Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 April to January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with no restrictions.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Reaction mass of N-[2-(2-Oxoimidazolidin-1-yl)ethyl] methacrylamide and methacrylic acid, other name: Sipomer wam II
- Stability under test conditions:
- Storage condition of test material: at room temperature and protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France.
- Age at study initiation: (P) 10 wks for males, 9 wks for females
- Weight at study initiation: (P) Males: 363-433 g; Females: 189-248 g
- Fasting period before study: no
- Housing: individually housed, except during pairing, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Toward the end of gestation and during lactation with their litter, autoclaved wood shavings (SICSA, Alfortville, France) were provided as nesting material, a few days before delivery and during the lactation period. Each cage contained an object (rat hut) for the enrichment of the environment of the rats. nor applicable
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 2626975 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 30 May 2012 To: 23 July 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water, treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle. The test item dose formulations were prepared on a daily basis and were stored and delivered at room temperature in brown flasks.

DIET PREPARATION
not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable
- Concentration in vehicle: 0; 13.3; 40 and 120/80 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5mL/kg bw/d
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until mating occurs or 14 days have elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations have been quantified by a validated analytical method.
The validation of the analytical method was conducted in CiToxLAB France/Study No. 38954 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report. The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1, 3 and 5 was determined.
Duration of treatment / exposure:
in the males:
- 2 weeks before pairing (from study days 1 to 14),
- during the pairing period (3 weeks) (from study day 15 until study days 16 to 28),
- until sacrifice (at least 5 weeks in total) (from study days 17 to 29 until study day 36).
in the females:
- 2 weeks before pairing (from study days 1 to 14),
- during the pairing period (3 weeks) (from study days 15 to 28),
- during gestation (from study days 16 to 29 until study days 36 to 49),
- during lactation until day 4 post-partum inclusive
(from study days 37 to 50 until study days 41 to 54).
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks for males, 11 weeks for females
Doses / concentrations
Remarks:
Doses / Concentrations:
0; 66.7; 200 and 600/400 mg/kg bw/d
Basis:
other: based on N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid content.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in a preliminary study, the test item was given to rats (3/sex/group), by daily oral administration (gavage) for 1 week at 0, 100, 300 or 1000 mg/kg bw/day. At 1000 mg/kg bw/day, all males had marked to severe clinical signs (ptyalism, piloerection, loud breathing, dyspnea, abdominal breathing, chromorhynorrhea, soiled snout and/or hypoactivity); 1/3 females had ptyalism. There was a minimal decrease in mean body weight (-6.7% vs. controls) at the end of the treatment period and a marked decrease in mean food consumption in males only (-25.7% vs. controls).
At necropsy, 1/3 males (1000 mg/kg bw/day) had a thymus reduced in size and stomach wall with red discoloration. One out of 3 females (300 mg/kg bw/day) had an uterus dilated and with a translucent content.
There were no treatment-related findings at 300 or 100 mg/kg bw/day.
Overall, 1000 mg/kg bw/day were considered to be an excessive dose-level. Therefore, 600 mg/kg bw/day were selected as the high-dose level. The low-dose and mid-dose have been selected using a ratio representing a three-fold interval (i.e. 66.7 and 200 mg/kg bw/day).

On study day 5 of the current study, 1/10 high dose males was euthanized for ethical reason and on study day 6, 3/9 surviving males from the same group had loud breathing, abdominal breathing and/or chromorhinorrhea and 1/10 females had piloerection and loud breathing. Therefore, 600 mg/kg/day was proved to be an excessive dose-level. It was thus decided to lower the dose administered to animals from the high-dose group from study day 6 (400 mg/kg/day instead of 600 mg/kg/day).
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: male: on the first day of treatment (day 1), then once a week until sacrifice; female: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p..

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females are mated.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis and epididymis weight for all tested males
Litter observations:
STANDARDISATION OF LITTERS
Not applicable

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 abnormalities. No tissues were preserved.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the end of the pairing period (at least 5 weeks of treatment in total)
- Maternal animals: All surviving animals on day 5 p.p..

GROSS NECROPSY
- Gross necropsy consisted of external and inernal examinations including the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 5 post-partum.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 7.8.1/1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
Animals were subjected to postmortem macroscopic examinations.

GROSS NECROPSY
- Gross necropsy consisted of external examinations only. No tissues were preserved.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not applicable as no tissues were preserved.
Statistics:
Data are compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).

PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
Reproductive indices:
pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

post-implantation loss (manually calculated):
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females
Offspring viability indices:
live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups


Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality at 600/400 mg/kg bw/d. See more details below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight loss between days 1 and 8 for the males in the highest tested group. Lower body weight gain during the period of days 1 to 15 for the females treated with the top dose. Then mean body weight was comparable to the one of the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body weight loss between days 1 and 8 for the males in the highest tested group. Lower body weight gain during the period of days 1 to 15 for the females treated with the top dose. Then mean body weight was comparable to the one of the control group.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see details below
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Decrease in mean food consumption in boh sexes from the high dose-group during the premating period. During the lactation period, lower mean food consumption than in the control group in the high dose group (but not statistically significant).

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):
See tables 7.8.1/3 and 7.8.1/4 and details below

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): there were no unscheduled deaths in the control, 66.7 or 200 mg/kg bw/d groups. Deaths were recorded in the 600/400 mg/kg bw/d group. 2/10 males were euthanasied for ethical reason (day 5 and 8 respectively) and 3/10 females were found dead or sacrificed for ethical reason (day 8, 15 and 20 respectively). Before sacrifice or death, animals suffered of ptyalism, piloerection, hypoactivity, chromorhinorrhea, dyspnea, loud and abdominal breathing and presented a round back and emaciated appearance or were cold to the touch. In the surviving animals clinical signs were observed and listed in table 7.8.1/2. Piloerection, round back, emaciated appearance, loud and abdominal breathing were considered to be related to the test item treatment. Ptyalism was considered to be related to the treatment with the test item but of minor toxicological significance. All others clinical signs (areas of hair loss, cutaneous lesions, abnormal growth of teeth and/or chromodacryorrhea) are commonly observed in this species and strain. A treatment-related effect was considered unlikely.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): In the group treated at 600/400 mg/kg/day, the pre and post-implantation losses were higher than in the control group and resulted in a non-statistically significant lower mean number of pups delivered (12.7 vs. 13.2 in the Historical Control Data). This minor difference was mainly due to one litter (with 57.1% of post-implantation loss) and therefore a test item treatment-related effect was considered unlikely.

GROSS PATHOLOGY (PARENTAL ANIMALS): gas distension of the stomach/cecum was found in 3/5 decedents, associated in one male with red and depressed foci which correlated with microscopic gastric erosions. In another decedent male, the thymus was gelatinous, which correlated with interlobular edema, and there was stress-associated thymic lymphoid atrophy and increased apoptotic cell numbers microscopically. In the surviving animals, few macroscopic findings were noted at the end of the treatment period in males but were of those commonly recorded in the Sprague-Dawley rat and none were considered to be related to the test item administration. There were no macroscopic findings in females.

HISTOPATHOLOGY (PARENTAL ANIMALS): The cause of deaths/morbidity in these rats was not evident at microscopic examination. In the surviving animals, there were no test item-related changes in the testis, epididymis, ovaries and oviducts.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
other: Dose adjusted for N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid content.
Sex:
male/female
Basis for effect level:
other: Mortality, clinical signs and effects on body weight and food consumption observed at 600/400 mg/kg bw/d
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
279 mg/kg bw/day (actual dose received)
Based on:
other: Dose of substance as registered (including impurities residual water necessary for stability)
Sex:
male/female
Basis for effect level:
other: Mortality, clinical signs and effects on body weight and food consumption observed at 557/836 mg/kg bw/d
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
400 - 600 mg/kg bw/day (actual dose received)
Based on:
other: Dose adjusted for N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid content.
Sex:
male/female
Basis for effect level:
other: Non-statistically significant increase in post-implantation losses in the highest dose group which was mainly due to one litter and therefore considered not to be adverse.
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
557 - 836 mg/kg bw/day (actual dose received)
Based on:
other: Dose of substance as registered (including impurities residual water necessary for stability)
Sex:
male/female
Basis for effect level:
other: Non-statistically significant increase in post-implantation losses in the highest dose group which was mainly due to one litter and therefore considered not to be adverse.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details below and in Table 7.8.1/5.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See details below.
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:
no effects observed
Description (incidence and severity):
See details below and in Table 7.8.1/5.
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING): In the groups treated at 200 or 600/400 mg/kg/day, the viability indexes were significantly reduced when compared with controls. However, there was no dose-relationship and the viability index of the high-dose group was similar to the mean viability index of the Historical Control Data (95.5% vs. 94.9%). Therefore, theses findings were considered not to be adverse.

