Reaction mass of 5,5'-{(phenylmethanediyl)bis[benzene-4,1-diyl-diazene-2,1-diyl]}bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride and 5,5’-[3,4’-(phenylmethanediyl)diphenylene]bis(diazene-2,1-diyl)bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride

Toxicity to reproduction

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:

from 2010-03-11 to 2010-10-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

registered substance

IUPAC Name:

registered substance

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals: 40 males (10 per group) and 40 females (10 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males (292 to 334 g) and females (181 to 215 g)
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS

Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 83/09).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared every four days using the test item as supplied by the Sponsor.

Test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


STORAGE OF DOSE FORMULATIONS

Dose formulations were stored at room temperature (20 ± 5 °C) in brown glass beakers.


TREATMENT

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group); Group 2: 30 mg/kg/day; Group 3: 80 mg/kg/day; Group 4: 200 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study C70880 (non-GLP), using dose levels of 30, 100 and 300 mg/kg/day, resulting in a NOEL of 30 mg/kg/day.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males (minimum 4 weeks); females (approximately 6 weeks)

Details on mating procedure:

MATING, GESTATION AND LACTATION
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day of mating was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 4 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Harlan Laboratories Ltd., Itingen / Switzerland and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

The application formulations investigated during the study were found to comprise the test item in the range of 88.3% to 94.9% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 1.5% (<15%) from the corresponding mean. In addition, the test item was found to be stable in application formulations when kept 4 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 6 weeks

Frequency of treatment:

Once daily

Details on study schedule:

Acclimatization: 7 days (males and females)
First Test Item Administration: Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 14 days maximum (males and females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day before sacrifice (males); on day 3 post partum (females)
Necropsy: After a minimum of 28 days treatment (males); on day 4 post partum (females)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
80 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
200 mg/kg/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 previous dose-range-finder study in Han Wistar rats
- Rationale for animal assignment (if not random): random

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Viability / Mortality: Twice daily

Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

Food Consumption: Males (weekly during pre-pairing and after pairing periods); females (pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum). No food consumption was recorded during the pairing period.

Body Weights: Recorded daily from treatment start to day of necropsy.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION OF THE STUDY
Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum. When birth did not occur on the expected date (day 21 post coitum), the dams were sacrificed on day 25 post coitum.

NECROPSY
All parent animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all parent animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. Kidneys, liver and spleen of all males and females of each dose group were also weighed.

TISSUE PRESERVATION
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution. In addition, kidneys from five males and females per group were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions and to all animals, which died spontaneously. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Possible test item-related morphologic changes were detected in kidneys of any high-dose animal, thus kidneys from the mid- and low-dose group were examined to establish a possible no-effect level. Histological examination of ovaries was carried out on any females that did not give birth (nos. 60, 61 and 69). In addition, microscopic examination of the reproductive organs of all infertile males was made (nos. 20, 21 and 29). A histopathology peer review was performed by Anapath GmbH.

Postmortem examinations (offspring):

TERMINATION OF THE STUDY
Pups were sacrificed on day 4 post partum.

NECROPSY
All pups sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all pups were killed by an injection of sodium pentobarbital. Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated:
fertility indices, mean precoital time, post-implantation losses, mean litter size.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated:
dead/live pups at first litter check,
pup sex ratios and postnatal loss (up to day 4 post partum).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg, soft feces were noted in all males during the second week of the pre-pairing period until the end of the study; effect considered not to be adverse.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day in males food consumption, body weights and body weight gain were lower; effect considered to be adverse. At 200 and 80 mg/kg bw/day in females body weights and body weight gain were lower; effect considered not to be adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day in males food consumption, body weights and body weight gain were lower; effect considered to be adverse. At 200 and 80 mg/kg bw/day in females body weights and body weight gain were lower; effect considered not to be adverse.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg, test item-related changes were observed in the kidney in six males; not considered as relevant for humans.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

1 IN-LIVE DATA - PARENTAL ANIMALS

1.1 VIABILITY / MORTALITY
In group 4, male no. 36 was found dead on day 13 of pairing period and female no. 74 on day 22 of the gestation period. In the male, soft feces were noted starting on day 7 of the pre-pairing period until the death and food consumption was lower during the first week of the pre-pairing period. At necropsy, beginning of autolysis and pelvis discolored dark red of both kidneys were noted. Histologically, congestion without reactive change was noted. For the female, no clinical signs and no abnormal findings at necropsy were noted. However, the cause of death could not be established.

