[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29-Jul-2009 To 04-Mar-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). There were no circumstances that may have affected the quality or integrity of the data.

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:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Molecular formula:

R-O-(CH2CH2O)nCH2CO-NH-CH2CH2OH

IUPAC Name:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Test material form:

liquid

Details on test material:

Colour: Yellowish
Storage conditions: at room temperature at about 20±5°C, away from direct sunlight
Stability of test item in vehicle: 7 days
Expiry date (Retest Date): 23-July-2010

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanRcc: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: (P) 11 weeks
- Weight at study initiation: (P) Males: 298 - 342 g; Females: 187 - 207 g
- Fasting period before study: no
- Housing: 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 (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 (batch no. 15/09) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimation period: 7 days minimum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): range: 22 ± 3 °C
- Humidity (%): range: 30 - 70%)
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12-hour dark cycle with music during the light period.

IN-LIFE DATES: From: 29-Jul-2009 To: 28-Sep-2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle : had been prooved as appriopriate in previous studies
- Concentration in vehicle: 200 g/ml
- Amount of vehicle: dose volume = 5 mL/kg body weight
- Lot/batch no.: 130022563006152

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor. The stability of the test item formulations was considered to be sufficient for weekly preparation based on the dose formulation analyses performed for non GLP study: Dose Range-Finding for Reproduction/ Developmental Toxicity Screening Test in the Wistar Rat. Stability analyses were repeated for this study.
The substance was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Test item formulation was stirred at least 15 minutes until a homogenous solution was prepared. Afterwards test item formulation was stirred very gently or incubated at room temperature without steering until discarding any foam.
Homogeneity of the test item in the vehicle was maintained during the daily administration periodusing a magnetic stirrer.
Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days maximum
- Proof of pregnancy: if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male of the same group which had already mated successfully. The reproductive organs of infertile females were examined histopathologically in order to ascertain the reason for the infertility.
- After successful mating each pregnant female was caged : 9/10 females were pregnant in groups 1 (contrl) and 2 (100 mg/kg bw/day) and 10/10 in groups 3 (300 mg/kg bw/day) and 4 (1000 mg/kg bw/day)
- 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 sample 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 (7 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 Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an evaporative light scattering detector (ELSD) following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

The substance peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of AA 15 and, therefore, it was confirmed that only PEG 300 was administered in the control part of the experiment.

The application formulations investigated during the study were found to comprise
The substance in the range of 95.7% to 104.5% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of the substance in the preparations was approved because single results found did not deviate more than 3.4% (<15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept 7 days at room temperature due to recoveries in the range of 2.6% to 6.1% which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and PEG 300 as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Males: 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Details on study schedule:

Acclimatization: 7 days minimum (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 (males); 14 days maximum (females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day before sacrifice (males); on day 3 post partum (females)
Necropsy: After 28 days of treatment (males); on day 4 post partum (females)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

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

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations : see attached document

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Males (weekly during pre-pairing period); females (pre-pairing period; days 1 - 8 and 8 - 14 of the pre-pairing period; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum). No food consumption was recorded during the pairing period.

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):

SACRIFICE

Males were sacrificed after they had been treated for 28 days. Dams were sacrificed on day 4 post partum.

Females which did not give birth on the expected date (day 21 post coitum) were sacrificed on day 25 post coitum.


NECROPSY

All parent animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes at the scheduled necropsy.

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

The testes and epididymides of all parental males were weighed as pairs.


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.


HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis were stained by PAS–hematoxylin.


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 principal investigator (histopathology phase). The same applied to all occurring gross lesions and to the female which had to be terminated for ethical reason.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Histological examination of ovaries was carried out on females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.

Postmortem examinations (offspring):

SACRIFICE

Pups were sacrificed on day 4 post partum.


NECROPSY

All pups were sacrificed by an injection of sodium pentobarbital.

Dead pups, except those excessively cannibalized, were examined macroscopically.

All pups were examined macroscopically for any structural changes at the scheduled necropsy.

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):

Pushing head through the bedding material and salivation in both sexes at 300 and 1000 mg/kg bw/day; diarrhea in males at 300 and 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day; activity reduced for 1-3 days in males at 1000 mg/kg bw/day.

CONCLUSION: No adverse effects

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Except for 1 female (no. 66) at the dose level of 300 mg/kg bw/day, all animals survived until the scheduled necropsy.

CONCLUSION: No adverse effects

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reversible and not adverse reduction of body weights and body weight gain in males at 300 and 1000 and in females at 1000 mg/kg bw/day.