CLINICAL SIGNS AND GROSS PATHOLOGY (OFFSPRING): There were dose-related increases in the percentages of litters with pups having clinical signs from 200 mg/kg/day. These increases were mainly due to litter Y23635 (200 mg/kg/day) and litter Y23648 (600/400 mg/kg/day) and there were no dose-related increases in the percentage of litters with pups having gross external abnormalities. Therefore a test item treatment-related effect was considered unlikely.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
toxicity on progeny
Generation:
F1
Effect level:
400 - 600 mg/kg bw/day (actual dose received)
Based on:
other: Dose adjusted for N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid content.
Sex:
male/female
Basis for effect level:
other: Viability indexes were slightly reduced in the 200 and 600 mg/kg bw/d groups but still comparable to historical control and therefore considered not to be adverse.
Dose descriptor:
NOAEL
Remarks:
toxicity on progeny
Generation:
F1
Effect level:
557 - 836 mg/kg bw/day (actual dose received)
Based on:
other: Dose of substance as registered (including impurities residual water necessary for stability)
Sex:
male/female
Basis for effect level:
other: Viability indexes were slightly reduced in the 557 and 836 mg/kg bw/d groups but still comparable to historical control and therefore considered not to be adverse.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 7.8.1/2 : Clinical signs observed in the surviving animals

Sex

Male

Female

Dose-level (mg/kg/day)

0

66.7

200

600/400

0

66.7

200

600/400

Piloerection

0

1

0

4

0

0

0

0

Round back

0

0

0

2

0

0

0

0

Emaciated appearance

0

0

0

2

0

0

0

0

Aggressive behaviour

1

2

0

1

0

0

0

0

Loud breathing

0

0

0

5

0

0

2 (P/G)

2 (P/G)

3 (L)

Abdominal breathing

0

0

0

2

0

0

0

0

Ptyalism

0

1

0

8

0

0

0

2 (P)

5 (G)

Aera of hair loss on forelimb, abdomen or neck

1

2

1

0

0

1 (P/G/L)

0

0

Cutaneous lesion on neck

1

0

2

1

1 (G)

0

0

0

Abnormal growth of teeth

0

0

1

0

0

0

0

0

Chromodacryorrhea

0

0

0

0

1 (P/G)

0

0

0

P: premating and mating periods, G: gestation period, L: lactation period.

Table 7.8.1/3:Mating and fertility data results

Dose-level (mg/kg/day)

0

66.7

200

600/400

Number of animals paired (M + F)

10 + 10

10 + 10

10 + 10

8 + 8 (a)

Number of males mated

10

10

10

7(b)

Number of females mated

10

10

10

7(b)

Mean number of days taken to mate

3.1

3.8

2.4

3.6

Number of pregnant females

10

10

10

7

Male fertility index(%)

100

100

100

100

Female fertility index(%)

100

100

100

100

(a): one female was sacrificed the first day of pairing period and another one was found dead before the start of the pairing period.

(b):one female was sacrificed after a 5-day pairing period with no evidence of mating.

Table 7.8.1/4:Delivery data results

Dose-level (mg/kg/day)

0

66.7

200

600/400

Number of pregnant females

10

10

10

7

Number of females which delivered

10

10

10

7

Mean duration of gestation (days)

21.2

21.1

21.1

21.3

Mean number ofcorpora lutea

16.4

17.0

15.2

15.9

Mean number of implantations

16.2

16.8

14.8

15.1

Mean pre-implantation loss (%)

1.1

1.3

2.9

5.1

Mean number of pups delivered

14.6

15.0

13.5

12.7

Mean post-implantation loss (%)a

10.8

10.9

10.1

17.5

a: manually calculated, no statistics performed.

Table 7.8.1/5: Clinical signs and gross necropsy results in the offspring 

 

Clinical signs

Gross external abnormalities

Dose-level (mg/kg/day)

0

66.7

200

600/400

0

66.7

200

600/400

Scab on tail

 

Y23621-3

 

 

 

 

 

 

Dehydration

 

Y23621-13

 

 

 

 

 

 

Emaciated appearance

 

Y23621-13

Y23635-6

Y23635-10

Y23635-12

Y23639-14

Y23644-6

 

 

Y23635-6

 

Hind limb necrosis

 

 

Y23631-8

Y23648-7

 

 

Y23631-8

Y23648-7

Generalized pallor

 

 

Y23635-6

Y23635-12

 

 

 

 

 

Cold to the touch

 

 

Y23635-6

Y23635-12

Y23639-14

Y23648-12

 

 

 

 

Hematoma on back/abdomen

 

 

 

Y23643-9

 

 

 

 

Hematoma on hind limb

 

 

 

Y23648-12

 

 

 

 

 

 

 

 

 

 

 

 

 

Number of affected pups

0

2

5

4

0

0

2

1

Number of litter examined

10

10

10

7

10

0

10

7

Number of affected litters

0
(0.0)

1
(10.0)

3
(30.0)

3
(42.9)

0
(0.0)

0
(0.0)

2
(20.0)

1
(14.3)

In brackets: percentage (%) of affected litters.