1.2 CLINICAL SIGNS OR OBSERVATIONS

In group 4, soft feces were noted in all males during the second week of the pre-pairing period until the end of the study. One male (no. 37) had diarrhea on one day during the pre-pairing period. This was considered to be due to the treatment with the test item. Signs of discomfort such as bedding in mouth and salivation were observed in two males (nos. 33 and 39) during the pre-pairing period. Female no. 77 had uncoordinated movements between days 11 and 21 and ruffled fur between days 14 and 22 of the gestation period and tilted head between day 2 and 4 of the lactation period. Female no. 73 had hair loss starting on day 2 of the gestation period until day 2 of the lactation period. Due to single occurrences, these clinical signs were considered to be incidental.

1.3 FOOD CONSUMPTION OF MALES

Pre-pairing and After Pairing Periods

In group 4, mean food consumption was lower during the pre-pairing period (-9.1% compared to the control). This reduction was considered to be a test item-related effect although not statistically significant.

In order of ascending dose levels, the overall differences in food consumption were: ±0.0%, +0.8% and -9.1% during pre-pairing period and +9.0%, +11.9% and +11.0% during the after pairing period (percentages refer to the respective values of the control group).

1.4 FOOD CONSUMPTION OF FEMALES

Pre-pairing, Gestation and Lactation Periods

Mean food consumption was not considered to be affected by the treatment with the test item during the entire duration of the study.

In order of ascending dose levels, the overall differences in food consumption were: +1.1%, 2.3% and -5.2% during the pre-pairing period, +4.2%, -1.9% and -2.3% during the gestation period and +4.9%, +2.6% and +9.8% (percentages refer to the respective values of the control group).

1.5 BODY WEIGHTS OF MALES

Pre-pairing, Pairing and After Pairing Periods

In group 4, mean body weight gain was statistically significantly lower on day 2, 3 and 4 of the pre-pairing period and on day 2 of the pairing period. At the end of the pre-pairing period, mean body weight gain was lower when compared to the control (about 24% less than the control group). This was due to the lower food consumption which was observed during the pre-pairing period.

In groups 2 and 3, no effects were noted. The statistically significantly lower body weight gain on day 2 of the pre-pairing period was considered to be incidental since it occurred only on this occasion.

In the order of ascending dose levels, the overall mean body weight gains were: +10%, +10%, +10% and +8% during the pre-pairing period, +7%, +8%, +8% and +5% during the pairing period and +1%, +2%, +3% and +3% during the after pairing period (percentages refer to the respective time intervals).

1.6 BODY WEIGHTS OF FEMALES

Pre-pairing, Gestation and Lactation Periods

In groups 3 and 4, mean body weight gain was statistically significantly reduced during most of the pre-pairing period and mean body weight was lower at the end of the pre-pairing period. Afterwards, mean body weight gain recovered and no effects were noted.

In group 2, mean body weight gain was statistically significantly lower on days 2, 6, 8, 10, 11, 12 and 14 of the pre-pairing period. This was a transient reduction, since mean body weight gain recovered and it was considered not to be adverse.

In the order of ascending dose levels, the overall mean body weight gain was +10%, +7%, +7% and +7% during the pre-pairing period and +60%, +56%, +55% and +53% during the gestation period and +6%, +6%, +7% and +11% during the lactation period (percentages refer to the respective time intervals).

2 REPRODUCTION AND BREEDING DATA

2.1 MATING PERFORMANCE AND FERTILITY

The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.7, 5.0, 4.3 and 4.9 days in order of ascending dose level. The median precoital time was 3, 4, 3 and 3 days in order of ascending dose level.

One female in group 2 and two females in group 3 were not pregnant. Thus the fertility and conception indices were 100.0%, 90.0%, 80.0% and 100.0% in groups 1, 2, 3 and 4.

2.2 DURATION OF GESTATION

The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.5, 21.6, 21.6 and 21.4 days, in order of ascending dose level.

2.3 CORPORA LUTEA COUNT

The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (13.5, 14.0, 13.9 and 14.5 in order of ascending dose level) and gave no indication of a test item-related effect.