CONCLUSION: No adverse effects

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reversible and not adverse reduction of food consumption in males at 1000 and in females at 300 and 1000 mg/kg bw/day

CONCLUSION: No adverse effects

Ophthalmological findings:

not specified

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:

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 CLINICAL SIGNS OR OBSERVATIONS

See attached tables on pp. 1-3

Except for 1 female (no. 66) at the dose level of 300 mg/kg bw/day, all animals survived until the scheduled necropsy.

In female no. 66, rales, ruffled fur and rhinorrhea were observed on days 2 and 3 of the lactation period. Because these symptoms were accompanied by a significant body weight loss (it lost 8 and 10% of body weight on days 2 and 3, respectively) an application error was assumed and this female was killed for ethical reason on day 3 of the lactation period.

No clinical signs were observed in the control group and at the dose level of 100 mg/kg bw/day.

At the dose levels of 300 and 1000 mg/kg bw/day, pushing head through the bedding material and salivation after application were observed in all males and females at both dose levels during the most of the treatment period. These clinical signs were considered not to be adverse but a result of discomfort caused by the test item administration.

During the pre-pairing period a slight diarrhea was observed in 3 males (nos. 23, 24 and 30) at the dose level of 300 mg/kg bw/day and in 6 males (nos. 31, 33, 36, 37, 38 and 39) and 2 females (nos. 72 and 79) at the dose level of 1000 mg/kg bw/day.
In addition, a reduced activity was observed in 7 males (nos. 31, 32, 33, 34, 35, 37 and 39) at the dose level of 1000 mg/kg bw/day.
Both these effects were considered to be test item-related but not adverse since in all affected animals they were transient (occurred only for a period of 1 to 3 days) and had no observable influence on the wellbeing of the animals.

Because of isolated occurrence following symptoms: rales in 2 males (nos. 35 and 37) at the dose level of 1000 mg/kg bw/day observed for 1 day during pre-pairing period and a watery discharge in 1 female (no. 73) at the dose level of 1000 mg/kg bw/day observed on days 8 and 9 of the pre-pairing period were considered not to be test item-related.


1.2 FOOD CONSUMPTION OF MALES

See attached tables on pp. 4, 8

Pre-pairing Period

No effects on mean food consumption were observed at the dose levels of 100 and 300 mg/kg bw/day.

Treatment of males with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of mean food consumption. From day 1 to 8 of the pre-pairing period, mean food consumption at the high dose was 15.5 compared to 20.5 g/animal/day in the control group. After this period mean food consumption recovered and was similar to the respective control value. Therefore the reduction of food consumption was considered not to be adverse.

The overall differences in mean food consumption of males during the pre-pairing period were: +2.3%, ±0.0% and -13.0% at the dose levels of 100, 300 and 1000 mg/kg bw/day respectively (percentages refer to the respective value in the control group).


1.3 FOOD CONSUMPTION OF FEMALES

See attached tables on pp. 5-7; 9-11

Pre-pairing, Gestation and Lactation Periods

No effect on mean food consumption was observed at the dose level of 100 mg/kg bw/day during the entire treatment period.

Treatment of females with the test item at the dose level of 300 mg/kg bw/day resulted in mean food consumption slightly lower than the respective control values from day 1 to 8 of the pre-pairing period. After this period mean food consumption increased and was similar to the respective control values during the second week of the pre-pairing period as well as during the gestation period. Therefore the reduction of food consumption was considered not to be adverse.
During the lactation period mean food consumption was statistically significantly lower at this dose level when compared to the respective control value. Because no dose-dependent reduction of the mean food consumption through all dose levels was observed, the alteration observed at the mid dose was considered not to be test item-related but a result of biological variability.

Treatment of females with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of mean food consumption. From day 1 to 8 of the pre-pairing period mean food consumption at the high dose was 11.4 compared to 15.5 g/animal/day in the control group. After this period mean food consumption recovered and during the second week of the pre-pairing period was even higher than the respective control value (not statistically significant). Therefore the reduction of food consumption was considered not to be adverse. During the gestation period mean food consumption was similar to the respective control values.

The overall differences in mean food consumption of females at the dose levels of 100, 300 and 1000 mg/kg bw/day were respectively: +1.2%, -4.3% and -8.0% during the pre-pairing period, +1.4%, -3.7% and ±0.0% during the gestation period and -2.1%, -15.7% and +4.3% during the lactation period (percentages refer to the respective values in the control group).


1.4 BODY WEIGHTS OF MALES

See attached tables on pp. 12, 13, 19, 20, 26, 27

Pre-pairing and Pairing Periods

No effects on mean body weight gain and mean body weights were observed at the dose level of 100 mg/kg bw/day during the entire treatment period.