Applicant's summary and conclusion

Conclusions:
Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg/day (based on mortality, clinical signs and effects on body weight and food consumption recorded for both sexes at 600/400 mg/kg/day),
- the NOAEL for reproductive performance (mating, fertility and delivery data) was considered to be 600/400 mg/kg/day,
- the NOAEL for toxic effects on progeny was considered to be 600/400 mg/kg/day.
Executive summary:

The potential effects of the Reaction mass of N-[2-(2-Oxoimidazolidin-1-yl)ethyl] methacrylamide and methacrylic acid, on reproductive and developmental parameters were assessed in an OECD 421 compliant study following daily oral administration (by gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 4post-partum (p.p.).

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Reaction mass of N-[2-(2-Oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid (batch No. MWAM12059A), daily, by oral administration (gavage), 2 weeks before mating, during mating and, for the males, until sacrifice, for the females, throughout gestation until day 4p.p., at dose-levels of 66.7, 200 or 600/400 mg/kg/day (i.e, 93, 279 and 836/557 mg/kg bw/day in terms of registered substance). An additional group of ten males and ten females received the vehicle control, drinking water, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5p.p..

The males were sacrificed after completion of the mating period. Dams were sacrificed on day 5p.p.. Body weights and selected organs weights were recorded and a complete macroscopicpost-mortemexamination performed, with particular attention paid to the reproductive organs.

Microscopic examination was performed on the reproductive organs from control and high-dose males and females at terminal sacrifice and from premature decedents, and on all macroscopic observations in all groups.

Pups, including those found dead before study termination, were also submitted for a macroscopicpost-mortemexamination.

.

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 5 remained within an acceptable range of -9.0% to -0.4% when compared to the nominal values. Reaction mass of N-[2-(2-Oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid was not detected in control samples

 

As death occurred in one male exposed to the dose of 600 mg/kg bw/d on day 5 (animal sacrificed for ethical reason), the high-dose level was lowered to 400 mg/kg bw/d for the rest of the study period. An another male was euthanized for ethical reason on day 8 and 3/10 females were found dead or sacrificed on day 8, 15 and 20 respectively, at the highest dose-level. Before sacrifice or death, animals suffered of ptyalism, piloerection, hypoactivity, chromorhinorrhea, dyspnea, loud and abdominal breathing and presented a round back and emaciated appearance or were cold to the touch. In the surviving animals clinical signs were observed (piloerection, round back, emaciated appearance, loud and abdominal breathing) and were considered to be related to the test item treatment. In the surviving animals, few macroscopic findings were noted at the end of the treatment period in males but were of those commonly recorded in the Sprague-Dawley rat and none were considered to be related to the test item administration.

 

With regards to reproductive and developmental parameters, the following observations were made:

 

Mating and fertility data: there were no effects on the mean number of days taken to mate. All mated females were pregnant.

 

Delivery data: there were no obvious treatment related effects. In the group treated at 600/400 mg/kg/day, the post-implantation loss was higher than in the control group but with no statistical significance. This difference was mainly due to one litter (with 57.1% of post-implantation loss).

 

Pups mortality: there were no obvious treatment-related effects on pup mortality.

 

Pups clinical signs and external abnormalities: there were no test item treatment-related findings.

 

Pup viability: there were no treatment-related effects on live birth and lactation indexes. In the groups treated at 200 or 600/400 mg/kg/day, the viability indexes were statistically significantly reduced (94.8% and 95.5% respectively,vs.controls) but still comparable to Historical Control Data (94.9%) and therefore considered not to be adverse.

 

Pup body weight: there were no effects on mean pup body weights and mean pups body weight changes when compared with controls.

 

Pup sex ratio: there were no treatment-related effects on sex ratio (% of male pups) both on days 1 and 5p.p..

 

Pathology: At terminal sacrifice, the test item administration did not induce any organ weight or macroscopic changes. There were no significant microscopic findings in the testis, epididymis, ovaries and oviducts.

 

In conclusion, based on the experimental conditions of this study:

- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 200 mg/kg/day (based on mortality, clinical signs and effects on body weight and food consumption recorded for both sexes at 600/400 mg/kg/day),

- the NOAEL for reproductive performance (mating, fertility and delivery data) was considered to be 600/400 mg/kg/day,

- the NOAEL for toxic effects on progeny was considered to be 600/400 mg/kg/day