2.4 IMPLANTATION RATE AND POST-IMPLANTATION LOSS

The mean number of implantations per dam and of post-implantation losses did not give any indication of a test item-related effect.

The mean numbers of implantations per dam were 12.9, 12.6, 13.6 and 13.6 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 8.5, 5.3, 8.3 and 9.8% in order of ascending dose level.

2.5 LITTER SIZE AT FIRST LITTER CHECK

The number of live pups at first litter check was not affected by treatment with the test item. The mean number of live pups per litter was 11.8, 11.9, 12.5 and 12.2 in order of ascending dose level.

2.6 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

The total numbers of pup loss during the first four days were 0, 2, 0 and 0 in order of ascending dose level, corresponding to 0.0, 1.9, 0.0 and 0.0% of living pups.

The resulting viability indices were 100.0%, 98.1%, 100.0% and 100.0% in order of ascending dose levels.

3 TERMINAL FINDINGS - PARENTAL ANIMALS

3.1 ORGAN WEIGHTS

Males

In group 4, absolute and relative weights of kidneys were statistically significantly increased. This correlated with the microscopic change noted during the histological evaluation and therefore considered to be test item-related. Statistically significantly higher absolute weights of spleen and of testis in group 4 were within the range of the historical control data. In groups 3 and 4, statistically significantly higher absolute and relative weight of liver were considered not to be adverse since there were no histological findings.

Females

In group 4, the statistically significantly higher absolute and relative weight of spleen was within the range of the historical control data. In groups 3 and 4, absolute and relative weight of liver were statistically significantly increased. Since no histological findings were noted, this was not considered to be adverse.

3.2 MACROSCOPICAL FINDINGS

Males

In group 4, beginning of autolysis and dark red discoloration of pelvis of both kidneys were noted in male which was found dead. Another male was noted to have kidney discolored tan. No other macroscopical findings were noted.

Females

In group 3, one female was noted to have a diaphragmatic hernia on lateral liver lobe. The histopathology examination revealed to be a hepatodiaphragmatic nodule. No other macroscopical findings were noted.

3.3 HISTOPATHOLOGY FINDINGS

Kidney

In group 4, test item-related changes were observed in the kidney in six males. The alterations consisted of minimal to moderate tubular cell degeneration and necrosis, increased incidence and severity of tubular basophilia, slight tubular cell hyperplasia (intratubular), minimal to moderate tubular dilation, minimal to moderate granular casts, minimal to slight hyaline casts, increased incidence and severity of mononuclear cell infiltration, increased incidence and severity of hyaline droplets, minimal granuloma, and minimal focal transitional hyperplasia were observed. In one female minimal tubular cell degeneration and necrosis was noted.

Liver

In group 3, one female was noted to have a hepatodiaphragmatic nodule. No other histopathological finding was noted.

No test item-related findings were noted in testes, epididmydes, prostate, seminal vesicles, coagulating glands and ovaries.

Treatment with the test item did not reveal effects on the completeness of stages or cell populations. There was no indication for maturation arrest or any other degenerative type. All findings recorded were within the range of normal background alterations.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: NOAEL = NOEL for effects on reproduction/development in this study.

Remarks on result:

other: Generation: reproduction/development (migrated information)

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:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

1 LITTER DATA - F1 PUPS

1.1 EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

At first litter check, one male pup in control group was noted to have the head discolored blue, two male pups in group 2 to have both nostrils closed and one male pup in group 3 to have a right wound on the mouth. During the lactation, one male pup in group 4 was noted to have the mouth injured. Type and incidence of these findings did not give any indication of a test item-related effect.

1.2 SEX RATIOS

Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.

The proportion of males on day 4 post partum was 53%, 48%, 49% and 43% in order of ascending dose level.

1.3 PUP WEIGHTS TO DAY 4 POST PARTUM

No statistical significances were observed in mean pup weights on day 1 and on day 4 post partum. On day 1 post partum mean pup weights were 6.4, 6.3, 6.2 and 6.1 g for combined data of male and female pups and on day 4 post partum were 9.5, 9.6, 8.8 and 8.7 g for combined data of male and female pups in order of ascending dose level.