Treatment of males with the test item at the dose level of 300 mg/kg bw/day caused reduction of body weight gain during the pre-pairing period. This reduction was more pronounced at the beginning of the treatment (statistically significant from day 2 to 5 of the pre-pairing period). Afterwards body weight gain recovered and during the pairing period was similar to the respective control values. Alteration of the body weight gain had no apparent impact on the body weights of males. During the entire pre-pairing and pairing periods mean body weights at this dose level were similar to the respective control values. Therefore the reduction of body weight gain was considered not to be adverse.

Treatment of males with the test item at the dose level of 1000 mg/kg bw/day caused statistically significant reduction of body weight gain during the entire pre-pairing period. This resulted in a statistically significantly lower mean body weights noted from day 3 to 7 of the pre-pairing period. Both, body weight gain and body weights recovered and during the pairing period were similar to the respective control values. Therefore the reduction of body weight gain and reduction of body weights were considered not to be adverse.

The overall mean body weight gains in the control group and group treated with the test item at the dose levels of 100, 300 and 1000 mg/kg were respectively: +9.0%, +8.8%, +7.2% and +4.4% during the pre-pairing and +9.6%, +9.6%, +9.5% and +9.74% during the pairing periods (percentages refer to the respective time intervals).


1.5 BODY WEIGHTS OF FEMALES

See attached tables on pp. 14-18; 21-25; 28-30

Pre-pairing, Gestation and Lactation Periods

No effects on mean body weight gain and mean body weights were observed at the dose levels of 100 and 300 mg/kg bw/day during the entire treatment period.

Treatment of females with the test item at the dose level of 1000 mg/kg bw/day caused reduction of body weight gain during the pre-pairing period. This reduction was more pronounced at the beginning of the treatment (statistically significant from day 3 to 6 of the pre-pairing period) and resulted in the statistically significant reduction of the mean body weights noted from day 3 to 8 of the pre-pairing period. Afterwards body weight gain and body weights recovered and toward the end of the pre-pairing period as well as during the entire gestation and lactation periods were similar to the respective control values. Therefore the reduction of body weight gain and reduction of body weights were considered not to be adverse.

The overall mean body weight gains in the control group and group treated with the test item at the dose levels of 100, 300 and 1000 mg/kg were respectively: +7.0%, +7.0%, +7.1% and +7.1% during the pre-pairing, +57.1%, +53.7%, +48.1% and +57.1% during the gestation and +3.9%, +4.8%, +3.7%, and +6.5% during the lactation periods (percentages refer to the respective time intervals).


2 REPRODUCTION AND BREEDING DATA

2.1 MATING PERFORMANCE AND FERTILITY

No effects of the treatment with the test item on mating performance or fertility were observed at any dose level. Mean precoital times were 2.4, 2.7, 2.8 and 2.5 days in order of ascending dose levels.

One female (no. 50, paired with male no. 10) in the control group and 2 females (no. 63, paired with male no. 23 and no. 67, paired with male no. 27) at the dose level of 300 mg/kg bw/day did not mate during the first pairing period. All these females mated after the change of the male on the first day of the second pairing period.

Except for 1 female (no. 43, mated with male no. 3) in the control group and 1 female (no. 51, mated with male no. 11) at the dose level of 100 mg/kg bw/day, all females were pregnant and gave birth to live pups.

Consequently, fertility index and conception rate were 90% in the control group and at the dose level of 100 mg/kg bw/day and 100% at the dose levels of 300 and 1000 mg/kg bw/day whereas gestation index was 100% in all groups.


2.2 DURATION OF GESTATION

No effect of the treatment with the test item on duration of gestation was observed at any dose level. In order of ascending dose levels, mean duration of gestation was 21.1, 21.4, 21.4 and 21.4 days.


2.3 CORPORA LUTEA COUNT

No effect of the treatment with the test item on the number of corpora lutea was observed at any dose level.

The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups: 15.6, 14.0, 13.6 and 14.9 in order of ascending dose levels.


2.4 IMPLANTATION RATE AND POST-IMPLANTATION LOSS

No effects of the treatment with the test item on the number of implantation sites or incidence of pre- and post- implantation loss were observed at any dose level.

No significant differences in the number of implantation were observed. Mean number of implantation sites per dam were: 12.4, 11.2, 9.7 and 11.2 in order of ascending dose level.

No marked post-implantation loss was observed in any group. The mean incidence of post-implantation loss as a percentage of total implantations was 1.8%, 2.0%, 1.0% and 0.0% in order of ascending dose levels.


