats of the high dose group (1000 mg MPA/kg b.w./day), which was more pronounced for the female rats.

Body weight at autopsy was accordingly reduced for the male and female rats of the high dose group (1000 mg MPA/kg b.w./day).

A reduction in food consumption was noted for the female rats of the high dose group (1000 mg MPA/kg b.w./day) during the whole study.

A test item-related thickened cardia part, squamous cell hyperplasia and hyperkeratosis were noted in 3 or rather 4 stomachs from the female rats of the high dose group (1000 mg MPA/kg b.w./day) during macroscopic and microscopic examination.

No influence was noted on the reproductive organs and the stages of spermatogenesis.

 

Reproductive Toxicity

A prolonged gestation length, reduced numbers of implantation sites per dam, of pups (born alive and dead) per dam, of pups (born alive) per dam and an increased pre-implantation loss were noted in the intermediate and the high dose group (300 and 1000 mg MPA/kg b.w./day).

Additionally, an increased post-implantation loss and an increased number of stillbirth were noted in the high dose group (1000 mg MPA/kg b.w./day), leading to a a reduced birth and live birth index.

Effects on the development of the F1generation (pups)

A reduced survival index and a reduced body weight were noted for the pups of the high dose group (1000 mg MPA/kg b.w./day).

 

 

The following no-observed-adverse-effectlevels were established:

 

Parental toxicity

NOAEL (no-observed-adverse-effect level):                      300 mg/kg b.w./day, p.o.

 

Reproductive toxicity

a) adverse effects on sexual function and fertility

NOAEL (no-observed-adverse-effect level):                      100 mg/kg b.w./day, p.o.

b) adverse effects on development of the offspring

NOAEL (no-observed-adverse-effect level):                      300 mg/kg b.w./day, p.o.