1.4 MACROSCOPICAL FINDINGS

In group 2, autolysis was observed in one male pup which was found dead on day 1 at first litter check. No other abnormal finding was noted.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (30)	3 (80)	4 (200)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated	10	10	10	10
Number of pregnant females (A)	10	9	8	10
Number of females found dead (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	10	9	8	9

 

(A) Female No. 60 in group 2, Female Nos. 61 and 69 in group 3 were not pregnant.

(B)  Female No. 74 in group 4 was found dead on day 22 of the gestation period.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL (= NOEL in this study) for reproduction/ developmental toxicity was considered to be 200 mg/kg/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the registered substance on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 10 males and 10 females were treated by gavage with the test item once daily. The animals were treated with the registration substance at dose levels of 0 (control), 30, 80 and 200 mg/kg body weight/day. A standard dosevolume of 10 mL/kg body weight with a daily adjustment to the actual bodyweight was used. Control animals received the vehicle (highly purified water) alone. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

Under the conditions of this study, the NOAEL (= NOEL in this study) for reproduction/ developmental toxicity was considered to be 200 mg/kg/day.

The following detailed results were obtained:

 

PARENTAL ANIMALS

Mortality and General Tolerability

At 200 mg/kg, one male and one female were found dead before scheduled necropsy. For the male, soft feces were noted starting on the second week of the pre-pairing period, onwards and lower food consumption during the first week of the pre-pairing period. At necropsy, the animal was autolytic and a dark red discoloration of the pelvis of both kidneys was observed. Histologically, congestion without reactive change was noted. For the female, no clinical signs and no abnormal macroscopical finding was observed. There was no clear indication that these deaths were caused by the treatment with the test item.

At 200 mg/kg, soft feces were noted in all males during the second week of the pre-pairing period until the end of the study.

Food Consumption

 

At 200 mg/kg, mean food consumption was lower in males during the pre-pairing period.

 

Body Weights

 

At 200 mg/kg, when compared to the control mean body weight was lower in males at the end of the pre-pairing period.

 

In females at all dose levels, mean body weight gain was statistically significantly lower during the pre-pairing period. This was considered to be of transient nature since mean body weight recovered and no effects were noted during the following periods.

 

Reproductive Data

 

Mating performance, fertility and duration of gestation were not affected by the treatment with the test item. Relevant reproduction parameters such as mean number of corpora lutea, mean number of implantations per dam, post-implantation losses and litter size were also not affected by the treatment with the test item.

Organ Weights

At 200 mg/kg, absolute and relative weight of kidneys was statistically significantly higher in males. This correlated with the histological findings and was therefore considered to be test item-related. Absolute weight of spleen was statistically significantly higher in males and in females, relative weight of spleen was also higher in females. However, the weights were within the range of the historical control data and not considered to be adversely affected.

 

At 80 and 200 mg/kg, absolute and relative weight of liver was statistically significantly increased in males and females. Although this increase was dose-dependent, the histological examination did not reveal any microscopic change. Therefore it was not considered to be adverse.

Macroscopical Findings and Histopathological Examinations

Kidney:
At 200 mg/kg, test item-related changes were observed in the kidney in six males. The alterations consisted of minimal to moderate tubular cell degeneration and necrosis, increased incidence and severity of tubular basophilia, slight tubular cell hyperplasia (intratubular), minimal to moderate tubular dilation, minimal to moderate granular casts, minimal to slight hyaline casts, increased incidence and severity of mononuclear cell infiltration, increased incidence and severity of hyaline droplets, minimal granuloma, and minimal focal transitional hyperplasia were observed. In one female minimal tubular cell degeneration and necrosis was found. However, it is scientifically well established, that the male rat is prone to hyaline droplet formation in the kidneys and that this unique male rat-specific kidney effect has no relevance for human health

 

No test item-related findings were noted in liver, testes, epididimydes, prostate, seminal vesicles, coagulating glands and ovaries. Sperm stages: Treatment with the test item did not reveal effects on the completeness of sperm stages or cell populations. There was no indication for maturation arrest or any other degenerative type. All findings recorded were within the range of normal background alterations.

 

LITTER DATA - F1 PUPS

 

Mean litter size at first litter check and on day 4 post partum was not affected by the treatment with the test item. Mean pup weight was also not affected.