2.5 LITTER SIZE AT FIRST LITTER CHECK

No effect of the treatment on the number of live pups at first litter check was observed at any dose level.

The mean number of live pups per litter was similar in all groups: 12.2, 11.0, 9.6 and 11.2 in order of ascending dose level.


2.6 POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

No effect of the treatment on the postnatal loss was observed at any dose level.

The total numbers of pup lost during the first 4 days of lactation were 3, 1, 12 and 3 in order of ascending dose levels, which corresponded to 2.7%, 1.0%, 12.5% and 2.7% of living pups, respectively.

The postnatal loss at the dose level of 300 mg/kg bw/day was statistically significantly higher when compared to the control group. However, all 12 pups lost at this dose level came from a single litter (no. 66). These pups were killed for ethical reason after termination of the parent female on day 3 post partum. Therefore the higher incidence of postnatal loss at this dose level was not test item-related.


3 TERMINAL FINDINGS - PARENTAL ANIMALS

3.1 ORGAN WEIGHTS

No effect of the treatment on the weights of testes and epididymides was observed at any dose level.

Mean values of the organ weights (absolute and relative to body weight) were similar in all groups.


3.2 MACROSCOPICAL FINDINGS

No test item-related macroscopical findings were noted at any dose level neither in males nor in females.

The incidences of testes reduced in size in 1 male (no. 3) and seminal vesicles reduced in size in 1 male (no. 9), both noted in the control group, were considered to be of spontaneous occurrence.


3.3 HISTOPATHOLOGY FINDINGS

A thorough examination of the reproductive organs both in males and females did not reveal any change related to the treatment with the test item.

No microscopical findings were noted in any female in the control group or at the high dose level as well as in the infertile female (no. 51) at the dose level of 100 mg/kg bw/day.

In males microscopical findings were found in 5 males in the control group, and 1 male each at the dose levels of 100, 300 and 1000 mg/kg bw/day. All these findings (listed below) were considered to be incidental.

In the control group:
- the infertile male no. 3 (with macroscopical changes) had pronounced atrophy and edema in the testes, aspermia and mixed cell infiltration in epididymides and a slight atrophy of prostate gland and seminal vesicles,
- male no. 2 had mixed cell infiltration in prostate gland,
- male no. 6 mixed cell infiltration in epididymides,
- male no. 9 (with macroscopical changes) had mixed cell infiltration in epididymides, slight atrophy of seminal vesicles and inflammation of prostate gland,
- the infertile male no. 10 had mixed cell infiltration in prostate gland and epididymides,

At the dose level of 100 mg/kg bw/day:
- the infertile male no. 11 had mixed cell infiltration in epididymides and prostate gland.

At the dose level of 300 mg/kg bw/day:
- the infertile male no. 23, had inflammation of prostate gland.
No findings were noted during the microscopical examination of the second infertile male (no. 27) at this dose level.

At the dose level of 1000 mg/kg bw/day:
- two males, nos. 32 and 34 had mixed cell infiltration in epididymides.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on observations the NOAEL (No Observed Adverse Effect Level) for general parental toxicity and for reproduction and development was considered to be 1000 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the dose level of 1000 mg/kg bw/day, body weight gain and body weights of pups were lower then the respective control values. Because of the minor nature of this effect it was considered not to be adverse.

CONCLUSION: No adverse effects

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

No findings were noted at first litter check at any dose level.


1.2 SEX RATIOS

No effect of the treatment on the fetal sex ratio were noted at any dose level.

The proportion of male pups at first litter check was 54%, 42%, 51% and 47% in order of ascending dose levels.


1.3 PUP WEIGHTS TO DAY 4 POST PARTUM

See attached tables on pp. 31, 32

No effect of the treatment on pup body weights on days 1 and 4 post partum were noted at the dose levels of 100 and 300 mg/kg bw/day. Also body weight gain of pups during the 4 days of the lactation period at these dose levels was similar to the control values.

At the dose level of 1000 mg/kg bw/day, body weights of pups on day 1 post partum were by 3.4% lower then the respective control value. Because the body weight gain of pups during the first 4 days of the lactation period was lower at this dose level, the difference between the body weights of pups at the high-dose and in the control group increased. Consequently, mean body weights of pups on day 4 post partum at the high-dose was by 6.0% lower than the respective control value. The effects on body weight gain and body weights of pups at this dose level were not statistically significant. The possibility that they are caused by the treatment of the parent females with the test item was not excluded. Because of the minor nature of this effect it was considered not to be adverse.

On day 1 post partum mean body weights of pups were 5.8, 6.0, 6.0 and 5.6 g in order of ascending dose levels.
Differences in mean pup body weight gain from day 1 to 4 of the lactation period were +44.8%, +46.7%, +46.7% and +41.1% in order of ascending dose levels.


1.4 MACROSCOPICAL FINDINGS

No macroscopical findings were noted during the necropsy of pups at any dose level.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

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 were observed

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 (100)	3 (300)	4 (1000)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated (A)	10	10	10	10
Number of pregnant females (B)	9	9	10	10
Number of females which reared their pups until day 4 post partum (C)	9	9	9	10

(A)  Female nos. 50, 63 and 67 were mated during the second pairing period

(B)  Female nos. 43 and 51 were not pregnant.

(C)  Female no. 66 and its pups were killed for ethical reason on day 3 post partum. 

See attached document for tables of results.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL (No Observed Adverse Effect Level) for general parental toxicity and for the reproduction and development was considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the 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 substance once daily. Males were treated over a 14-day pre-pairing period and during the pairing periods up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

The following dose levels were used:

Group 1:                0 mg/kg body weight/day (control group)

Group 2             100 mg/kg body weight/day

Group 3:            300 mg/kg body weight/day

Group 4           1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (PEG 300).

 

The following results were obtained:

 

 

PARENTAL ANIMALS

 

Mortality and General Tolerability

 

No test item-related deaths occurred during the study.

 

No clinical signs were observed in the control group and at the dose level of 100 mg/kg bw/day.

 

At the dose levels of 300 and 1000 mg/kg bw/day in both sexes pushing head through the bedding material and salivation after application were observed during the most of the treatment period. These symptoms were considered to be signs of discomfort caused by the treatment with the test item but not adverse effect. A short-lasting diarrhea was observed in males at the dose levels of 300 and 1000 mg/kg bw/day and females at the dose level of 1000 mg/kg bw/day and activity reduced for 1 to 3 days was observed in males at the dose level of 1000 mg/kg bw/day.

These symptoms were considered to be test item-related but not adverse effects.

 

Food Consumption

 

Treatment with the test item at the dose levels of 100 mg/kg bw/day caused no effects on food consumption neither in males nor in females.

 

Treatment with the test item at the dose level of 300 mg/kg bw/day caused in females a not statistically significant reduction of mean food consumption at the beginning of the treatment period. Afterwards mean food consumption increased and was similar to the respective control values.

 

Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of mean food consumption in males and females at the beginning of the treatment. Afterwards mean food consumption increased and was similar (in males) or even slightly higher (in females) than the respective control values.

 

Because food consumption recovered after the initial reduction and in all groups remained similar to the respective control values until the completion of the treatment, the reduction of food consumption was considered not to be adverse.

 

Body Weights

 

Treatment with the test item at the dose level of 100 mg/kg bw/day caused no effects on body weight gain and body weights neither in males nor in females.

 

Treatment with the test item at the dose level of 300 mg/kg bw/day caused in malesa statistically significant reduction of body weight gain at the beginning of the treatment period. Afterwards body weight gain increased and was similar to the respective control values.

 

Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of body weight gain in males and females at the beginning of the treatment. This resulted in a statistically significant reduction of body weights in both sexes. Afterwards body weight gain and body weights increased in both sexes and were similar to the control values during the remaining treatment period.

 

Because body weight gain and body weights recovered after the initial reduction and in all groups remained similar to the respective control values until the completion of the treatment, the reduction of body weight gain and body weights was considered not to be adverse.

 

Reproductive Data

 

The relevant reproductive data: mating performance, fertility, gestation, number of corpora lutea, implantation rate and post-implantation loss were not affected by the treatment with the test item.

 

Organ Weights

 

No effects on the weight of testes or epididymides were observed at any dose level.

 

Macroscopical Findings and Histopathological Examinations

 

No test item-related macroscopical findings were noted at any dose level neither in males nor in females.

 

LITTER DATA - F1 PUPS

 

No clinical signs were noted at first litter check and during the 4 days of the lactation period.

 

No macroscopical findings were noted during the necropsy of pups at any dose level

 

No effects on body weights of pups were observed at dose levels of 100 and 300 mg /kg bw/day.

 

At the dose level of 1000 mg/kg bw/day, body weight gain and body weights of pups were lower then the respective control values. The effects were not statistically significant. The possibility that the treatment of the parental females with the test item at this dose level caused a disturbance in the body weight of their pups was not excluded. Because of the minor nature of this effect it was considered not to be adverse.

Based on the results of this study, the NOAEL (No Observed Adverse Effect Level) for general parental toxicity and for the reproduction and development was considered to be 1000 mg/kg bw/day.